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Fungal Allergy and Pathogenicity Chemical Immunology Vol. 81

Series Editors Luciano Adorini, Milan Ken-ichi Arai, Tokyo Claudia Berek, Berlin Anne-Marie Schmitt-Verhulst, Marseille

Basel · Freiburg · Paris · London · New York · New Delhi · Bangkok · Singapore · Tokyo · Sydney Fungal Allergy and Pathogenicity

Volume Editors Michael Breitenbach, Salzburg Reto Crameri, Davos Samuel B. Lehrer, New Orleans, La.

48 figures, 11 in color and 22 tables, 2002

Basel · Freiburg · Paris · London · New York · New Delhi · Bangkok · Singapore · Tokyo · Sydney Chemical Immunology Formerly published as ‘Progress in Allergy’ (Founded 1939) Edited by Paul Kallos 1939–1988, Byron H. Waksman 1962–2002

Michael Breitenbach Professor, Department of Genetics and General Biology, University of Salzburg, Salzburg Reto Crameri Professor, Swiss Institute of Allergy and Asthma Research (SIAF), Davos Samuel B. Lehrer Professor, Clinical Immunology and Allergy, Tulane University School of Medicine, New Orleans, LA

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus.

Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.

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XII Foreword M. Breitenbach, Salzburg; R. Crameri, Davos; S.B. Lehrer, New Orleans, La.

1 Introduction A.M. Chiu, J.N. Fink, Milwaukee, Wisc.

3 References

5 Impact of Current Genome Projects on the Study of Pathogenic and Allergenic Fungi M. Breitenbach, Salzburg; R. Crameri, Davos; S.B. Lehrer, New Orleans, La.

6 Candida albicans Genome Project 7 Cryptococcus neoformans Genome Project 7 Aspergillus fumigatus Genome Project 8 The Postgenomic Era 8 References

10 Fungal Aerobiology: Exposure and Measurement E. Levetin, W.E. Horner, Atlanta, Ga.

11 Sampling Equipment 13 Analysis 15 Patterns of Variation 19 Measurement Problems 22 Clinical Implications 24 Conclusions 25 References 28 Allergy to Basidiomycetes A. Helbling, K.A. Brander, Bern; W.E. Horner, Atlanta, Ga.; S.B. Lehrer, New Orleans, La.

28 Prevalence of Airborne Basidiospores Including Indoor Exposure 30 Prevalence of Sensitization 31 Clinical Aspects 31 Respiratory Allergy 32 Bronchial and Nasal Challenges 32 Atopic Eczema 33 Contact Dermatitis 33 Food Allergy 33 Invasive Mycosis 34 Source Materials 35 Basidiomycetes as Allergens 35 B. edulis (cèpe) 35 Calvatia spp. (Puffballs) 36 Coprinus spp. (Inky Cap) 37 Ganoderma spp. 37 Pleurotus spp. (Oyster Mushroom) 38 Psilocybe spp. 39 Cross-Reactivity 40 Molecular Biological Approaches to Basidiomycete Allergens 40 Screening of C. comatus Phage Display Library – 7 Putative Allergens 41 Cop c 1, the First C. comatus Allergen 44 Cop c 2 44 Conclusions 44 References

48 The Allergens of Cladosporium herbarum and Alternaria alternata M. Breitenbach, B. Simon-Nobbe, Salzburg

49 Problems with Reproducibility of Mold Extracts and the Study of Mold Allergens 51 Experience with Specific Immunotherapy in the Treatment of Mold Allergies 54 Importance of Molecular Cloning Techniques 54 Cloning, Analysis, Production and Clinical Testing of the Allergens of Cladosporium and Alternaria 54 Cloning Methods 57 The Major Allergen of A. alternata, Alt a1 59 The Major Allergen, Cla h1 59 Enolase, an Important Allergen in C. herbarum and A. alternata, Exhibits Cross-Reactive Properties 61 Epitope Mapping of C. herbarum Enolase 62 Other Allergens 63 A. alternata

Contents VI 63 C. herbarum 66 Production of Highly Purified Alt a1 and A. alternata Enolase for Clinical Use 66 Alt a1 66 A. alternata Enolase 67 Clinical Study with Recombinant A. alternata Allergens 68 Discussion and Outlook 68 Acknowledgments 69 References

73 Molecular Cloning of Aspergillus fumigatus Allergens and Their Role in Allergic Bronchopulmonary Aspergillosis R. Crameri,Davos

74 Host Defense Mechanisms and A. fumigatus-Related Diseases 75 A. fumigatus-Related Diseases 76 Diagnosis and Epidemiology of Allergic Bronchopulmonary Aspergillosis 77 Cloning and Molecular Characteristics of A. fumigatus Allergens 82 Clinical and Diagnostic Evaluation of Recombinant A. fumigatus Allergens 83 The Role of A. fumigatus Allergens in Allergic Bronchopulmonary Aspergillosis 86 Utilizing the Allergenic Repertoire of A. fumigatus Identified with Advanced Technologies 87 Conclusions 87 Acknowledgments 88 References

94 Defense Mechanisms of the Airways against Aspergillus fumigatus: Role in Invasive Aspergillosis H.F. Kauffman, J.F.C. Tomee, Groningen

95 Overview of Manifestations of Aspergillosis 95 Nonpathogenic Saprophytic Colonization 96 Aspergilloma 96 Hypersensitivity-Induced Aspergillosis 96 Aspergillus Asthma 97 Allergic Bronchopulmonary Aspergillosis 98 Hypersensitivity Pneumonitis 99 Invasive Pulmonary Aspergillosis 99 Host defense against Aspergillus 99 Innate Defense Strategies of Airways against Fungi 105 The Adaptive Immunological Response 108 Effect of Immunosuppressing Agents on the Innate and Adaptive Defense Mechanisms 109 Concluding Remarks 110 References

Contents VII 114 Secreted Proteinases and Other Virulence Mechanisms of Candida albicans M. Monod, Lausanne; M. Borg-von Zepelin, Göttingen

115 Site-Directed Mutagenesis and Genomics to Investigate Virulence Factors 116 Dimorphism 116 Switching System of C. albicans 118 Adherence 119 Secreted Hydrolases 119 Phospholipases 120 Secreted Aspartic Proteinases 121 Aspartic Proteinases in the Adherence Process 123 Aspartic Proteinases in Deep-Seated Candidiasis 124 Acknowledgment 124 References

129 Cutaneous Mycology T. Hawranek, Salzburg

130 Pathogenesis of Cutaneous Fungal Infection 132 Clinical Mycology 132 Superficial Mycoses 132 Pityriasis versicolor 133 White Piedra 133 Black Piedra 134 Tinea nigra 134 Cutaneous Mycoses 134 Dermatophytosis (Ringworm, Tinea) 134 Special Clinical Patterns 134 Tinea corporis 138 Tinea inguinalis (Tinea cruris) 138 Tinea capitis 141 Tinea barbae 141 Tinea pedis (‘Athlete’s Foot’) 142 Tinea manuum 142 Onychomycosis 143 Skin and Nail Infections by Molds 143 Scytalidium Species 144 Onychomycosis Caused by Scopulariopsis brevicaulis 144 Onychomycosis Caused by Other Molds 144 Tinea incognito 144 ‘Superficial’ Candidiasis 145 Oral Candidiasis (Thrush) 146 Pseudomembranous Candidiasis

Contents VIII 146 Erythematous (Atrophic) Candidiasis 146 Candida Leukoplakia (Chronic Plaque-Like or Hyperplastic Candidiasis) 146 Genital Candidiasis 146 Candida Balanitis 146 Vaginal Candidiasis 147 Scrotal and Perianal Candidiasis 147 Candida Paronychia and Onychomycosis 147 Congenital Candidiasis 148 Chronic Mucocutaneous Candidiasis 148 Other Forms of Candidiasis 149 Subcutaneous Mycoses 149 Sporotrichosis 149 Chromoblastomycosis 150 Mycetoma (Madura Foot) 150 Subcutaneous Zygomycosis 150 Entomophthoromycosis 150 Localized Mucormycosis 151 Lobomycosis 151 Rhinosporidiosis 151 Systemic (Deep) Mycoses Caused by Pathogenic Fungi 152 Coccidioidomycosis 152 Paracoccidioidomycosis 152 Histoplasmosis 153 Blastomycosis 153 Systemic (Deep) Mycoses Caused by Opportunistic Fungi 153 Candidiasis 154 Cryptococcosis 154 Systemic Zygomycosis 155 Aspergillosis 155 Pseudallescheriasis 155 Rare Mycoses 155 Hyphomycosis 156 Pheohyphomycosis 156 Hyalohyphomycosis 156 Protothecosis 156 Penicillium marneffei Infection 157 Laboratory Diagnosis 157 Collection of Material 157 Direct Examination (Potassium Hydroxide Test) 158 Culture and Additional Tests 159 Histopathology, Serology, Identification of Yeasts and Molds 163 Principles of Therapy 163 References

Contents IX 167 Toxins of Filamentous Fungi D. Bhatnagar, J. Yu, K.C. Ehrlich, New Orleans, La.

168 Mycotoxicosis 170 Mycotoxicology 170 Natural Occurrence of Mycotoxins 171 History of Mycotoxins 173 Classification of Mycotoxins 173 Economic Impact of Mycotoxins 176 Detection and Screening of Mycotoxins 176 Selected Mycotoxins 177 Aflatoxins 179 Toxic Polyketides Other Than Aflatoxins 179 Ochratoxins 181 Cyclopiazonic Acid 181 Patulin 182 Penicillic Acid 182 Citrinin 183 Sterigmatocystin 183 Zearalenone 184 Fumonisins 187 Trichothecenes 190 Alternaria Toxins 191 Neurotropic Mycotoxins 191 Ergot Alkaloids and Related Toxins 192 Other Neurotropic Mycotoxins 193 Management of Mycotoxin Contamination 193 Preharvest Control 194 Postharvest Control 196 Dietary Consideration 196 Summary 197 References

207 Phylogeny and Systematics of the Fungi with Special Reference to the Ascomycota and Basidiomycota H. Prillinger, K. Lopandic, W. Schweigkofler, Wien; R. Deak, Budapest; H.J.M. Aarts, Wageningen-UR; R. Bauer, Tübingen, K. Sterflinger, G.F. Kraus, Wien, A. Maraz, Budapest

210 The Kingdom Mycobionta (Eumycota) or True Fungi 212 Morphological Differentiation within the Kingdom Mycobionta 215 Sexual Differentiation within the Kingdom Mycobionta 224 Phylogenetic Relationships among the Chytridiomycota and Zygomycota 226 Phylogenetic Relationships among the Ascomycota and Their Anamorphs 228 Hemiascomycetes 231 Protomycetes

Contents X 233 Euascomycetes 234 Chaetothyriales 235 Eurotiales 237 Onygenales 239 Hypocreales 242 Ophiostomatales 242 Phyllachorales 243 Sordariales 244 Microascales 244 Dothideales 246 Pleosporales 248 Leotiales 249 Pezizales 249 Phylogenetic Relationships of the Basidiomycota and Their Anamorphs 253 Urediniomycetes 255 Cystobasidiales 255 Microbotryales 256 Uredinales 257 Ustilaginomycetes 258 Malasseziales 258 Georgefischerales 259 Microstromatales 259 Ustilaginales 260 Hymenomycetes 261 Tremellales 263 Cantharellales 264 Gomphales 264 Thelephorales 265 Polyporales 267 Hymenochaetales 268 Russulales 268 Boletales 269 Schizophyllales 270 Agaricales 272 Genotypic Identification 275 Acknowledgments 275 References 294 Notes added in proof 296 Glossary 302 Author Index 303 Subject Index

Contents XI Foreword

Fungal organisms (yeasts and molds) are an increasing public health problem (Chiu and Fink) worldwide for several reasons. After transplantation surgery immunosuppressed patients frequently suffer from fungal infections which can become fatal; the AIDS pandemic, which is still on the increase, also leads to fungal infections that are difficult to treat and there is an increase in real and perceived allergic diseases which, in some developed countries, involve 20–30% of the population; a proportion of these allergies is due to inhaled fungal spores and other fungal material. Some of the older patients suffering from allergic or ‘nonallergic’ asthma are particularly prone to fungal infections of the lung. Moreover, fungal toxins (Bhatnagar et al.) still represent an important health problem both in humans and farm animals. Exposure to fungal spores is ubiquitous and, therefore, of pivotal importance for the development of mycoses acquired via the respiratory tract. This situation has also led to increased public awareness of the importance of indoor air quality and to the emergence of aerobiology, which has established itself as a major environmental academic discipline (Levetin and Horner). Fungal infections have led to an increasing demand for antifungal drugs. Compared to the well-known antibacterial antibiotics, these are generally less than satisfactory, because it is more difficult to combat eukaryotic than pro- caryotic pathogens, and severe side effects are still frequent. It is expected that fungal whole genome sequences (Breitenbach et al.), which are now being determined, will be very helpful in devising new antifungal drugs. Fungal genetics plays an increasing role in the study of clinically relevant fungi. Genome sequences will also lead the way to the discovery of new virulence factors which are important as drug targets. There are good reasons for hope, but at present no antifungal drug developed on the basis of fungal genomics is yet on the market. Fungi are different from bacterial pathogens also because they are mostly opportunistic, that is they are present all the time in healthy individuals (for instance, on the skin), and only become dangerous in certain situations. A variety

XII of pathological conditions, including impaired immune function, are believed to cause host susceptibility to fungal infections. The major reason for the increase in systemic mycoses is undoubtedly related to an increased number of patients with congenital or acquired immunodeficiency. Therefore, it is important to study not only the fungal pathogens but also the host factors that contribute to fungal infec- tivity (Kauffman and Tomee, and Monod and Borg-von Zepelin). Until recently, the study of fungal allergy was still in its infancy. It is no exaggeration to say that modern cDNA cloning techniques caused a major breakthrough in this field. Before the advent of allergen cloning, it was difficult, for several reasons, to identify the relevant fungal allergens unequivocally. Commercially available fungal extracts were for the most part not satisfactory for a reliable diagnosis and were not authorized for specific immune therapy (‘hyposensitization’) in many countries. This situation will improve with the advent of well-defined pure recombinant fungal allergens (Helbling et al., Breitenbach and Simon-Nobbe, and Crameri). Nearly all major systematic groups of fungi are now known to contain allergenic and/or pathogenic species (Prillinger et al.). Among the Ascomycota, the new sequence-based phylogeny defines three large groups: the Hemiascomycetes, the Protomycetes (Archiascomycetes) and the Euascomycetes. Presently there is some debate on whether the Protomycetes are primitive Ascomycota (Archiascomycetes) or a derived group of Ascomycota with similarities to the Basidiomycota (Prillinger et al.). Until recently, some Ascomycota (Aspergillus, Alternaria, Cladosporium, Penicillium, Candida and others) were called ‘fungi imperfecti’ (Deuteromycota), but are now recognized as close relatives of ‘perfect’ Ascomycota based on molecular systematics. The Hemiascomycete, Candida albicans, is a very important pathogen and allergen, aspects of which are treated by Monod and Borg-von Zepelin. Among the Euascomycetes, Trichophyton rubrum causes superficial skin infections (Hawranek); Histoplasma capsulatum is an intracellular parasite of the monocyte/macrophage system occasionally causing fatal infections, and Aspergillus fumigatus is a very important allergen and pathogen that is able to colonize the human lungs. Some aspects of Aspergillus infections are the topic of the chapters by Crameri, and Kauffman and Tomee. Cladosporium herbarum and Alternaria alternata (Breitenbach and Simon-Nobbe) are Euascomycetes. They are important causes worldwide of allergies. Pneumocystis carinii, an important pathogenic member of the Protomycetes, is the most common cause of lung infection in AIDS patients. Among the major groups of the Basidiomycota, only the Uredinomycetes contain practically no known pathogens and/or allergens. Rhodotorula mucilaginosa and Rhodotorula glutinis have occasionally been isolated from patients. Interestingly, Malassezia (previously called Pityrosporum) furfur, a human

Foreword XIII pathogen infecting the skin and hair follicles (Hawranek), is a member of the Ustilaginomycetes, a large group of plant pathogens. The most highly developed group of the Basidiomycota, the Hymenomycetes, contain a number of recently recognized important allergy-causing fungi: Coprinus cinereus, Psilocybe cubensis, and puffballs of the genus Calvatia, among others (Helbling et al.). Another Hymenomycete, Cryptococcus neoformans, is an important human pathogen causing the often fatal cryptococcosis of the lung and meningitis. This volume clearly shows the importance of correct fungal systematics to understand the clinically relevant fungi. For this reason, we have included an extensive chapter on the new molecular fungal systematics. This chapter (Prillinger et al.) should be consulted whenever questions appear as to the correct systematic nomenclature and the older synonyms of a fungal organism. We are including a glossary because we feel that the highly specialized terminology, especially of clinical mycology and of systematic mycology, should be explained for nonspecialists. This book addresses not only clinicians who want to learn more about clinically important fungi, but also allergologists, mycologists, biologists and lawyers concerned with the increasing number of lawsuits because of fungal spores in indoor air, which are claimed to be a major reason for ‘sick building syndrome’. We are very grateful to Thomas Nold and the members of the team of Karger (Basel, Switzerland) for their excellent cooperation and patience during the time of collecting and editing the chapters of this book. We are also very grateful to the authors of this book who have spent many days checking and rechecking every chapter, especially to Birgit Simon-Nobbe and to Hansjörg Prillinger. Finally, we thank our families for their support and confidence in the final success of this project.

M. Breitenbach, R. Crameri, S.B. Lehrer

Foreword XIV Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 1–4

Introduction

Asriani M. Chiu, Jordan N. Fink

Medical College of Wisconsin, Department of Pediatrics and Medicine (Allergy and Immunology), Milwaukee, Wisc., USA

Allergy is a hypersensitive response of the immune system to inhaled or ingested proteins, or allergens [1]. Since only some individuals will develop allergy even though many are exposed, a genetic predisposition along with environmental exposure is necessary for the atopic state to occur. The allergic response can involve different organ systems, and symptoms can include nasal congestion, shortness of breath or skin rash, resulting in rhinoconjunctivitis, asthma, urticaria and angioedema, or even anaphylaxis and death. Over 20% of the population has one form of allergic disease or another, and a number of allergens associated with different forms of allergy have been reported all over the world [2]. The concentration of environmental allergens depends on many variables, including climate, vegetation, and air quality. The outdoor allergens are predominantly plant pollens and fungal spores. The indoor allergens include proteins from dust mite, cockroach, animal dander, and also fungal spores. Indoor allergen concentrations are affected by humidity, ventilation, and the presence or absence of pets, carpets, or houseplants. Avoidance or reduction of exposure may be important factors in decreasing the development of allergy to these indoor and outdoor allergens. Fungi are eukaryotic, unicellular (yeasts), dimorphic or filamentous, and usually spore-bearing organisms. A number of biochemical and genetic markers clearly distinguish them from plants and algae. They lack chlorophyll, and dendrograms derived from rDNA sequence comparisons group them outside the plants and animals in a kingdom ‘fungi’. Existing as parasites, saprobes or symbionts they depend on outside sources for nourishment. There are currently over 80,000 described species of fungi, including the yeasts and the molds. The yeasts typically are single-celled-organisms and multiply asexually by budding. The yeasts can also reproduce sexually by the formation of ascospores or basidiospores. Molds are comprised of branching tubular (siphonal or trichal) hyphae, and collectively, all the hyphae of one fungal organism are called the mycelium. Certain pathogenic fungi are dimorphic and able to exist in nature as hyphae and convert to either yeasts or spherules after they infected the body of a human or animal. Under special environmental conditions, some yeasts can form pseudohyphae as well as blastospores, arthrospores, and chlamydospores, depending on the genus and species of yeast. Mycotic diseases include infections – cutaneous, localized, or invasive; mycotoxicoses, which result from the ingestion of the toxic metabolites of fungi and allergy, which will be the main topic of this monograph. Fungi can also produce potent carcinogens. The airborne spores of fungi are usually considered to be etiologic agents of allergic rhinitis and asthma. The prevalence of respiratory allergy to fungi is estimated to be 20–30% among atopic individuals, and 6% of the general population in hot and humid areas of the world. These numbers can be somewhat smaller in other climatic zones. The major allergic manifestations induced by fungi include asthma [3], rhinitis, allergic sinusitis, allergic bronchopulmonary mycoses, and hypersensitivity pneumonitis. Allergy to Alternaria is a risk factor for death in patients with asthma. Allergic bronchopulmonary aspergillosis is a pulmonary disease characterized by immunologic reactions to antigens of Aspergillus fumigatus, and diagnostic criteria include a history of asthma, elevated total serum IgE, immediate cutaneous reactivity to Aspergillus, elevated serum IgE and/or IgG antibodies to A. fumigatus, and central bronchiectasis. Hypersensitivity pneumonitis is a syndrome with a spectrum of clinical features related to the immunologic pulmonary inflammation after the inhalation of a variety of organic dusts, and can present in an acute, subacute, or chronic form depending on the frequency and intensity of the exposure. Other disorders that have been attributed to molds include sick building syndrome [4], and pulmonary hemorrhage and hemosiderosis related to Stachybotrys atra [5]. Sick building syndrome is a term used to describe a constellation of symptoms that include rhinitis, difficulty breath- ing, headaches, flu-like symptoms, and watery eyes, and are associated with poor indoor air quality. S. atra exposure has long been known to cause respiratory symptoms in animals, due to its mycotoxic effects. In 1993–1994, there was a cluster of 10 cases of pulmonary hemorrhage and hemosiderosis in Cleveland, Ohio, USA, that was possibly related to exposure to toxigenic S. atra. Among the groups of fungi, there are three that contain all the species that are important causes of allergic disease. These are the Zygomycota, which include the bread molds (e.g. Rhizopus and Mucor); the Ascomycota, many of which are soil fungi (e.g. Penicillium and Aspergillus) and some that are leaf surface fungi or endophytes (e.g. Alternaria and Cladosporium); and the third group, the Basidiomycota, which include the mushrooms, puffballs, plant rusts and smuts. There have been problems obtaining adequate quantities of fungal extracts that are qualitatively and quantitatively reproducible in terms of allergen content.

Chiu/Fink 2 There are different considerations when selecting source materials for a fungal extract, and these include whether fungi should be grown in liquid culture medium, or harvested from the wild, and whether spores or hyphae should be used. There also is variability in allergen content and in the amount of allergen produced from fungal sources, even if grown in the laboratory environment. The allergenicity of the extract or antigen fraction can be evaluated by skin prick or intradermal testing of allergic subjects. Specific in vitro tests, radioallergosorbent test or enzyme-linked immunosorbent assay, may also be used. In addition to standardization difficulties with fungal extracts, it is also often difficult to identify mold-allergic patients [6]. This may be due to the sizable number of mold species, and the difficulty in deciding which are clinically relevant and important, as well as the fact that no allergy-testing materials may be available for evaluation. Furthermore, cross-reactivity among fungi may make the interpretation of the skin tests more difficult. In the last several years there has been progress in the area of characterization of fungal allergens. It is now possible to grow allergenic fungi in synthetically defined media with less variability. The common fungal allergens have been characterized, and can be cloned and expressed by molecular biologic techniques [7]. Based on the primary structure of the fungal allergens, it appears that the majority of the identified allergens are proteins associated with essential basic fungal metabolism, such as protein synthesis or glycolysis. Recombinant allergens currently available from fungi include allergens from Alternaria alternata, including Alt a 1, Cladosporium herbarum, including Cla h 6, and A. fumigatus, including Asp f 1. T cell and B cell epitope mapping has been studied with Asp f 1 from A. fumigatus, and B cell epitope mapping with Asp f 2 and Cla h 6. The increasing knowledge of the three-dimensional structures and the T cell and B cell epitopes of fungal allergens may provide information to help develop newer therapeutic strategies including immunotherapy with synthetic peptides, recombinant mutant allergen isoforms, or other modified allergens. As more research is done in the area of molecular biology of fungal allergens, we will hopefully begin to have better quality and more standardized allergen extracts, and thus have better ways to diagnose and treat patients. This text will provide a comprehensive overview of the current knowledge of fungal allergy and its immunopathogenicity.

References

1 Blumenthal MN, Rosenberg A: Definition of an Allergen (Immunobiology); in Lockey RF, Bukantz SC (eds): Allergen and Allergen Immunotherapy. New York, Dekker 1999, pp 39–51. 2 Wüthrich B: Epidemiology of allergic disease: Are they really on the increase? Int Arch Allergy Appl Immun 1989;90:3–10.

Introduction 3 3 O’Hollaren MT, Yunginger JW, Offord KP, Somers MJ, O’Connell EJ, Ballard DJ, Sachs MI: Exposure to an aeroallergen as a possible precipitating factor in respiratory arrest in young patients with asthma. N Engl J Med 1991;324:359–363. 4 Cooley JD, Wong WC, Jumper CA, Straus DC: Correlation between the presence of certain fungi and sick building syndrome. Occup Environ Med 1998;55:579–584. 5 Etzel RA, Montana E, Sorenson WG, Kullman GJ, Allan TM, Dearborn DG, Olson DR, Jarvis BB, Miller JD: Acute pulmonary hemorrhage in infants associated with exposure to Stachybotrys atra and other fungi. Arch Pediatr Adolesc Med 1998;152:757–762. 6 Karlsson-Borga A, Jonsson P, Rolfsen W: Specific IgE antibodies to 16 widespread mold genera in patients with suspected mold allergy. Ann Allergy 1989;63:521–526. 7 Kurup VP, et al: Molecular biology and immunology of fungal allergens. Indian J Clin Biochem 2000;12(suppl):31–42.

Prof. Jordan N. Fink, MD, Medical College of Wisconsin, Department of Pediatrics and Medicine, Allergy and Immunology, Milwaukee, WI 53226 (USA) Tel. ϩ1 414 266 6840, Fax ϩ1 414 266 6437, E-Mail [email protected]

Chiu/Fink 4 Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 5–9

Impact of Current Genome Projects on the Study of Pathogenic and Allergenic Fungi

M. Breitenbacha, R. Cramerib, S.B. Lehrerc

a Department of Genetics and General Biology, University of Salzburg, Salzburg, Austria; b Swiss Institute of Allergy and Asthma Research, Davos, Switzerland, and c Department of Medicine, Tulane University Medical Center, New Orleans, La., USA

The present era of biomedical research is characterized by a predominance of genetic approaches and models to investigate the basis and causes of disease. Although this tendency is not without dangers (such as underestimating lifestyle and environmental influences on the development of disease or underestimating the importance of biochemical thinking), application of the genetic paradigm has undeniably been highly successful and will remain so for decades to come. The chapters in this book testify to this fact. While modern molecular genetics has been at the center of the biomedical revolution in research for about 25 years now, its latest development, genomics, has only just started to influence medical research. It is therefore appropriate to briefly describe the potential of studying whole genome sequences as a new tool in biomedical research. In particular, the ongoing Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans genome-sequencing projects are very useful examples to illustrate the importance of whole genome sequences. Other genome- sequencing projects of pathogenic fungi are also under way or will soon be started (for instance, Pneumocystis carinii). Generally speaking, the progress of sequencing technology and of bioinfor- matics (data management, gene prediction algorithms, functional analysis in silico) has made feasible the sequencing and analysis of many genomes which have approximately the size of the genome of Saccharomyces cerevisiae (12.5 millions of base pairs), the first eukaryotic genome to be sequenced completely [1]. The genome sequence of S. cerevisiae, a prototypic and nonpathogenic fungal organism is therefore of utmost importance for systematic comparison with the fungal pathogens. Most known fungal genomes are in the maximal range of 15–25 million base pairs and many sequencing projects are under way [2]. Why is a whole genome sequence a useful tool for medical research? What are the immediate and long-term benefits for our understanding and for the diagnosis and treatment of disease? Parasites and pathogens generally have a specialized genetic outfit to successfully interact with the host. Frequently those genes are not housekeeping genes but specialized genes which (1) are not found in closely related free- living microorganisms and (2) are transcriptionally induced during interaction with the host. Knowing these genes is of great importance for understanding the pathomechanisms, for finding and identifying new antibiotic targets and for the development of new drugs [3]. The methods used to achieve these goals include annotation of all genes, genome-wide transcript analysis, comparisons of closely related pathogenic vs. nonpathogenic organisms (like C. albicans vs. S. cerevisiae), the systematic deletion and phenotypic analysis of candidate genes coding for new drug targets, and testing of deletion mutants in an animal model of the disease. The development of new antifungal drugs is extremely important as the major classes of currently used antifungal drugs suffer from numerous problems like development of resistance and/or severe side effects. The morbidity and mortality rates associated with infections caused by fungal pathogens are high, and prevention, diagnosis and treatment of these infections remain quite difficult. At the same time, typical fungal infections are rapidly increasing due to an increasing number of immune-suppressed patients after organ transplantation, after HIV infection and as a consequence of certain cancers of the immune system (leukemias). C. albicans is presently considered the most important fungal pathogen in terms of frequency of infection and total cost of treatment.

Candida albicans Genome Project

‘Shotgun sequencing of the diploid C. albicans genome … is complete with the sequencing of 10.4 haploid genome equivalents, which is sufficient to ensure identification of all of the genes in this organism’ [4]. A complete (or even partial) annotation of the Candida genes has not been published at the time of writing. Current information about the ongoing project can be found at two websites: http://www-sequence.stanford.edu/group/candida and http://alces.med.umn.edu/Candida.html. The genome sequence has so far revealed a very close relationship between C. albicans and S. cerevisiae including the discovery of the Candida mating type locus [5] and of many of the

Breitenbach/Crameri/Lehrer 6 genes needed for meiosis and recombination as indicated by close sequence similarity to the respective S. cerevisiae genes [4]. Low-frequency mating has been achieved experimentally [6, 7], but meiosis has so far not been observed. About 80% of the genes found are closely related in sequence to S. cerevisiae, but the remaining genes have no counterpart in the well-known S. cerevisiae genome. It can be assumed that these 20% of the genome have to do with the parasitic lifestyle of C. albicans and are a rich ground for the discovery of new drug targets and of genes coding for new virulence factors. One aspect of the virulence of C. albicans that became very clear in recent years is the formation of hyphae of this dimorphic yeast [8]. The genome project showed that formation of hyphae is under the control of a hierarchy of signaling systems and transcription factors, homologues of which are well known from S. cerevisiae studies, in particular the MAP-kinase and the cAMP/PKA pathways [Alistair J.P. Brown, pers. commun.] [9]. The genome of Candida tropicalis, a human pathogen that is closely related to C. albicans, has been partially sequenced, and about 1,000 new genes have been found [10].

Cryptococcus neoformans Genome Project

Current information about the ongoing project [11] can be found at the website http://www-sequence.stanford.edu/group/C.neoformans/index.html. At the time of writing (December 2001) sequencing of the C. neoformans serotype D genome is ‘nearing completion’ [12]. A detailed bioinformatic analysis of the C. neoformans genome sequence is not yet available. C. neoformans is a member of the Basidiomycota and the only known pathogen from this group. Still, the pathogen-specific genes, for instance genes responsible for the formation of the protective coat that is of prime importance for virulence [12], have no counterpart in closely related basidiomycetes. This situation is very similar to the one encountered in C. albicans and its close relative, S. cerevisiae. Formation of the protective coat is controlled by the cAMP-dependent protein kinase, PKA [13].

Aspergillus fumigatus Genome Project

Plans for sequencing the complete genome of this important ascomycete human pathogen were announced in early 2000 [14]. The sequencing project is a joint effort of the University of Manchester, the Sanger Centre and the Institut Pasteur. Details about the current progress of this project can be found at their website http://www.aspergillus.man.ac.uk. A parallel project is under way at the

Genome Projects of Pathogenic and Allergenic Fungi 7 Institute of Genomic Research (TIGR) in the USA (http://www.tigr.org). Comparison with the genetically well-characterized and nonpathogenic species Aspergillus nidulans will certainly help in the identification of virulence factors and drug targets.

The Postgenomic Era

The genome sequence of S. cerevisiae has been completed, providing us with a powerful tool for comparisons between pathogenic and non-pathogenic fungi. Sequence projects involving pathogenic fungi have been nearly completed or started recently. Embedded within these genomes are the sequences of both, essential genes governing the metabolic pathways and survival of the organisms and genes involved in pathogenesis. One of the central goals of the postgenome era will be to relate individual genes to specific functions and particularly to identify genes responsible for the pathogenic behavior of certain fungal organisms. This approach will lead to the identification of target genes for the development of new generations of antifungal drugs. However, we should keep in mind that most fungi are opportunistic pathogens and therefore depend on specific underlying conditions of the human host to be able to become pathogens. In spite of the progress achieved in fungal genomics, we should not forget that fungal infections have been shown to be influenced by host-specific conditions and that many drug responses of single individuals are influenced by inherited differences in our genes. Clearly, this means that human genomics and genetics also have a strong impact on the study of fungal pathogenicity. To win the fight against fungal pathogens both, genome projects and an in-depth investigation of host-pathogen interactions will be required.

References

1 Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG: Life with 6000 genes. Science 1996;274:546–567. 2 Souciet J, Aigle M, Artiguenave F, Blandin G, Bolotin-Fukuhara M, Bon E, Brottier P, Casaregola S, de Montigny J, Dujon B, Durrens P, Gaillardin C, Lepingle A, Llorente B, Malpertuy A, Neuveglise C, Ozier-Kalogeropoulos O, Potier S, Saurin W, Tekaia F, Toffano-Nioche C, Wesolowski-Louvel M, Wincker P, Weissenbach J: Genomic exploration of the hemiascomycetous yeasts. 1. A set of yeast species for molecular evolution studies. FEBS Lett 2000;487:3–12. 3 Black T, Hare R: Will genomics revolutionize antimicrobial drug discovery? Curr Opin Microbiol 2000;3:522–527. 4 Tzung KW, Williams RM, Scherer S, Federspiel N, Jones T, Hansen N, Bivolarevic V, Huizar L, Komp C, Surzycki R, Tamse R, Davis RW, Agabian N: Genomic evidence for a complete sexual cycle in Candida albicans. Proc Natl Acad Sci USA 2001;98:3249–3253.

Breitenbach/Crameri/Lehrer 8 5 Hull CM, Johnson AD: Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans. Science 1999;285:1271–1275. 6 Hull CM, Raisner RM, Johnson AD: Evidence for mating of the ‘asexual’ yeast Candida albicans in a mammalian host. Science 2000;289:307–310. 7 Magee BB, Magee PT: Induction of mating in candida albicans by construction of MTLa and MTLalpha strains. Science 2000;289:310–313. 8 Madhani HD, Fink GR: The control of filamentous differentiation and virulence in fungi. Trends Cell Biol 1998;8:348–353. 9 Whiteway M: Transcriptional control of cell type and morphogenesis in Candida albicans. Curr Opin Microbiol 2000;3:582–588. 10 Blandin G, Ozier-Kalogeropoulos O, Wincker P, Artiguenave F, Dujon B: Genomic exploration of the hemiascomycetous yeasts: 16. Candida tropicalis. FEBS Lett 2000;487:91–94. 11 Heitman J, Casadevall A, Lodge JK, Perfect JR: The Cryptococcus neoformans genome sequencing project. Mycopathologia 1999;148:1–7. 12 D’Souza CA, Heitman J: Dismantling the Cryptococcus coat. Trends Microbiol 2001;9:112–113. 13 D’Souza CA, Alspaugh JA, Yue C, Harashima T, Cox GM, Perfect JR, Heitman J: Cyclic AMP- dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans. Mol Cell Biol 2001;21:3179–3191. 14 Brookman JL, Denning DW: Molecular genetics in Aspergillus fumigatus. Curr Opin Microbiol 2000;3:468–474.

Dr. M. Breitenbach University of Salzburg, Department of Genetics and General Biology, Hellbrunnerstrasse 34, A–5020 Salzburg (Austria) Tel. ϩ43 662 8044 5786, Fax ϩ43 662 8044 144, E-Mail [email protected]

Genome Projects of Pathogenic and Allergenic Fungi 9 Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 10–27

Fungal Aerobiology: Exposure and Measurement

Estelle Levetina, W. Elliott Hornerb

a Faculty of Biological Science, University of Tulsa, Tulsa, Okla., and b Air Quality Sciences, Inc., Atlanta, Ga., USA

The earliest well-known aerobiology experiment was likely Pasteur’s demonstration of the causes of putrefaction. Pasteur was attacking the paradigm of spontaneous generation, but also established that airborne dust included germs – or bioaerosols. Even with the relatively simple equipment available in the 19th century, some basic principles of aerobiology were established. One of these was the value of volumetric sampling. As early as 1900, at least one microbiology textbook stated that settle plates were unreliable indicators of airborne microbial concentrations because the volume of air sampled was unknown [1]. Perhaps surprisingly, there were several volumetric air samplers available even then. These were cumbersome, but useful. Known sample volumes were collected in one sampler by siphoning a liquid from one container to another. The displacement of the siphoned liquid drew air into the sampler and the volume of air sampled equaled the volume of the liquid. Quantitative data were produced without batteries, power sources or calibrations. Several methods were also available to capture airborne particles. An agar- coated cylinder was the collector in one sampler. After being filled with air and capped, the spores were allowed to settle onto the inner walls of the cylinder. Packed cylinders of sterile sand were also used as filters that could then be plated onto culture media. Although inventive, these samplers were cumbersome, as a result extensive sampling was impractical. The renaissance of aerobiology was essentially the mid 20th century when samplers were developed that made repeat observations feasible. The two chief samplers developed at this time were the Hirst spore trap and the Andersen culture impactor [2, 3]. The half-century since their development has seen aerobiology greatly expand and support fields such as plant pathology, industrial hygiene, palynology and allergy. Numerous other samplers have been developed and improvements on some of these original designs are available [4]. The more important developments at this time however were synthetic. Gregory [5] was involved early on in the renaissance of aerobiology. He contributed to and also first compiled the fundamental descriptive work of aerobiology and the basic science of the behavior of small particles in fluids such as air. Later, Lacey’s work was fundamental to the early application of aerobiology sampling and the aerodynamics of biological particles to the problems of human health associated with exposure to bioaerosols [6]. These are among the giants upon whose shoulders current workers are standing. Presently, emphasis is on the application of immunochemical, molecular, and chemical analyses of air samples. Monoclonal antibodies have been developed for selected agents and these have been used to detect these agents in air [7]. Gene probes and primers for specific agents have also been developed. In addition, the improved sensitivity of some chemical analyses from biological materials show promise of being able to detect biological agents of significance to human health. Perhaps the most notable of these is endotoxin.

Sampling Equipment

Every exposed surface, either indoors or outdoors, likely is contaminated with fungal spores from the air. Although the function of most airborne spores is to colonize new surfaces (substrates), these spores are often remarkably adapted to staying airborne, which means that effort is required to capture them. More importantly, different spores are adapted to different ways of being removed from the air. Spores of leaf surface fungi or foliar pathogens may be adapted to interception by or impaction onto leaves under turbulent conditions. Spores of litter decay fungi, on the other hand, may be adapted to settling onto surfaces in quite still air. Air normally contains spores of both types of fungi, often in addition to spores adapted to other dispersal mechanisms, such as water splashes. Thus, capturing a representative sample of spores from air is a challenge since these spore types differ in their ability to stay airborne, or conversely differ in their ease of capture. Passive sampling by collecting spores that settle onto a surface (settle plate, or gravity plate) has two critical flaws. Unless precautions are taken to assure perfectly still air over the surface, the volume of air that is sampled cannot be determined and the method is thus nonvolumetric. Volumetric sampling is essential if a concentration of spores is to be determined, which is crucial for rigorously comparing results from different samples. More importantly, passive

Fungal Aerobiology: Exposure and Measurement 11 Table 1. Impactor samplers with their advantages and weaknesses

Sampler Advantage Weakness

Rotorod Independent of wind orientation Relatively inefficient for spores Formerly was less expensive Tauber No power requirements Not suitable for fungal spores Burkard 7 day Time discrimination Expensive Personal Burkard Ease of use Single grab sample Self-contained fan Variable (user) slide preparation Air-O-Cell Inexpensive, ease of use Single grab sample Less efficient for small spores Andersen Very efficient for spores Requires externally powered pump SAS Portable Expensive

sampling cannot compensate for spores that are more difficult to recover from air, and hence favors the recovery of spore types that are easier to recover. This produces a bias in the type of fungi that are recovered, in addition to the nonvolumetric nature of passive sampling. Active sampling is any sampling that uses artificial force (not gravity) to help collect spores from air. This usually means some type of fan or pump to induce and control airflow through the sampler. If properly calibrated, this can provide samples from a known volume of air (volumetric) that allows results to be expressed as concentrations and therefore comparable with other results. More importantly, an artificial force can largely compensate for differences among spore types in ease of recovery (resistance to capture). This provides a far more representative sample of the spore mix that is present in the air sampled. The samplers most often used for aeroallergen monitoring and for indoor air studies are impactors and filters, although there are samplers based on other principles, such as impingers and electrostatic precipitators [4]. Filter samplers have been used to collect spores as well as antigens from outdoor and indoor air. Studies using filter samplers helped establish that pollen allergens are not always associated with pollen grains. Starch grains released from pollen and other pollen fragments have been shown to contain allergens [8]. Filter samplers are also used in indoor allergen studies. Although not widely used, impingers have several advantages. The sample is collected in a liquid, which can protect spores for culture analysis. The impinger fluid can also be divided and analyzed in different ways. The true workhorses of aeroallergen samplers however, are all impactors (table 1). The rotorod, the Burkard (Hirst-type) spore trap, the personal Burkard, the Air-O-Cell, and the Andersen (several types) are all impactor

Levetin/Horner 12 samplers. The surface to agar system (SAS) sampler is a portable type of sieve plate impactor sampler. Each of the samplers mentioned above has a limitation; each has an advantage and a favored use. The Andersen is a sampler for culturable (sometimes called viable) mold spores, the others sample for particles (spores and/or pollen) without a need for culture. The principle that all impactors use to capture particles is to sharply deflect an airstream so that particles in the airstream break free due to inertia and impact onto the sampling surface. In the rotorod sampler, the surface (sampling rod) is accelerated rapidly through the air, which is deflected around the rod. With the other samplers mentioned, a pump or fan is used to direct an airstream through a sharp 90° turn in front of a sampling surface at a critical (short) distance from the air inlet of the sampler. As the airstream is forced to turn sharply, any entrained particles (pollen grains and mold spores) break free of the airstream and impact onto the sampling surface. Tauber traps are nonvolumetric sedimentary samplers used by scientists wishing to study the deposition of pollen over weeks, months, or even years. Pollen collected by Tauber traps is considered analogous to pollen incorporated into sediments. In general these traps are not suitable for analysis of airborne fungal spores.

Analysis

Air samples collected by the equipment described above are analyzed by various methods. Culturing and microscopy are the most common methods; however, biochemical, immunochemical, and molecular techniques are finding greater applications. Culturing is the method of analysis required for several instruments, such as Andersen samplers, which impact air samples onto an agar surface. Culturing can also be used with samples from impingers, filter samples, or dust samples. Although a variety of culture media can be utilized for air sampling, malt extract agar is generally suggested for mesophilic fungi [9] and DG-18, which contains dichloran (2,6-dichloro-4-nitroaniline) and 18% glycerol for xerophilic fungi, especially from dust. Samples are usually incubated for 5–10 days at room temperature. Identification often depends on observing reproductive structures and methods of spore development, which can be seen in culture. As a result, the major advantage of culture analysis is that many fungi can be precisely identified provided trained personnel are available. The disadvantage is that not all fungi are able to grow or reproduce on the culture medium used. Some fungi are not able to grow at all in culture, while others require specialized media to grow or reproduce. Also, some airborne spores lose viability

Fungal Aerobiology: Exposure and Measurement 13 quickly. Although these dead spores obviously cannot grow in culture, they may still retain their allergenicity. Consequently, results derived from culture methods only reflect a portion of the air spora. Samples collected by Burkard (Hirst-type) spore traps, rotorod samplers, Air-O-Cell samplers, and MK-3 samplers are usually analyzed by microscopy. This is the most important method of analysis for outdoor air samples, which usually contain both fungal spores and pollen. Samples are typically stained with basic fuchsin or phenosafranin to assist in pollen identification although fungal spores are seldom stained by these chemicals. Wet mounts can be prepared and samples examined immediately; however, many investigators prepare permanent slides especially for Burkard samples, and this may take up to 24 h for the mounting medium to harden. The major advantage of this method is that microscopy permits the visualization and counting of all spores, both viable and nonviable. The disadvantage of this method is that some spores cannot be identified. Small spherical spores that lack distinctive morphological features cannot be identified. In addition, Penicillium and Aspergillus spores, which can be common in indoor samples, cannot be distinguished from each other by this method. Microscopy also requires highly skilled personnel to identify the many types of spores and pollen found in air samples. Additionally, for accurate counting and identification, the slide should be analyzed at a magnification of 1,000ϫ; this requires considerable time. Biochemical methods can be used to detect specific fungal compounds, such as ergosterol or ␤-glucans. Ergosterol is a sterol found in fungal cell membranes, whereas ␤-glucan is a fungal wall carbohydrate. Assays for these compounds can be used with samples collected by an impinger or on filters as well as dust samples. These assays provide an estimate of total fungal biomass, but they cannot supply any identification. Immunochemical and molecular methods are normally used when specific allergens or pathogens are the focus of study, because only the target organism will be detected by these techniques [7]. With the large variety of airborne fungi that occur in the atmosphere it is not practical to use these methods for routine analysis of outdoor air samples. However, they are of value when the presence of a specific fungus is suspected in an indoor environment. Air samples collected by an impinger, on filters, or even on spore trap samples can be analyzed with these procedures as can dust samples. For immunochemical analysis, it is necessary to have monoclonal or polyclonal antibodies specific for the fungus under investigation. These antibodies can be used in an ELISA assay or tagged with a fluorescent marker. The polymerase chain reaction (PCR) is an example of a molecular method that can also be used to identify specific fungi. Segments of DNA from an environmental isolate are amplified. Nucleic acid probes specific for the target organism are added and will bind to the amplified

Levetin/Horner 14 DNA if the organism of interest is the same as the environmental isolate. Both immunochemical and molecular methods are very sensitive and specific, and results can be obtained in a shorter period of time than through culturing. The disadvantage is that only predetermined fungi can be detected if present. Despite this limitation, these methods are finding greater applications for analysis of air samples [10].

Patterns of Variation

Daily pollen and spore counts provide a snapshot of the atmosphere in a given area and typically represent the average daily concentration from a single air sampler. However, a great deal of variability exists within the atmosphere and physicians should be aware of the clinical implications of this variability. Major causes of variability relate to the diurnal rhythms of spore discharge, discharge related to weather events, seasonal effects, and spatial effects. Also contributing to variability are the ways that different spore types respond to environmental conditions. The major groups of fungi important in aerobiology are the Ascomycota (the ascomycetes or sac fungi), the Basidiomycota (the basidiomycetes or club fungi) and asexual fungi (deuteromycetes or imperfect fungi). The ascomycetes include numerous microfungi as well as larger cup fungi and morels. They produce sexual spores known as ascospores within a sac-like structure called an ascus. Generally there are eight ascospores within each ascus, and they are explosively shot from the ascus when moisture is available. The basidiomycetes include mushrooms, bracket fungi and puffballs. They produce sexual spores called basidiospores on a club-like structure, which is known as a basidium. Typically, four basidiospores are produced by each basidium, and numerous basidia line the gills of mushrooms and the pores of bracket fungi. Like the ascospores, basidiospores are actively released when moisture is available. Also included within the basidiomycetes are the smut fungi and the rust fungi, two groups of plant pathogens that produce enormous numbers of airborne spores. The asexual fungi reproduce through the formation of asexual spores called conidia. Although sexual reproduction is rare or absent entirely in this group, the majority of these fungi are ascomycetes, with a small percent being basidiomycetes. The asexual fungi are extremely successful and are the most abundant microfungi in the environment. Many asexual fungi are leaf surface or soil saprobes, and on most days their conidia dominate the air spora. During dry weather, the spores of many asexual fungi show an early to mid-afternoon peak in spore discharge and an early morning low [5, 11, 12]. The daily peak usually occurs when temperatures and wind speeds are high, and

Fungal Aerobiology: Exposure and Measurement 15 20 20 16 16 Boletus Coprinus 12 12 8 8 4 4 0 0 Percent of average daily total Percent of average daily total 0:00 2:00 4:00 6:00 8:00 0:00 2:00 4:00 6:00 8:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00

20 20 16 16 Ganoderma Agaricus 12 12 8 8 4 4 0 0

Percent of average daily total Percent of average daily total 0:00 2:00 4:00 6:00 8:00 0:00 2:00 4:00 6:00 8:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00

Fig. 1. Diurnal variation in airborne levels of basidiospores from four genera of basidiomycetes.

humidity is low. These spores are often referred to as the ‘dry air spora’ and include conidia of Cladosporium, Alternaria, Drechslera, Epicoccum, Curvularia, Botrytis, and Pithomyces as well as other genera of asexual fungi and smut spores. Release of these spores is passive and related to the meteorological conditions, especially wind speed. In temperate areas the dry air spora dominates the atmosphere from spring through fall except during periods of rain. Basidiospores usually show a strong diurnal rhythm with peak concentrations during late night to early morning hours and lowest levels in the late afternoon. The spores are actively discharged from the basidium by a mechanism that requires atmospheric moisture but not rainfall. As a result peak atmospheric levels occur when humidities are high. In a recent study of spores in the Tulsa atmosphere total basidiospores showed peaks from 4:00 a.m. to 6:00 a.m. [13]. At a second site south of Tulsa, total basidiospore levels also showed a 4:00 a.m. peak; however, when individual genera of basidiomycetes were examined, the time of the peak varied with the spore type (fig. 1). Agaricus basidiospores showed a peak at midnight, Boletus spores at 2:00 a.m., Ganoderma at 4:00 a.m. and Coprinus basidiospores showed an 8:00 a.m. peak. Although the dispersal of basidiospores does not depend on rain, basidiomycete fruiting bodies, such as

Levetin/Horner 16 mushrooms and puffballs, frequently develop within a few days after rainfall. As a result, high concentrations of airborne basidiospores released from these fruiting bodies generally occur during seasons when rainfall is frequent [14]. Ascospores also require moisture for discharge. Some types of ascospores are released during periods of high humidity and show a diurnal rhythm similar to basidiospores; however, most ascospores are only released during or after rainfall [15]. Ascomycetes vary in their response to rainfall. Some species begin releasing spores within 10–15 min after the start of rain, while other species release spores only when the reproductive structures begin to dry following a rain event [13, 15, 16]. The rainfall requirement for many ascomycetes tends to obscure any diurnal rhythm; however, when rain is present some ascomycetes show rhythms with either daytime or nighttime peaks depending on the species [15]. Besides triggering the active release of ascospores, rain can cause the passive dispersal of other spores by various mechanisms. Members of the dry air spora can be dispersed by raindrops as they strike leaf surfaces and other substrates [17]. The initial raindrops that hit the leaf can cause vibrations that result in a puff of conidia being dislodged from the surface. This mechanism may explain some anomalous air-sampling data that showed increases of Cladosporium, Alternaria, and other conidia during rain events [11, 18, 19]. Rain splash is another method of spore dispersal for some fungi [20]. Spores dispersed by rain splash are generally formed in mucilage, which inhibits dispersal by wind. During rain, the initial drops dissolve the mucilage. This leaves a spore suspension available for dispersal by subsequent raindrops. Although these mechanisms describe dispersal during rain, rain also cleanses spores from the atmosphere, especially members of the dry air spora [21]. In addition to rainfall, other meteorological events can cause variability in atmospheric spore levels. Wind gusts and other conditions associated with approaching weather systems can have dramatic effects on the air spora. Although spore levels in Tulsa are often high, remarkable increases (spore plumes) in spore levels have occurred on some days. These spore plumes last for a short time, and hourly concentrations have exceeded 100,000 spores/m3. One of these events occurred on 30 September 1998. Between 10:00 a.m. and noon on that day, the hourly concentrations increased from about 27,000 to 144,000 spores/m3. The spore plume coincided with increases in both wind speed and barometric pressure. The daily average of approximately 40,000 spores/m3 on 30 September 1998 clearly did not reflect variability during the day nor the magnitude of the exposure risk at noon and the early afternoon hours. Meteorological factors are also responsible for seasonal variability in the air spora. In most temperate areas, spore concentrations tend to increase in late spring often showing a significant correlation with increasing temperatures [13]. Many studies have shown highest levels of airborne spores typically

Fungal Aerobiology: Exposure and Measurement 17 occurring from late spring through early fall [12, 22–26]. In tropical and subtropical areas, airborne spores may be abundant year round or follow seasonal rainfall patterns. Some components of the air spora are produced by plant pathogenic fungi, and their seasonal peaks may parallel the flowering or harvesting of various crop plants. The dependence on the life cycle of the host is especially pronounced for smut fungi, which only have a single reproductive phase each year. Although smut spores are found in the Tulsa atmosphere for at least 6 months of the year, different smuts are prevalent at different seasons. The most common types in the spring include smut species that infect Bermuda grass, Johnson grass, oats, and wheat, while in the fall the most common spores were produced by the fungus that causes corn smut and those infecting some native grasses [27]. Pathogenic ascomycetes may also show parallels with the host plant life cycles. For example, in the spring Venturia ascospores are produced on dead apple leaves that have overwintered in orchard [28, 29]. The asexual spores of this pathogen are produced later in the growing season. The atmosphere contains a heterogeneous mixture of fungal spores from many different sources. Although there are major similarities in the air spora within an area, at any one time or place the air spora may be dominated by nearby sources of spores [30]. Air samples collected from a single roof top sampler do not reflect the exposure closer to a source. Concentrations of airborne basidiospores from the bracket fungus Ganoderma were compared from two sites in Tulsa using Burkard spore traps. The central sampling station was located on the roof of a building on the University of Tulsa campus at 12 m above ground. The second station was in a residential neighborhood; the intake orifice at this site was at nose level, 1.5 m above ground. Ganoderma basidiospores are typically in the atmosphere for 6 months of the year [31]. During the season peak in 1987 (July to September), the average daily concentration from the roof top sampler was 94 spores/m3, while the nose level sampler had a significantly higher mean of 180 spores/m3 (p Ͻ 0.05). Although both samplers showed parallel day-to-day fluctuations, the nose level sampler had consistently higher concentrations of Ganoderma spores (fig. 2). By contrast total basidiospore levels showed little difference during this period [14]. These data suggest that the higher Ganoderma concentrations from the nose level sampler reflected nearby sources. Although no fruiting bodies were seen in the immediate vicinity of the sampler, they were common in the surrounding area. The atmospheric variability has also been examined in previous studies. Solomon et al. [32] examined intrasampler variance and found small but consistent differences between the recoveries of four adjacent rotorod samplers and also between the recoveries of three adjacent rotoslide samplers. Although the authors suggested that the intrasampler variance reflected minor differences in

Levetin/Horner 18 1000 900 800 700 3 600 500 400

Spores/m 300 200 100 0 Site B 7/1/87 8/1/87 Site A 9/1/87

Fig. 2. Average daily concentration of airborne Ganoderma spores from two locations in Tulsa, Okla., during the months of July, August, September 1987.

the functioning of the samplers, it is possible that the inherent variability of atmospheric bioaerosols also contributed to the variance. Hall [33] specifically examined the question of atmospheric variability using an experimental array of nine Tauber traps located approximately 6 m apart. He found that the mean annual pollen deposition from the sampler array had a standard deviation of 10.5% [33]. Tauber traps are sedimentary samplers that rely on gravity to assess the composition of the atmosphere. As a result, the variance seen in that study could not be attributed to differences in sampler function. While it is not possible to routinely analyze multiple samples, the clinical significance of the atmospheric variability should always be considered.

Measurement Problems

The variability in the atmosphere means that the average spore count does not apply to all times of the day or to all parts of a city (or region). There are additional sources of variance not related to the atmosphere that the physician should also recognize. Although the Burkard (Hirst-type) spore trap is one of the most widely used air-sampling instruments, there is no standard counting method used in analyzing the spores or pollen captured by this type of sampler. The most accurate method of analysis would be to count the entire exposed surface; however, this is not practical because of time limitations. As a result only a portion of the daily capture is counted. Because the sampler drum with attached tape moves at 2 mm per hour by the intake orifice, microscopic

Fungal Aerobiology: Exposure and Measurement 19 a

Fig. 3. Three counting methods frequently used for analysis of slides from a Burkard spore trap. a Twelve transverse sweeps. b Single longitudinal sweep. c Four longitudinal sweeps. analysis of the slide at 4-mm intervals (perpendicular to the direction of movement) can provide information on the concentrations every 2 h throughout the day. This allows determination of diurnal rhythms for the various spore or pollen types. Conversely, one or more longitudinal sweeps parallel to the direction of movement will provide information on the average daily concentration. Three counting methods are widely used by aerobiologists: 12 transverse sweeps (fig. 3), a single longitudinal sweep and 3 or 4 longitudinal sweeps. The accuracy of these counting methods has been addressed in several studies [34–36]. Each counting method resulted in errors when compared to the totals for the whole slide. Kapyla and Penttinen [34] found that the 12 transverse sweep method gave reliable estimates of daily concentrations of airborne pollen, whereas 1 or 2 longitudinal sweeps gave unreliable estimates. Comtois et al. [35] showed that both 12 transverse and 4 longitudinal sweeps had lower percent errors than the single longitudinal sweep. These two studies specifically addressed pollen counting, but Sterling et al. [36] compared the single longitudinal sweep with the 12 transverse method for the major spore types found in the atmosphere. They found that the 12 transverse method generally had higher concentrations, but both methods sensed parallel fluctuations in daily concentration. Comparisons with the counts for the total slide showed that neither

Levetin/Horner 20 method was equivalent to the actual count but the 12 transverse sweep method gave slightly better approximations. These studies suggest that the values obtained for pollen and spore concentrations should be used as good indicators of the outdoor bioaerosol concentrations, but not considered as absolute values. Physicians should also be aware of the reporting lag often involved with disseminating air sampling data through the local media. Typically, individuals involved in daily pollen and spore counting will change the sampler in the morning and then analyze the sample, which was exposed to the atmosphere for the previous 24 h. After analysis the sampling data may be reported to the local media or to a city, county, or state agency. It may be late afternoon or evening before these day-old counts are available to the general public, and atmospheric concentrations may have changed dramatically since sample collection. Because of the time involved in analysis of air samples this delay is unavoid- able; however, aerobiologists are beginning to make the transition from descriptive aerobiology to predictive aerobiology [37]. Reliable forecasts for airborne allergens may be widely available in the future. Measurement problems also occur for air samples analyzed by culturing. Some widely used culture plate impactors pull air through holes in a ‘sieve plate’ which direct the air stream onto the agar plate. This sampler design constrains the trapped particles to land only under the sieve holes, which leads to multiple spores often landing close together. Since culture plate samplers are analyzed by culturing colonies that result from spores landing on the agar surface, any juxtaposed colonies will be indistinguishable and counted as one. Statistical cor- rections are available that will estimate the true count on the plate that is needed to give an observed number of colonies [38]. When using data from sieve plate impactors, such as the Andersen, SAS or similar type, it is important to note whether raw counts or corrected counts were used to calculate the airborne concentration being measured. There is an optimal density for counting particles on a surface. Too few particles give too sketchy a sample of the pollen and spore populations. But too much of a particle load on the sampling surface will decrease capture efficiency and also increase analysis errors due to crowding and for culturable analyses, incur risks of overgrowth and inhibition. Some samplers have a set sampling rate, so that adjusting the count density is not possible. On other samplers, the sample density is easily adjusted by varying sample volume, usually by changing the amount of time sampled. Unfortunately, it is often difficult to gauge the best sample volume until the sample is analyzed. Drawing on experience is perhaps the most practical way to strike a balance between these concerns. Culture plates should have at least 10–30 colonies to be representative. However, if there are more than 60–100 colonies, some colonies will be over- grown or inhibited by other colonies. On spore trap type samplers, sampling

Fungal Aerobiology: Exposure and Measurement 21 should not exceed a point where 50% of the sampling surface is covered, or additional particles will begin bouncing off previously trapped particles rather than sticking to open sampling surfaces. Conversely, enough particles should be trapped to permit a reasonable time of examination to attain counts above 100 particles.

Clinical Implications

Although fungal spores are well-known allergens, only a few epidemiological studies have been conducted that show the direct relationship between outdoor spore levels and symptoms. Recent studies have focused primarily on asthma symptoms. Targonski et al. [39] studied asthma deaths in Chicago from 1985 to 1989. They found that airborne spore levels were significantly higher on days when asthma deaths occurred than on days with no asthma-related deaths. O’Hollaren et al. [40] studied a group of 11 asthma patients with sudden respiratory arrest along with a group of 99 control patients with no history of arrest. They found a significant difference in sensitivity to Alternaria between the two groups. Their data showed 91% of the patients with respiratory arrest were sensitive to Alternaria while only 31% of the control patients were sensitive (p Ͻ 0.001). In addition serum IgE antibodies to Alternaria were elevated in 9 of the 11 patients (the other 2 patients were not tested) and all the respiratory arrest events occurred during the season when airborne Alternaria concentrations were usually high. Neukirch et al. [41] studied the association of skin test sensitivity with asthma severity. Subjects were tested with 11 common allergens, and asthmatics were categorized as having mild, moderate, or severe disease. Using multiple-regression analysis, the investigators found that only Alternaria was independently associated with severe asthma. Two studies by Delfino et al. [42, 43] focused on the effects of outdoor airborne allergens and air pollution on asthma symptoms. Subjects in each study were asthmatics who self-reported daily symptoms and inhaler use. Although both studies were carried out in California, the first was conducted in a coastal area during the fall, and the second was in an inland community in the spring. Both studies found that outdoor fungal spore levels were significantly associated with asthma severity. Among the spore types, the greatest effects on symptom scores were produced by atmospheric basidiospores, especially for children [43]. Similar studies in other parts of the world have also shown correlations between asthma exacerbation and atmospheric basidiospore levels [44, 45]. Basidiospore extracts are generally not included in routine skin testing; however, several studies using noncommercial extracts showed levels of sensitivity comparable to fungi typically used in skin test panels [46]. Also, Horner et al. [47] found

Levetin/Horner 22 that the relative risk of developing asthma was significantly related to basidiospore sensitivity. These studies clearly illustrate the clinical importance of basidiospores as well as other fungal spores as triggers of allergic disease. There is currently great interest and active debate about the health effects of indoor mold exposure. Over the last 10–15 years, numerous studies documented an association between damp and/or moldy indoor environments and an increased prevalence of respiratory complaints. A recent review [48] summarized 9 such studies that included some quantitative measure of indoor mold exposure. Seven of the 9 studies found a positive association between some exposure index and symptoms, although there were no significant associations with other exposure indexes. Interestingly, ‘total culturable counts’ (from either air or dust), i.e. with no attempt at identification, was prevalent among the exposure measures with no association. This is not surprising though, since measuring ‘total culturable mold’ is analogous to counting total pollen levels with no identification. A study on allergic rhinitis would demand counts of particular pollens to associate with symptoms in a population with known sensitization, rather than attempting to associate total pollen counts with rhinitis symptoms. Surprisingly, the allergy literature still tolerates ‘total’ mold counts. In addition to questions of how best to assess exposure, significant problems remain with the diagnostic reagents for assessing sensitization to fungi. This has constrained the ability to distinguish between allergic and other, nonallergic mechanisms possibly inducing mold-associated respiratory symptoms. The debate on mechanisms of mold/damp-associated respiratory complaints is focused on the indoor rather than the outdoor environment. This is at least partly due to the recent increased attention to the indoor environment in general. However, subjects with respiratory complaints that are associated with damp/moldy indoor environments often are not allergic to other indoor allergens and are skin test negative to the available mold allergen reagents. This suggests that affected subjects may be reacting to molds through nonallergic mechanisms. The principal nonallergic mechanisms that have been proposed involve nonspecific biologically active mold components such as ␤-glucans or spore- borne mycotoxins. ␤-Glucans have long been known to have immunomodula- tory effects [49], particularly on macrophages (cytokine production), but also on antibody production [50, 51]. Only a few studies have measured their levels, but increased ␤-glucans have correlated with greater nonspecific respiratory complaints [52]. Numerous molds that are common in damp buildings are capable of producing mycotoxins [53]. There is currently disagreement on the potential clinical effects of mycotoxin exposure from indoor molds, and this is unlikely to be resolved soon due to technical difficulties of measuring mycotoxin levels in

Fungal Aerobiology: Exposure and Measurement 23 indoor environments. A recent synopsis of the available literature, however, suggests that inhalation is a more potent route of exposure than ingestion, and the potential effects of mycotoxins should be critically studied rather than disregarded [54]. Any presumed causal relationship between a building exposure and a clinical effect should include some evaluation of the building rather than relying only on a subject’s assertion. It is important to note that there are several methods to sample indoor environments for dampness and/or mold growth. Source and reservoir sampling are often more valuable than air sampling due to the (often rapid) fluctuations inherent in airborne spore levels [10]. In some cases, a thorough inspection by an experienced building investigator may be sufficient. There should be a clear plan, however, to evaluate the building side of the equation, just as a differential diagnosis for the patient would be pursued. Debate and research continue on whether allergic reactions, nonspecific mechanisms, or their interaction underlie the association of respiratory complaints with damp/moldy buildings. Debate also continues on what method of exposure assessment is most reliable, and there are currently insufficient data to establish any risk-based guidelines for indoor mold levels. Despite these limitations on understanding the respiratory effects of being in damp/moldy buildings, there is ample evidence that documents the association [48]. Fortunately, these respiratory complaints are avoidable, since the engineering aspects of maintaining dry buildings are well known and entirely feasible.

Conclusions

• Aerobiology can be a valuable tool for estimating bioaerosol exposure; however, it is essential that volumetric sampling be used. • Samples collected by gravity plates or slides (passive samples) are biased toward larger spores and greatly underestimate the contribution of many different fungi with small spores. • Culturing and microscopy are standard methods of analysis for routine air sampling. Both methods require highly skilled individuals for spore identification. • Immunochemical and molecular methods are useful for detecting specific fungi from air samples. These techniques are finding greater applications as monoclonal antibodies and DNA probes become available for fungi with known health effects. • The atmosphere contains a mixture of spores that is heterogeneous both in morphology and in ecology. Variations in the air spora are due to many factors: the diurnal rhythms of spore release (ecology) that differ for various

Levetin/Horner 24 fungi, weather-related factors, and spatial factors. The clinical implications of this variability should always be considered. • Measurement problems resulting from counting errors, overloading, or undersampling can all affect the accuracy of air-sampling analyses. As a result, concentrations obtained from air sampling should not be considered as absolute values but as relative levels of exposure. • Only a few recent epidemiological studies have shown a direct relationship between spore levels and asthma symptoms. Although more research is needed, these studies have illustrated the importance of fungal spores both indoors and outdoors as triggers of allergic disease. • Indoor mold exposure is considered potentially very serious by some researchers, and by others to have only minor proven health effects, if any. Epidemiological studies argue strongly that respiratory complaints increase in damp or moldy buildings, though. • Indoor mold measures might include air sampling or source sampling. Interpretation of either should not rely on total counts, but rather on the types of molds recovered and the rank order of types of mold spores (or colonies) recovered.

References

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Levetin/Horner 26 43 Delfino RJ, Zieger RS, Seltzer JM, Street DH, Matteucci RM, Anderson PR, Koutrakis P: The effect of outdoor fungal spore concentrations on daily asthma severity. Environm Health Perspect 1997;105:622–635. 44 Epton MJ, Martin IR, Graham P, Healy PE, Smith H, Balasubramaniam R, Harvey IC, Fountain DW, Hedley J, Town GI: Climate and aeroallergen levels in asthma: A 12-month prospective study. Thorax 1997;52:528–534. 45 Dales RE, Cakmak S, Burnett RT, Judek S, Coates F, Brook JR: Influence of ambient fungal spores on emergency visits for asthma to a regional children’s hospital. Am J Resp Crit Care Med 2000;162:2087–2090. 46 Lehrer SB, Hughes JM, Altman LC, Bousquet J, Davies RJ, Gell L, Li J, Lopez M, Malling HJ, Mathison DA, Sastre J, Schultze-Werninghaus G, Schwartz HJ: Prevalence of basidiomycete allergy in the USA and Europe and its relationship to allergic respiratory symptoms. Allergy 1994;49:460–465. 47 Horner WE, Hughes JM, Lopez M, Lehrer SB: Basidiomycete skin test reactivity is a risk factor for asthma. Pediatr Asthma, Allergy Immunol 2000;14:69–74. 48 Verhoeff AP, Burge HA: Health risk assessment of fungi in home environments. Ann Allergy Asthma Immunol 1997;78:544–554. 49 DiLuzio NR: Update on the immunomodulating activities of glucans. Springer Semin Immunopathol 1985;8:387–400. 50 Okazaki M, Adachi Y, Ohno N, Yadomae T: Structure-activity relationship of (1→3) beta-D- glucans in the induction of cytokine production from macrophages, in vitro. Biol Pham Bull 1995; 18:1320–1327. 51 Rylander R, Holt PG: (1→3) Beta-D-glucan and endotoxin modulate immune response to inhaled allergen. Mediators Inflamm 1998;7:105–110. 52 Rylander R, Norrhall M, Engdahl U, Tunsater A, Holt PG: Airways inflammation, atopy and (1→3) beta-D-glucan exposures in two schools. Am J Respir Crit Care Med 1998;158:1685–1687. 53 Samson RA, Flannigan B, Flannigan ME, Verhoeff AP, Adan OCG, Hoekstra ES (eds): Health Implications of Fungi in Indoor Environments. Amsterdam, Elsevier, 1994. 54 Sorenson WG: Fungal spores: Hazardous to health? Environ Health Perspect 1999;107(suppl 3): 469–472.

Estelle Levetin Faculty of Biological Science, University of Tulsa, Tulsa OK 74104 (USA) Tel. ϩ1 918 631 2764, Fax ϩ1 918 631 2762 , E-Mail [email protected]

Fungal Aerobiology: Exposure and Measurement 27 Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 28–47

Allergy to Basidiomycetes

Arthur Helbling a, Karl A. Brander a, W. Elliott Horner b, Samuel B. Lehrer c

a Division of Allergology, Clinic for Rheumatology, Clinical Immunology and Allergology, Inselspital Bern, Switzerland; b Air Quality Sciences, Inc., Atlanta, Ga., and c Section of Clinical Immunology, Allergy and Rheumatology, Tulane University School of Medicine, New Orleans, La., USA

For many years, fungal spores have been recognized as potential causes of respiratory allergies [1–7]. Among the fungi, molds or microfungi, are well recognized as sources of allergens. Basidiomycetes, another group of fungi, that are physically the largest and morphologically the most complex fungi, can also be allergenic (table 1) [5, 7, 8]. There are about 14,000 basidiomycete species, including mushrooms, bracket fungi, puffballs, toad stools and jelly fungi, as well as the plant-pathogenic rusts and smuts (table 2) [9].

Prevalence of Airborne Basidiospores Including Indoor Exposure

Spores of basidiomycetes (fig. 1) are abundant throughout many parts of the world. Amounts range from 5 to 60% of the total spore load [6, 8, 10–16]. In several studies, basidiospores are among the top categories of airborne spores [10, 11, 13–16]. Atmospheric concentrations of basidiomycete spores vary according to daily and seasonal patterns [8]. For example, mushroom spore counts for a given day are usually highest after midnight and during periods of high humidity. In temperate zones, seasonal peaks of basidiospores are observed in spring and autumn, which coincide with the major periods of mushroom fruiting [11–14, 16]. However, some airborne basidiospores are usually present during any time of the year with temperatures above freezing and adequate moisture. Since many basidiomycetes will not readily grow on laboratory culture media, spore trap samplers such as the Burkard or Lanzoni are typically used to Table 1. Skin test reactivity to basidiomycetes

Authors1 Locality Selection Extract source Species Positive skin criteria material tested test reactors, %

Herxheimer et al. Cardiff, UK summer asthma spores 8 16 Gold et al. Ann Arbor, USA resp. allergy spores 5 64 Hasnain et al. Auckland, NZ resp. allergy spores/tissue 26 10 Lehrer et al. New Orleans, USA resp. allergy spores 15 32 Sprenger et al. Seattle, USA resp. allergy spores 15 30 Helbling et al. Bern, CH unselected spores/tissue 3 3.8

1 Individual references cited in Horner et al. [7, 8].

Table 2. Taxonomic distribution of the subphylum Basidiomycota (following Hawksworth et al. [9])

Class Basidiomycetes (32 orders, 140 families, 473 genera, 14,000 species) Phragmobasidiomycetidae Agarcostilbales (2 spp.) Atriactiellales (13 spp.) Auriculariales (16 spp.) Heterogastridiales (1 spp.) Tremellales (256 spp.) Holobasidiomycetidae1 Agaricales (6,000 spp.) Coprinus, Lentinus, Pleurotus, Psilocybe Boletales (727 spp.) Boletus Ganodermatales (81 spp.) G. lucidum, G. applanatum Lycoperdales (272 spp.) Calvatia Schizophyllales (46 spp.) S. commune Class Teliomycetes (2 orders, 15 families, 167 genera, 7,134 species) (allergenicity demonstrated by Wittich and Stakman, cited in Horner et al. [7]) Class Ustomycetes (7 orders, 10 families, 63 genera, 1,064 species) (allergenicity demonstrated by Cadham, cited in Horner et al. [7])

1 There are an additional 22 orders for which there are no allergen data.

sample airborne basidiospores rather than culture-based samplers. Basidiospores are found in large quantities in Burkard samples taken in the atmosphere of the city of Bern (Switzerland). For example, in 1997 spores of Ganoderma spp. were registered from mid June until the end of October with levels as high as 3,000 spores/m3 [17]. Ganoderma spp. spore load ranked second after

Allergy to Basidiomycetes 29 Fig. 1. Spores of P. pulmonarius (length ϫ width 5 ϫ 10 ␮m).

Cladosporium of all spores identified in this area. Although there is a significant exposure to basidiomycetes, since basidiospores are often not tallied separately in aerobiology studies, their allergenic significance may be underestimated. Basidiomycete antigens have also been demonstrated from indoor house dusts [18, 19]. In the study of Horner et al. [18], 4 of 9 houses had moderate to high levels of basidiomycete antigen in dusts collected on two or more sampling visits [18]. This could be readily explained by infiltration of basidiospores from outdoor sources, as occurs with pollen. Since basidiospores are smaller than pollen grains, their infiltration is likely even greater than that of pollen. Also, if wood becomes wet, such as through leaks, basidiomycetes will decay this wood, which can release basidiospores, often indoors. Mycelial antigens, can also be released, either as mycelial fragments or as secreted antigens which might become incorporated into dust. Thus, although basidiomycete exposure is predominantly outdoors, clearly indoor exposure may be important as well.

Prevalence of Sensitization

Comparisons of sensitization rates to various basidiomycetes, and between basidiomycetes and other groups of fungi have been made in the frame of skin test surveys [20–22]. In a multicenter study in Europe and the USA, it was shown that sensitization to basidiomycetes was as prevalent as sensitization to other well-established allergenic molds, such as Alternaria, Cladosporium, Aspergillus and Fusarium [21]. Generally, 25–30% of subjects with respiratory

Helbling/Brander/Horner/Lehrer 30 allergy were sensitized to at least one basidiomycete extract [8, 20, 21]. Since October 1996, more than 4,000 patients referred to the outpatient allergy Clinic in Bern were tested with extracts of the basidiomycetes Boletus edulis, Coprinus comatus and Pleurotus pulmonarius. Overall, 3.5% of the subjects were sensitized to at least one basidiomycete; 14% of the basidiomycete-positive individuals were mono-sensitized to oyster mushroom (P. pulmonarius), 8% reacted solely to cèpe (B. edulis) and 2% only to shaggy cap (C. comatus). Sensitization only to basidiomycetes was detected in 4% of these individuals. Among atopic subjects, the sensitization rate to basidiomycetes was 9.8% [22]. Thus, it is important to distinguish this study, which was based on unselected allergy clinic attendees, from prior studies that generally selected mold-sensitized or asthmatic subjects. It is estimated from these data that in Switzerland (7.5 million inhabitants) approximately 250,000 individuals are sensitized to at least one of these three mushrooms.

Clinical Aspects

Respiratory Allergy

Respiratory allergic symptoms, primarily of asthma, have been correlated with outdoor spore exposures such as Alternaria, Cladosporium or basidiomycetes [23–25]. The focus of basidiospore allergy studies on asthmatics is justified by the small particle size of basidiospores, many of which are 5–15 ␮m. Many basidiospores are also elliptic or oblong, so their aerodynamic diameter is closer to the smaller dimension that allows them to easily reach the lower airways and initiate an asthmatic reaction. The potential of basidiospores to induce allergic disease (shown in sensitization and provocation studies) is widely accepted; their significance as allergens is still debated though. The clinical relevance of basidiospore exposure and asthma was indicated in a study that correlated several climate and aeroallergen factors with asthma symptoms. Epton et al. [25] found that among the climate and aeroallergen factors measured, only elevated basidiospore levels were associated with aggravation of asthma. Clearly, there is a portion of the asthmatic population for whom basidiospore exposure is clinically relevant, and important. Besides respiratory allergies with hay-fever-like symptoms and asthma in atopic individuals, continuous occupational basidiospore exposure may result in hypersensitivity pneumonitis or extrinsic allergic alveolitis. The basidiomycetes Lentinus edodes, Pleurotus ostreatus, and Merulius lacrymans have all been shown to cause hypersensitivity pneumonitis from occupational exposure [26–28]. Actinomycetes in the compost used to grow button mushroom

Allergy to Basidiomycetes 31 (Agaricus bitorquis) also causes hypersensitivity pneumonitis, but basidiospores of Agaricus are usually not involved in these cases.

Bronchial and Nasal Challenges

Bronchoprovocation and nasal challenges indicate the clinical relevance of basidiomycete extracts to allergic disease. Lopez et al. [29] showed that 5 out of 8 skin-prick-test (SPT)-positive asthmatic subjects had a significant, dose- dependent decrease in forced expiratory volume in 1 s ranging from 20–47% after bronchial challenge with basidiospore extracts of Psilocybe cubensis, Ganoderma lucidum, or Coprinus quadrifidus [29]. More recently, nasal airflow was measured after nasal challenges with three different concentrations of P. pulmonarius spore extracts (0.1, 1.0, 10.0 mg protein/ml) on 12 skin-test- reactive subjects with respiratory allergies (9 asthmatic and 3 rhinitic) [22]. Whereas 6 skin-test-negative controls, including 2 pollen-allergic subjects, were unaffected, all 12 P. pulmonarius skin-test-reactive subjects had a significant reduction in nasal airflow (mean 73%) with subjective symptoms. In a 41- year-old mushroom collector who claimed to recognize Boletus spp. by acute hay fever symptoms, active anterior rhinomanometry demonstrated complete obstruction of the nasal air flow with accompanying conjunctivitis. A facial rash also occurred following nasal challenge with B. edulis extract at a concentration of 0.1 mg protein/ml [30]. Thus, basidiomycetes are widely accepted potential sensitizers that can induce clinically relevant respiratory allergic disease.

Atopic Eczema

Patch testing [31] of sensitized subjects supports the relevance of basidiomycete allergens in skin reactions. Atopy patch-test solutions from cap tissue or extracts of spore containing tissue of C. comatus were applied for 48 h in test chambers on clinically uninvolved skin on the back of 38 subjects with atopic dermatitis [32]. Twelve (32%) patients showed a positive skin reaction either to Coprinus spore extract or ground Coprinus cap in Vaseline. The lesions that were evoked were consistent by histologic and immunohistochemi- cal findings with acute atopic eczema. A few subjects reacted solely to the Coprinus extract. Similar results were obtained with P. pulmonarius spore extracts [30]. These data indicate that basidiomycete allergens can induce eczematous skin lesions in individuals with atopic dermatitis, although the clinical relevance of positive atopy patch tests to aeroallergens is not completely elucidated.

Helbling/Brander/Horner/Lehrer 32 Contact Dermatitis

Several reports have described allergic contact dermatitis from handling mushrooms of various species [33–35]. Although in most cases, extensive contact with the offending mushroom occurred, there is only 1 case reported after initial exposure to the mushroom species. This case involved a plant pathologist, with known contact dermatitis to other mushrooms; this suggests clinically relevant cross-reactivity among various species [33]. Generally, any component of the mushroom may cause cutaneous lesions, but in some cases only particular parts produce symptoms, e.g. elements of the cap but not spores [33]. Thus, taken together, these reports suggest that basidiomycete allergens can also induce clinically relevant dermatologic and respiratory allergic disease.

Food Allergy

In the past, reports of food allergies to mushrooms were largely anecdotal; however, there are 6 well-documented cases of food allergy to mushrooms [36–38]. B. edulis (known colloquially as cèpe, boler, steinpilz, porcini, or King bolete) caused IgE-mediated systemic allergic reactions in 5 occupationally and nonoccupationally exposed subjects. Symptoms ranged from generalized pruri- tus, abdominal pain and diarrhea [37] to urticaria, asthma and even anaphylaxis [36]. The authors are aware of 2 additional subjects with anaphylaxis following ingestion of Boletus spp. By SDS-PAGE IgE-immunoblotting, digestion-stable Boletus proteins between 75 and 80 kD were identified by reactivity in a nonatopic, B. edulis SPT-positive subject [30]. These IgE-binding bands were not detected in B. edulis skin-test-positive atopic subjects with respiratory allergies. Another report described a reproducible eosinophilia and gastrointestinal symptoms consistent with eosinophilic gastroenteritis following chronic shiitake (Lentinus edodes) intake in a 56-year-old male participant in a cholesterol-lowering study [38]. Eosinophilia caused by ingestion of shiitake powder was shown in 40% of healthy subjects [38]; this is a concern since shiitake powder is an increasingly popular dietary supplement.

Invasive Mycosis

Well-documented cases where filamentous basidiomycetes were responsible for mycosis in immunocompromised subjects have recently been reported [39–41]. Chronic sinusitis was the most frequent clinical symptom, but lung

Allergy to Basidiomycetes 33 lesions, brain abscesses, allergic pulmonary mycosis, ulcerative buccal lesions of the mucosa and endocarditis have also been described [39–41]. These cases involved Schizophyllum commune (naturally occurs on decomposing branches), Ustillago spp. (a plant parasite), and Coprinus spp. (turf, dung, rotting wood). De Hoog et al. [42] demonstrated the advantage of PCR to identify the correct species of basidiomycetes in invasive mycosis [42]. These cases emphasize that basidiomycetes are also involved in nonallergic disease.

Source Materials

Only limited data directly compare the allergenic potential of different source materials of fungi. Among basidiomycetes, comparisons have been made for Coprinus, Pleurotus, Ganoderma and Psilocybe [43–46]. In Coprinus quadrifidus, allergens were present in caps, spores, and stalks; in some cases, the caps and stalks exhibited greater allergenic activity than did spores. Pleurotus extracts from washed mycelium (broth-cultured), caps, and spores each contained allergens [44]. Crossed radioimmunoelectrophoresis (CRIE) detected spore allergens that were not in the other tissues, although the total allergenic activity of the spore extract was not greater than that of other tissues, and spore and mycelial extracts of Pleurotus ostreatus were comparable in skin test activity [44]. These observations that fruiting bodies of Coprinus, Ganoderma, and Pleurotus all contain allergens support recent reports of food-induced allergic reactions from eating mushrooms which suggest that edible parts of the mushroom contain clinically relevant food allergens [36–38]. Thus, allergens of basidiomycetes may be present throughout the mushroom, but no data are available on allergen expression in various tissues or developmental stages. Spores are the presumed source for sensitization to fungal allergens. In most skin test surveys, basidiomycete extracts originate from disrupted spores. Though such extracts favor the release of soluble proteins, spore disruption may also release components, which are not normally released. Little is known about the kinetics of allergen release from intact fungal components. Horner et al. [47] compared allergen release in (control) extracts of homogenized spores with that released from intact (nonhomogenized) spores. Calvatia cyathiformis and Psilocybe cubensis both have very thick spore walls. In contrast to the strongly melanized and rather easily wetted walls of P. cubensis, spores of C. cyathiformis are not heavily melanized but are very hydrophobic. Intact spores of these species did not release as much allergen as homogenized spores and generally required at least 24-hour elutions to obtain any significant release of allergens. Conversely, Lentinus edodes and P. ostreatus both have

Helbling/Brander/Horner/Lehrer 34 thin-walled hyaline spores; spores from these species released substantial allergenic activity rather quickly, with little additional release beyond 1 h. This suggests that allergen release is related to spore wall characteristics, at least in some species. The spores with thick, or melanized cell walls are well protected from environmental stress, but also release minimal allergen very slowly. The spores of Lentinus and Pleurotus are probably not well protected by their cell wall from environmental stress, and their allergen content is readily released into the environment.

Basidiomycetes as Allergens

Of the many basidiomycetes to which we are exposed, only 50 or so species have been tested for allergenicity, and about 25 are recognized as significantly allergenic [7, 8]. Allergens identified at the DNA or protein level have been described for six genera of basidiomycetes, and are discussed below.

B. edulis (cèpe) (fig. 2a)

Aqueous extracts of dried (cèpe) mushroom, contain several allergens from 14 to approximately 90 kD by SDS-PAGE/Western blot [36, 37]. The serum of a woman with a food allergy to B. edulis identified three IgE- binding bands at 14, 26, and 39 kD [38]. In a nonatopic, B. edulis SPT-positive subject with recurrent anaphylaxis following the ingestion of B. edulis, a triplet of IgE-binding proteins of molecular weights between 75 and 80 kD were detected [30]. An IgE-binding component of 75 kD has been shown to be resistant to pepsin digestion after 60 min. However, this component could not be detected with sera of Boletus SPT-reactive individuals who otherwise could eat it without clinical symptoms.

Calvatia spp. (Puffballs) (fig. 2b)

Immunoprints with a panel of 19 sera from SPT-reactive subjects to Calvatia cyathiformis crude extracts revealed at least 21 different protein-binding bands [48]. Two of these proteins (pI 9.3 and pI 6.6) reacted with 68 and 63%, respectively, of the sera tested. The most reactive allergen had an estimated isoelectric point (pI) of 9.3 and was particularly labile [49]. Preparative isoelectric focusing was used to isolate the pI 9.3 allergen, which has a molecular weight

Allergy to Basidiomycetes 35 a b

c d

Fig. 2. a B. edulis (photographed by G. Martinelli, Dietikon, Switzerland). b C. cyathiformis (photographed by G. Martinelli, Dietikon, Switzerland). c C. comatus (photographed by G. Martinelli, Dietikon, Switzerland). d G. applanatum (photographed by G. Martinelli, Dietikon, Switzerland).

of 16 kD [50]. A number of the Calvatia allergens were cross-reactive with allergens from 4 other species of Calvatia [51].

Coprinus spp. (Inky Cap) (fig. 2c)

The allergenic activity of different Coprinus tissues was demonstrated by RAST inhibition using separate extracts of spores, caps and stalks [43]. Mycelial extracts of C. comatus and C. quadrifidus had allergenic activity both by skin test and by RAST [52, 53]. IgE-binding bands from 22 to Ͼ100 kD were detected by immunoblots in C. comatus; the most intense were at 29, 33 and 55 kD [22]. Recently, C. comatus allergens have been isolated using a molecular phage display approach [54]. Since several Coprinus spp. are common, and at times abundant, in lawns and gardens, these more ‘domestic’ rather than strictly woodland species obviously cause greater exposure in humans.

Helbling/Brander/Horner/Lehrer 36 Ganoderma spp. (fig. 2d)

Ganoderma spores are morphologically very distinct. Consequently, these spores are easily identified and more frequently reported in spore trap studies than many other basidiospores. Spore and cap extracts of Ganoderma applanatum are skin-test reactive, and spore counts have been generally correlated with patient symptoms [55, 56]. Using 6 sera from skin-test and RAST-positive subjects, SDS-PAGE immunoblots of Ganoderma meredithae spore and cap extracts revealed 10 IgE-binding proteins from 14 to 66 kD [45]. Seven of the 10 allergens occurred in both spore and cap extracts; the remaining allergens of 28, 50 and 66 kD were unique to the cap. IEF immunoprints revealed a total of 16 proteins (pI Ͻ3.5–6.6) of which 8 were found in both the cap and the spore [45]. In G. applanatum spore extracts, 14 allergens were detected by CRIE and additional immunoblots revealed several IgE-binding bands between 18 and 82 kD [57]. Immunoblot inhibition studies with Ganoderma and other basidiomycetes indicated that Ganoderma had little cross-reactivity with other basidiomycetes [58]. Despite the fact that Ganoderma spp. have been studied by more laboratories than any other basidiomycetes, no allergens have yet been isolated or characterized.

Pleurotus spp. (Oyster Mushroom) (fig. 3a)

Since Pleurotus ostreatus is a cultivated mushroom to which significant occupational exposures occur, the allergenicity of this mushroom and inhalation of its spores resulting in hypersensitivity pneumonitis were recognized early [26]. In an interesting contrast, there is significant occupational exposure to spores of Pleurotus mushrooms but essentially none to the commonly cultivated button mushroom (Agaricus bitorquis). Pleurotus releases spores as soon as the cap begins to expand, and prodigious quantities are released before the caps grow to merchantable size. These exposures can lead to hypersensitivity pneumonitis. In contrast, caps of the ordinary button mushrooms are generally harvested before the caps open and release spores, which minimizes exposure. Button mushrooms are grown in compost, which, however, is heavily colonized by actinomycetes, and these spores can induce hypersensitivity pneumonitis in workers. Oyster mushroom spores contain potent allergens capable of inducing rhinitis and bronchoconstriction [59, 60]. At least 5 allergens have been shown by CRIE in spore extracts of Pleurotus [44]. Immunoblots revealed IgE-binding proteins of 10, 29, 30 and 33 kD, as well as some larger IgE-binding molecular components. Using mycelial extracts, at least 13 allergens have been

Allergy to Basidiomycetes 37 a b

Fig. 3. a Pleurotus ostreatus (photographed by G. Martinelli, Dietikon, Switzerland). b Psilocybe cubensis (photographed by A. Helbling).

identified [61]. In a timed test of allergen release from intact spores, allergens were released rapidly from Pleurotus spores [47]. This was in marked contrast to other spore types and may increase the clinical relevance of Pleurotus and other species with thin-walled spores.

Psilocybe spp. (fig. 3b)

Although exposure of most people to Psilocybe spp. is undoubtedly minimal, skin test reactivity to P. cubensis spore extracts is the most significant of all basidiomycetes tested in Europe and the USA [20, 21]. This reactivity probably results from exposure to related species. P. cubensis occurs naturally on the northeastern coast of South America, the Caribbean and the southeastern coast of North America. Although related species occur throughout the temperate northern hemisphere, the distribution of these mushrooms is probably less important clinically than the cross-reactivity patterns of its allergens. Several species in genera closely related to Psilocybe are common lawn

Helbling/Brander/Horner/Lehrer 38 mushrooms, including several Coprinus spp., which are also known allergens that cross-react. Spore and mycelial extracts of P. cubensis (magic mushroom) have been thoroughly characterized; 13 and 11 allergens, respectively, were identified by SDS-PAGE immunoblot analysis [46, 62]. RAST inhibition demonstrated common IgE-binding components, although the spore extract consistently had greater inhibitory potency than the mycelial extract [46]. Using a panel of 11 individual sera (from SPT- and RAST-positive subjects), the allergens that bound IgE from most sera were the 16-kD (82% reactive), the 35-kD (100%) and the 76-kD (91%) proteins. IEF immunoprints indicated that the most reactive proteins were at pI 5.0 (80%), 5.6 (87%), 8.7 (80%), and 9.3 (100%). P. cubensis extracts showed the greatest inhibitory activity of all fungal species tested by RAST inhibition in an evaluation of cross-reactivity [61]. Hence Psilocybe allergens are potent as well as cross-reactive. Monoclonal antibodies were prepared against the 48-kD allergen and may allow future structural analysis [63]. A cDNA clone, the first of a recombinant allergen from a basidiomycete, was isolated that codes for the 16-kD allergen [64]. The cDNA sequence and deduced amino acid sequence indicate homology with cyclophilin which is an abundant and widespread peptidyl prolyl-isomerase of the lumen of the endoplasmic reticulum. In contrast to human cyclophilin, which is not allergenic, fungal cyclophilin seems to be a major allergen in P. cubensis.

Cross-Reactivity

Only three well-documented studies have addressed cross-reactivity among or involving basidiomycetes [46, 58, 61]. These studies all used allergen extracts to inhibit IgE-specific immunochemical assays. RAST inhibition was first used to detect the cross-reactivity of various basidiospore allergen extracts [61]. The second study included spores from six different mushrooms, and the results were concordant with the taxonomic classification of basidiomycetes. Four species from related orders (C. quadrifidus, C. cyathiformis, P. ostreatus, P. cubensis) were significantly cross-reactive; species from two unrelated orders (Ganoderma meredithae, Pleurotus tinctorinus) showed only minimal cross-reactivity with one another or the other four species [58]. Another RAST inhibition study assessed allergenic relationships between basidiomycete and conidial fungi [65]. In this study, there was minimal although detectable cross-reactivity between the two groups. Far stronger reactions were consistently observed though among species within each group. These results indicate that relatively broad cross-reactivity occurs among basidiomycetes;

Allergy to Basidiomycetes 39 this generally follows accepted taxonomic groupings, but far more than 6 species should be tested in order to properly support this conclusion. These observations may be important in future development of useful diagnostic as well as therapeutic basidiomycete reagents. They also could serve as a basis for monitoring basidiospore levels in the atmosphere.

Molecular Biological Approaches to Basidiomycete Allergens

The molecular analysis of basidiomycete allergens has lagged behind that of other aeroallergens in general and other fungi in particular. This is mainly due to a lack of sufficient source material and allergic sera and the fact that basidiomycetes have only recently been recognized as important fungal allergens compared with other fungi. Nevertheless, progress has been made with allergen characterization. Several basidiomycete allergens have been purified and/or produced as recombinant allergens as listed in table 3. The first DNA sequence identified for a basidiomycete allergen was for a 16-kD allergen of P. cubensis [64]. Only limited progress was made with P. cubensis allergens, but the 16-kD allergen was determined by cDNA sequence analysis to be homologous with cyclophilin, an abundant, widespread peptidyl prolyl isomerase. However, the mushroom C. comatus, (extracts from which showed SPT reactivity in 58% basidiomycete-sensitive individuals [22]) has recently been used successfully as a model system. Using the pJuFo filamentous phage display system of Crameri et al. [66], 7 cDNAs were sequenced and 2 C. comatus allergens (Cop c 1 and Cop c 2) were characterized.

Screening of C. comatus Phage Display Library – 7 Putative Allergens

The synthesis of a pJuFo library [66] of C. comatus with sera of basidiomycete-allergic individuals allowed the isolation of 7 putative allergens [30, 54]. Homology searches on various databases showed that generally no other homologies could be found except for Cop c 2, which encodes a C. comatus thioredoxin (TRX). Cop c 1 may represent a putative transcription factor because of the presence of two leucine zipper motifs. Cop c 1 and Cop c 2 were characterized in detail (table 4) [30, 54]. Altogether, 5 of the 7 different cDNAs were cloned into different bacterial expression systems. This substantiates the assertion that basidiomycete proteins are an important new spectrum of allergens in addition to the variety of known allergens from established plant, other mold and animal sources.

Helbling/Brander/Horner/Lehrer 40 Table 3. Cloned basidiomycete allergens

Species Allergen designation Homology Function Reference

P. cubensis Psi c 1 Helbling1 Psi c 2 cyclophilin isomerase Horner [64] C. comatus Cop c 1 leucine transcription Brander [54] zipper factor Cop c 2 thioredoxin redox Brander2 Cop c 3 ? ? Helbling1 Cop c 4 ? ? Helbling1 Cop c 5 ? ? Helbling1 Cop c 6 ? ? Helbling1 Cop c 7 ? ? Helbling1

1 Unpublished. 2 Manuscript in preparation.

Table 4. Characteristics of Coprinus cDNA coding for allergens

Coprinus Accession No. cDNA insert Open reading Homology designation size, bp frame, aa c 1 AJ 132235 463 81 putative transcription factor c 2 AJ 242791 463 115 TRX c 3 AJ 242792 985 342 ? c 4 not submitted 1500 227 ? c 5 AJ 242793 756 68 ? c 6 not submitted 250 49 ? c 7 AJ 242794 614 140 ?

Cop c 1, the First C. comatus Allergen (fig. 4)

Since Cop c 1 cDNA amplified most frequently during the screening process, Brander et al. [54] cloned this cDNA into the pQE-System (Qiagen) and produced recombinant protein in Escherichia coli (85 mg/l culture). The expressed N-terminal His-tagged fusion protein was purified over a Ni-matrix and dialyzed stepwise against decreasing concentrations of urea. The last step was dialysis against phosphate-buffered saline. Pure, recombinant Cop c 1

Allergy to Basidiomycetes 41 a

Fig. 4. a cDNA sequence of Cop c 1 and deduced amino acid sequence. b Deduced amino acid sequence of rCop c 1 fusion protein. Underlining ϭ N-terminal His-tag; italics ϭ amino acid sequence that derives from multicloning site of ␭-ZAPII phagemid SK. bold ϭ sequence encoded by Cop c 1. c The same deduced amino acid sequence is shown twice. Upper sequence: two leucine zipper motifs termed N- and C-terminal; lower sequence: two different repeats, 6 times L-4-L and 7 times P-5-L.

Helbling/Brander/Horner/Lehrer 42 a

Fig. 5. SPT series with (a) rCop c 1 or (b) rCop c 2 (photographed by F. Schweizer, Dermatology, Bern).

allergen (rCop c 1) was tested by SDS-PAGE, immunoblotting, ELISA, ELISA- inhibitions, proliferation assays and SPTs [54]. rCop c 1 is recognized by 25% of 92 tested sera from basidiomycete-sensitized subjects (fig. 5). Positive immunoblots and proliferative responses also support the conclusion that rCop c 1 is an IgE-binding component of C. comatus. As few as 11 fmol rCop c 1/␮l induce a clearly positive wheal-and-flare reaction in SPTs.

Allergy to Basidiomycetes 43 Cop c 2

Cop c 2 most likely represents a basidiomycete (C. comatus) TRX [30]. TRX participates in various redox reactions through reversible oxidation of its active center dithiol to a disulfide and catalyzes dithiol-disulfide exchange reactions. TRX of different sources is highly conserved in the amino acid sequence with typical conserved regions. IgE-ELISA data indicate that this ubiquitous enzyme also possesses allergenic potential, since it bound IgE from 20% of sera from basidiomycete-sensitive subjects. Positive ELISA inhibitions and proliferation assays, as well as strong skin reactions upon testing with 14 ng/ml of recombinant protein (fig. 5) confirm the allergenicity of this protein.

Conclusions

Basidiomycetes are important sources of aeroallergens and sensitization to various species is by far more frequent than previously accepted. Basidiospores have been demonstrated to cause respiratory allergy and are also suggested to trigger inflammatory skin eruptions in a subgroup of patients with atopic eczema. The clinical findings are supported by various in vitro methods demonstrating IgE-binding proteins. Through molecular biology techniques, several recombinant allergens have been produced and their biologic activity has been demonstrated in vivo by skin tests. To date, two recombinant C. comatus proteins have been shown to meet all criteria of allergens. Six of the 7 isolated C. comatus allergens are either coded in unknown genes, or genes not yet identified as coding for allergens [unpubl. data]. Recently, robot-based high- throughput screening of an enriched C. comatus phage surface display library has revealed the presence of at least 37 allergen-encoding cDNAs [67, 68]. Plants, other molds and other allergen sources all are known to have many cross-reactive allergens. Hence, it is reasonable to assume that many basidiomycete allergens would also be cross-reactive, and indeed the limited data available support this. This suggests that basidiomycetes may harbor an unknown array of new allergens. Clearly, isolation and characterization of basidiomycete allergens should be a major goal for future research.

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Prof. Samuel B. Lehrer, PhD, School of Medicine, Department of Medicine SL57, Section of Clinical Immunology and Allergy, 1700 Perdido Street, New Orleans, LA 70112 (USA) Tel. ϩ1 504 588 5578, Fax ϩ1 504 584 3686, E-Mail [email protected]

Allergy to Basidiomycetes 47 Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 48–72

The Allergens of Cladosporium herbarum and Alternaria alternata

Michael Breitenbach, Birgit Simon-Nobbe

Department of Genetics and General Biology, University of Salzburg, Salzburg, Austria

The two mold species Cladosporium herbarum and Alternaria alternata are important causes of allergies. Before 1990 little was known about the relevant allergens of these two worldwide-occurring fungi. Both molds are important sources of allergens for mold-allergic patients (perhaps after Aspergillus fumigatus [1]) and are not pathogens except, as reported in recent years, for a minority of immuno-compromised patients [2–4]. However, A. alternata can be cultured from hypersensitivity pneumonitis [5]. A question that is largely unanswered even today is: What is the most important way of sensitization of the patients? Several possibilities come to mind, among them inhalation of dried mycelia in house dust or inhalation of spores from outdoor or indoor sources. It is well known that the spores of the two molds are common in indoor and outdoor air [6]. The molds grow in soil, on decaying plant material and as plant pathogens. Recently it was found that both C. herbarum and A. alternata are also common plant endophytes. They grow within the extracellular space of plants without causing disease [H.J. Prillinger, pers. commun.]. Most of the allergens identified so far from the two molds are intracellular housekeeping proteins. Notable exceptions usually are the major allergens, which in the case of A. alternata and A. fumigatus are secreted proteins. It is an open question how the intracellular proteins are presented to the immune system. The spores of the two molds discussed here are too large to reach the alveoli of the lung, but they may still be important inhalant allergens. However, none of the allergens identified so far are spore specific. What is further missing are longitudinal studies on children allergic to A. alternata and C. herbarum, epidemiologic studies and a sufficient number of well-documented case histories to draw conclusions about the ‘typical’ symptoms and time course of the mold-allergic patient. Composting and mushroom-growing facilities have been suspected widely to be dangerous to the workers exposed to the emanating mold spores and ‘organic dust’. There seems to be a severe health risk of developing [7–10] allergic bronchopulmonary aspergillosis (ABPA) and other forms of pulmonary complications only in asthmatic and atopic patients, but these are mainly due to thermophilic actinomycetes and A. fumigatus. C. herbarum and A. alternata do occur in compost, however, in much smaller numbers than the species just mentioned. The incidence of C. herbarum and A. alternata sensitization varies in the different climatic zones of the world. In a European multicenter study [11] it was demonstrated that 3–20% of all allergic patients tested showed positive responses to A. alternata and/or C. herbarum in skin prick tests (SPT). A survey done in Israel [12] revealed that 3.3 and 12% of the allergic patients had positive SPT responses to C. herbarum and A. alternata, respectively. In Austria the number of patients with positive SPT responses to the two molds can be estimated as follows: in a large outpatient clinic, from where all sera used for the investigations of the authors [13–16] of the present review were obtained, about 15,000 new patients are diagnosed as allergics every year. About 3% of those patients usually show positive SPT and serum radioallergosorbent test (RAST) responses to a ‘mold mix’. Considering the low quality of the commercial mold extracts, the number may be underestimated, as assumed for all fungal allergies [1]. About 30% of these mold allergics proved to be sensitized to A. alternata and/or C. herbarum, as shown by ‘patient blots’ (IgE-specific immunoblots of mold extract) performed in our laboratory. Our own mold extracts usually produced many more bands and contained more undegraded allergens than commercially available mold extracts. The patients were rarely sensitized to just one mold species but in most cases to several mold species. One reason for this might be the presence of cross-reactive phylogenetically conserved allergens (discussed later in this chapter).

Problems with Reproducibility of Mold Extracts and the Study of Mold Allergens

We will now discuss some of the reasons why mold allergens are more difficult to standardize than other aeroallergens such as pollen allergens and why some of the commercially available extracts are very poor when analyzed for the presence of major allergens. These commercial extracts were also inferior to pure recombinant allergens when compared in clinical tests. Methods developed in our laboratory for optimal production and extraction of mold allergens will be described.

C. herbarum and A. alternata Allergens 49 It is difficult to unequivocally identify closely related species and strains by classical morphological criteria. Little is known about genetic variation below the species level in these organisms. No sexual forms (ascospores) of these molds are known, therefore identification on purely morphological criteria is difficult. The strains used by us were well-characterized strains from a major European strain collection (Institut für Gärungsgewerbe, Berlin, Germany). Identification was based on morphology and molecular criteria (rDNA sequences; see Prillinger et al. [17, this volume]). It is unknown at the moment how large intra species genetic variation is in C. herbarum and A. alternata. The Alt a 1 cDNA sequences isolated from cDNA libraries which originated from European and North American strains showed minor sequence differences at the nucleotide level but no sequence differences at the amino acid level [18] (also see ‘The major allergen of A. alternata, Alt a 1’ below). The total number of relevant allergens is larger in molds than in pollens and foodstuffs. The total number of IgE-reactive allergens from A. fumigatus may be as high as 100 [19, this volume]; in C. herbarum so far at least 36 allergens have been cloned (also see ‘Other Allergens’ below). This large number of partly comigrating proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) makes it nearly impossible to unequivocally identify a protein band in a so-called patient blot, particularly in the range of 30–60 kD. This difficulty can only be overcome using pure recombinant or highly purified natural allergens. However, based on data to be presented later it is likely that a small number of purified recombinant allergens of the two molds are sufficient to correctly diagnose patients sensitized to the two molds with a sensitivity and specificity superior to those obtained with commercial extracts. Growth conditions: The presence of specific allergens (including the major allergens!) depends very much on the growth conditions. For instance, we showed that when growing the molds on petri dishes containing complex medium, allergen content was optimal on day 5, at the beginning of extensive formation of conidiospores as tested by immunoblots. Complex medium contained 1% yeast extract, 2% peptone and 2% glucose solidified with 2% agar and supported vigorous growth of C. herbarum and A. alternata. Growing the molds in liquid medium with shaking or stirring resulted in much lower yields of allergens. It is well known that for industrial purposes and for the production of commercially available mold extracts, the fungi are batch grown for different periods of time in liquid tanks. This might partly explain the poor properties of these extracts. Protein extraction methods are critical. In previous studies various extraction procedures have been tested. Paris et al. [20] could show that after breaking A. alternata cells, the use of carbonate buffer supplemented with protease inhibitors (phenylmethylsulfonyl fluoride) and phenol-binding components

Breitenbach/Simon-Nobbe 50 (polyvinylpyrrolidone) resulted in better extracts than with the use of Coca’s solution containing sodium carbonate and phenol. Portnoy et al. [21] tested four different buffers and various extraction times in order to evaluate their effects on the protein composition and content of the respective A. alternata extracts. They could not determine an optimal extraction time valid for every allergen. With respect to the different extraction buffers they could not detect major differences between the buffers tested, except that low pH resulted in a lower yield. In our laboratory the following method for allergen extraction was developed. The fungal ‘mat’ was carefully removed from the surface of the petri dish after 5 days of growth and immediately put into a mortar filled with liquid nitrogen. The frozen material was ground under liquid nitrogen and the fine powder was extracted with extraction buffer containing a mixture of protease inhibitors and centrifuged at 4 °C [13]. Aliquots of the clear supernatant were stored at Ϫ70 °C. The use of these extraction methods allowed to generate extracts showing the maximum number and intensity of IgE-binding bands in immunoblots (fig. 1, 2).

Experience with Specific Immunotherapy in the Treatment of Mold Allergies

Specific immunotherapy is defined as the administration of increasing doses of an allergen extract to an allergic patient suffering from IgE-mediated hypersensitivity. As a prerequisite the allergy has to be determined by an SPT or an in vitro test like the RAST. Several questions arise in connection with immunotherapy of mold allergy: Which in vivo or in vitro tests yield the most reliable results in the diagnosis of fungal allergy? How can the problem of variability of fungal extracts be overcome? Is it possible and/or necessary to reduce the frequency of systemic side effects after specific immunotherapy in order to reduce the patients’ symptoms? In a placebo-controlled double-blind study [22] it was demonstrated that a negative SPT response to C. herbarum was always correlated with the absence of an allergy. In contrast a positive RAST always indicated clinical allergy; it was thus concluded that a combination of these two tests would result in 100% agreement with a positive clinical diagnosis. In this high-dose immunotherapy study 81% of the group hyposensitized with C. herbarum extract showed an improvement of their clinical symptoms, whereas 19% of the treated patients exhibited deterioration. In the untreated control group 73% of the patients showed aggravation of the symptoms whereas 27% improved. In a double-blind placebo- controlled study [23] 30 Cladosporium-allergic children suffering from asthma or

C. herbarum and A. alternata Allergens 51 Days of growth

kD M5 7 9 11 13 15 18 20 22

97 — 67 —

46 —

30 —

21 —

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Days of growth

kD 5 7 9 11 13 15 18 20 22

97 — 67 —

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Fig. 1. Time course of allergen expression in C. herbarum. a Extracts were prepared on the days indicated after growth at 28 °C on solid YPD. Fungal proteins were separated by SDS-PAGE and Coomassie stained. Equal amounts of fungal material were used for every experiment. Protein bands between 14 and 21 kD, but also Cla h 1 at 30 kD were most strongly expressed at day 5. b Aliquots of the same samples that were used in a were separated by SDS-PAGE under the same conditions, blotted onto Immobilon-P membrane and decorated with a serum pool of C. herbarum-allergic patients. Again it is shown that the region corresponding to Cla h 1 is most strongly stained on day 5 extracts. rhinoconjunctivitis and showing a positive SPT and RAST were studied. The authors observed an increase in tolerance to conjunctival or bronchial challenge in the 16 treated patients. Cantani et al. [24] reported the effects of Alternaria immunotherapy on asthma and rhinitis in 79 children in a 3-year study. They could show that immunotherapy was successful in 80% of the children sensitized

Breitenbach/Simon-Nobbe 52 Extraction time (h)

kD M1612243648

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Fig. 2. Time course of protein extraction from C. herbarum cells. C. herbarum was grown as described in figure 1, harvested on day 5, treated with extraction buffer for varying times and the protein extracts were separated by SDS-PAGE. Routine extraction was done overnight. to Alternaria with doses above 80,000 protein nitrogen units (PNU). In another double-blind placebo-controlled study [25] a ‘rush’ protocol was carried out with a standardized Alternaria extract. The patients actively treated (13 out of 24) and exclusively sensitized to Alternaria benefited from specific immunotherapy. The efficacy was determined by patients’ self-evaluation, global symptom-medication scores and nasal challenge tests. At the antibody level an unchanged specific IgE level and an increased specific IgG level were detected. Immunotherapy with fungal extracts has often been associated with a high frequency of systemic side effects. It was shown that high doses of Cladosporium extract (top doses of 100,000 biological units) resulted in an improved therapeutic effect [22]. According to preliminary data from Norman and Lichtenstein [26], the elicitation of mild side effects was even intended and considered to indicate that this would result in increased clinical efficacy. Tuchinda and Chai [27] reported similar observations for Alternaria. Although several studies about mold immunotherapy have been performed and have shown clinical efficiency [22–25], the number of patients included in the respective studies was rather small emphasizing the need for further studies. One major problem in mold immunotherapy is the lack of reliable fungal extracts [28–30]. In the last years, molecular biology has considerably contributed to the solution of this problem. Several allergens of C. herbarum and A. alternata

C. herbarum and A. alternata Allergens 53 have been identified by cloning techniques [13, 14, 31, 32]. Using recombinant allergens for skin tests Unger et al. [33] demonstrated that recombinant Alt a 1 and Alt a 5 (enolase) achieved a higher specificity and sensitivity than commercial extracts. Comparing specific allergen levels in mold extracts, Vailes et al. [33] used recombinant Alt a 1 (rAlt a 1) (Biomay, Vienna, Austria) as internal standard for the determination of the Alt a 1 content in the different commercial extracts. As a prerequisite Vailes et al. developed an Alt a 1-specific enzyme- linked immunosorbent assay (ELISA) with a monoclonal antibody directed to natural Alt a 1. ELISA dose response curves showed immunological equivalence of recombinant (rAlt a 1) and natural Alt a 1 (nAlt a 1). This result suggests that the biological properties of rAlt a 1 and nAlt a 1 are comparable. The rAlt a 1 used in this study may be suitable for immunotherapy. The use of recombinant allergens for immunotherapy instead of natural mold extracts is desirable as it was shown by Birkner et al. [35] that de novo sensitization after an immunotherapy with crude pollen extract can occur. An improvement could be achieved by injection of only those allergens against which the patient is sensitized. Therefore the use of recombinant proteins for the diagnosis and therapy of mold allergy seems desirable.

Importance of Molecular Cloning Techniques

As the importance of molecular cloning techniques for research on mold allergens has been reviewed several times [36–38], we will not deal with it extensively here. However, it is clear from the facts just mentioned that the importance of cloning methods can hardly be overestimated, especially in the field of mold allergy. Speaking about diagnostics, the allergogram of a patient often becomes clear only after pure recombinant allergens have been used. Immunotherapy with mold extracts has not been widely used in the past because of inherent problems and dangers related to commercially available mold extracts, but could be done with the well-characterized pure recombinant mold allergens that are available now.

Cloning, Analysis, Production and Clinical Testing of the Allergens of Cladosporium and Alternaria

Cloning Methods

In our first attempts to clone the most important allergens of the two molds, cDNA libraries were prepared in phage vectors and standard immunological

Breitenbach/Simon-Nobbe 54 screening methods were employed. A technical detail that seems to be important for the preparation of cDNA libraries from fungal materials is the presence of a large excess of carbohydrate polymers in the RNA preparation from fungal sources. Additionally, unidentified phenolic compounds that tend to bind to the RNA have to be removed prior to subsequent in vitro enzymatic steps. Special methods were developed to overcome these problems [13]. Growth conditions of the two molds were chosen exactly as described above for protein extraction, i.e. the molds were cultured on solid YPD for 5 days. The reasoning was that the material containing all of the allergens should also contain the corresponding mRNAs, which are the starting material for cDNA synthesis. Care was taken to ensure that the starting material contained vegetative hyphae as well as conidiospores, as we did not know beforehand whether any one of the important allergens was spore specific. In reality it was found that only a small minority of the allergens were actually spore specific while the majority of the allergens were so-called housekeeping proteins. PolyAϩmRNA was purified from the mold material, and double-stranded cDNA was prepared [13]. The cDNA was cloned directionally (EcoRI/XhoI) into the cloning site of the phage vector, ␭-ZAP II (Stratagene, La Jolla, Calif., USA) following the manufacturer’s instructions. Using ␭-ZAP II instead of the more conventional ␭-gt11 has three major advantages: (1) directional cloning improves the chance to create a correct in frame fusion; (2) the vector contains a much smaller fusion part (the ␣-fragment of Escherichia coli ␤-galactosidase) of the recombinant protein as compared to ␭-gt11, enabling much easier purification and immunological testing of the recombinant fusion protein, and (3) ␭-ZAP II offers the possibility of ‘in vivo excision’ after obtaining a positive clone, thus greatly speeding up the cloning procedure. The number of plaque- forming units of the primary libraries was always greater than 1 million. After amplification the libraries were used for immunological screening with appropriate patient sera. It is our experience that in principle the majority of the important allergens from one mold source can be cloned by this method. However, there are exceptions that will be discussed. In all cases, the primary immunopositive phage clones were plaque purified to homogeneity. In practice, this required up to 4 rounds of plaque purification. After in vivo excision and plasmid DNA preparation the cDNA clones were sequenced [39] and analyzed for the presence of a complete open reading frame. If an N-terminal deletion was found, the library was rescreened by plaque hybridzation with a 32P-labeled probe prepared from the primary clone. Complete cDNA clones could be found in every case. The complete open reading frame of the allergen in question was then recloned in pMW172 [40–42] resulting in a protein sequence identical to the natural one. In case of secreted allergens (for instance, Alt a 1) the recombinant

C. herbarum and A. alternata Allergens 55 non-fusion (rnf) protein was expressed without the hydrophobic targeting sequence. Methods were then developed to optimize expression in E. coli and to purify the rnf protein for laboratory immunological tests and for clinical use. In two cases (Alt a 1 and Alt a 5) these methods will be described in detail. For all other allergens, analogous methods were used. We deliberately decided to produce only rnf proteins (instead of tagged or fusion proteins) to be able to produce proteins that are structurally and immunologically identical to the natural proteins thus avoiding a concern for clinical use. Although the methods described above were clearly successful, we envisaged a number of problems with immunological screening of phage cDNA libraries for the cloning of mold allergens. The number of clones corresponding to a given allergen depends on the relative abundance of the mRNA in the given population of mRNAs which in turn depends on the physiological status of the cell from which these mRNAs were derived. In addition, it depends on a number of minor factors like the possible loss of specific sequences during library amplification. The clone representing a given allergen might therefore be rare in the library and its identification by screening might become difficult. If the allergen to be cloned is a secreted protein, which is the case for some major allergens of fungi, like Alt a 1 [17] and Asp f 1 [43], the natural form of the allergen may be different from the primary translation product. Posttrans- lational modification, such as proteolytic processing, oxidation to form disulfide bridges and glycosylation, might occur in the natural product. Postsynthetic modifications of proteins pose a special problem for allergen research. To test whether postsynthetic modifications were present on the highly purified natural proteins, sugar analysis [44] as well as physicochemical measurements, in particular mass spectrometry (MALDI-TOF-mass spectrometry [45]) of the highly purified natural and recombinant proteins were employed. Some of the postsynthetic modifications may have a strong influence on IgE binding. While it is relatively easy to mimic the proteolytic trimming and oxidation in vitro, glycosylation remains a problem. These items will be discussed, considering, as an example, Alt a 1. The clinical importance of glycosylation for allergenicity and for IgE binding is presently an open question. Finally, the reducing milieu in the E. coli cell together with the absence of postsynthetic modifications can in some cases be an obstacle for the correct folding of a protein, thus preventing the isolation of the correct clone by immunological screening with patient sera. At least in principle, many of the problems just described can be overcome by using the phage display technique [46], a method described in detail elsewhere in this volume [19]. Only a very short summary of the advantages of the method is given here: The system produces filamentous phage particles which

Breitenbach/Simon-Nobbe 56 Fig. 3. Alignment of N-terminal sequences of Alt a 1. N-terminal peptide sequences of Alt a 1 [48, 50, 51] were compared with the complete open reading frame of Alt a 1 [16, 18]. Identical amino acids are marked in grey. The numbering of the amino acids is according to the complete open reading frame. Curran et al. [48] as well as Aden [50] identified 2 different variants with minor differences in the respective N-terminal sequences. Dashes represent gaps in the sequence. Dots indicate that this part of the protein was not sequenced. The first 5 sequences were determined by solid-phase Edman degradation of nAlt a 1. The last two sequences are deduced from cDNA sequences. carry both the genetic information for a clone in their genome and the protein encoded by the insert on their surface. This makes possible repeated rounds of enrichment by bio-panning. Rare clones (say, 1 positive clone in 1 million clones) can be isolated using this system [47]. In principle, the assembly of the phage in the oxidizing milieu of the periplasmic space of E. coli should enable disulfide bridges to be formed and should improve the folding of the recombinant protein and, therefore, its immunological reactivity. The results obtained so far show that relatively large numbers of new allergen-encoding cDNAs of C. herbarum can be cloned and that all of the clones tested so far code for IgE-binding proteins.

The Major Allergen of A. alternata, Alt a 1

A 30-kD protein was recognized on nonreducing SDS-PAGE that reacted with IgE of the majority of A. alternata-sensitized patients. Attempts to purify this protein in several laboratories resulted in N-terminal amino acid sequences determined by Edman degradation [17, 48–51]. A listing of these sequences (fig. 3) shows rather large variability, which is, however, not due to genetic variability of the protein, but, rather, to inaccurate sequencing. It will become clear from the following discussion why we can make such a statement. All attempts to isolate a cDNA clone coding for Alt a 1 by screening standard ␭gt11 or ␭-ZAP II expression libraries with patient sera failed [Vijay, pers. commun. and own experience]. Finally, a cDNA clone was isolated in Vijay’s laboratory by screening a cDNA library in ␭gt11 prepared from mRNA of a North American isolate of A. alternata with a rabbit polyclonal antiserum raised against highly purified

C. herbarum and A. alternata Allergens 57 natural Alt a 1 [18]. In our own laboratory, a cDNA clone coding for Alt a 1 was isolated by PCR cloning from a cDNA library prepared from mRNA of a European isolate of A. alternata [16, 33]. The cDNA sequences were remarkably similar, with only 3 silent nucleotide differences [16] in the coding region leading to identical deduced amino acid sequences. A comparison of the cDNA sequence with the published N-terminal amino acid sequences showed that the mature natural protein starts at aspartic acid 26. Computer analysis of the deduced amino acid sequence gave clear evidence for an N-terminal hydrophobic leader sequence typical for ER targeting and also correctly predicted the mature N-terminal amino acid. Thus the molecular weight of the protein is predicted to be 14.6 kD, which is observed under reducing conditions (SDS-PAGE in the presence of ␤-mercaptoethanol). However, under nonreducing conditions, the protein migrates with an apparent molecular weight of 29–30 kD. The conclusion is that the natural allergen is a secreted protein, which presumably resides in the periplasmic space of A. alternata cells, and must be either a homodimer or a heterodimer consisting of two similar subunits. There is now strong evidence that the allergen is, in fact, a homodimer: Firstly, the 14.6-kD subunit cloned by us and De Vouge et al. [18] spontaneously dimerized in air, and the resulting 29-kD protein shows full immunological reactivity with a large number of patient sera. Cross-inhibition experiments showed that preincubation of serum with the recombinant homodimer completely inhibited the appearance of the Alt a 1 band in the patient blots performed with A. alternata extract [16]. Secondly and more importantly, this homodimer was used to raise monoclonal antibodies and to create a quantitative immunological test (ELISA). The ELISA dose-response curves for highly purified natural and recombinant Alt a 1 were shown to be superimposable, thus showing that these two proteins exhibit identical affinity and avidity for the antibody used [34]. Thirdly, it was claimed that the second subunit present in the presumed heterodimer Alt a 1 should have a very similar but not identical amino acid sequence to Alt a 1 based on the different N-terminal amino acid sequences obtained (fig. 3). We used the cDNA clone coding for the 14.6-kD Alt a 1 subunit for hybridization screening of the library, resulting in 12 new cDNA clones, all of identical sequence. This renders the existence of a closely related but not identical second subunit improb- able and also shows that different isoforms of Alt a 1 probably do not exist. PCR methods were used to obtain a genomic clone of Alt a 1 (GenBank AF288160) which displayed a short intron of 60 nucleotides after bp 344, as shown by DNA sequence determination. In the same experiment, we showed by genomic Southern blots that just one gene coding for the Alt a 1 subunit is present on the haploid genome of A. alternata [16]. It is remarkable that extensive homology searches up to now revealed no Alt a 1 homolog from other species and thus no clue as to the biological function of this major allergen.

Breitenbach/Simon-Nobbe 58 The Major Allergen, Cla h 1

Cla h 1, is the most important allergen of C. herbarum in terms of frequency of sensitization. Performing IgE blots of C. herbarum extract with about 100 C. herbarum-sensitized individuals revealed that 61% of the patients tested reacted specifically with a single, 30-kD protein band [13]. We termed this protein Cla h 1. In a first attempt it was not possible to clone the Cla h 1-encoding cDNA by screening of a C. herbarum cDNA expression library with patient sera. There are various possible explanations for this result: on the one hand it is conceivable that the mRNA coding for this allergen was short-lived or for other reasons underrepresented in the library. On the other hand it might be that the overall three-dimensional structure of the protein expressed in E. coli is different from the native structure and, therefore, not recognized by human IgE antibodies. This could be due to posttranslational modifications of the protein or simply to the reducing milieu in the E. coli cell and/or denaturing conditions during plaque lifting hampering correct folding of the protein. We met similar problems during our research on Alt a 1, the major allergen of A. alternata (see the previous section of this chapter). This allergen is not recognized by IgE after plaque lifting and the disulfide-bridged homodimeric protein could therefore not be cloned using patient sera. In two-dimensional IgE immunoblots, Cla h 1 was detected as a well- defined major spot with a minor spot probably corresponding to an isoform of the allergen [15]. We have now purified the protein to homogeneity by preparative SDS-PAGE and raised a polyclonal rabbit antiserum against Cla h 1. This will be used as described for Alt a 1 to clone the cDNA coding for Cla h 1 by immunoscreening of the C. herbarum library. As a complementary strategy, we will reclone the inserts of our new ␭-ZAP II library into the modified pJuFo vector pGA [46 and Achatz et al., unpubl. obs.] and select for IgE-binding phagemids as well as for phagemids binding our polyclonal rabbit serum.

Enolase, an Important Allergen in C. herbarum and A. alternata, Exhibits Cross-Reactive Properties

The glycolytic enzyme enolase (EC 4.2.1.11) catalyzes the interconversion of 2-phospho-D-glycerate and phosphoenolpyruvate. Due to its essential role in glycolysis, enolase is a rather highly conserved enzyme, which has been identified and characterized from diverse sources ranging from bacteria to

C. herbarum and A. alternata Allergens 59 higher vertebrates. The active form of the enzyme is a dimer either forming a heterodimer (e.g. Saccharomyces cerevisiae) [52] or a homodimer (e.g. Candida albicans) [53]. Enolase was first identified as an allergen in S. cerevisiae [54–56] and C. albicans [57]. Subsequently, we cloned the enolases of C. herbarum (Cla h 6) [13] and A. alternata (Alt a 5) [14]. Testing the IgE-binding properties of Cla h 6 and Alt a 5 revealed that 22% of the C. herbarum- and/or A. alternata- allergic patients specifically reacted with the respective enolase. Investigation of the protein sequence homologies between enolases from C. herbarum, A. alternata, S. cerevisiae and C. albicans revealed identities ranging from 73 to 89%, the latter being observed between Cla h 6 and Alt a 5. The sequence data together with observations of immunological cross-reactivity [54, 58] made it very probable that enolase represents a fungal pan-allergen. Therefore we performed a rather detailed investigation of the IgE-based cross-reactivity between the enolases from C. herbarum, A. alternata, S. cerevisiae, C. albicans and A. fumigatus. Analysis of IgE cross-reactivity was performed by cross- inhibition tests as described [14]. All cross-inhibition experiments were carried out with several patient sera stressing that the results obtained are not only true for a single patient, but are of general relevance. Taken together, our results clearly show extensive IgE cross-reactivity between fungal enolases. Comparison of the IgE reactivities between Cla h 6 and Alt a 5 revealed that preincubation of patient sera with rnfCla h 6 completely abolished specific IgE reactivities to rnfAlt a 6. On the contrary, the sera depleted with rnfAlt a 5 exhibited residual IgE binding to rnfCla h 6. Based on these experiments we conclude that Cla h 6 contains additional IgE-reactive epitopes as compared with Alt a 5. A similar result was obtained when the cross-reactivity between the enolases of C. herbarum and A. fumigatus was investigated. Performing cross-inhibition experiments between Cla h 6 and the enolase of S. cerevisiae we could show that both enolases seem to have the same IgE- reactive epitopes, as no residual IgE reactivity was observed when the enolase of S. cerevisiae was incubated with rnfCla h 6-depleted sera and vice versa. Investigation of IgE cross-reactivities between Cla h 6 and the enolase of C. albicans showed that there exist cross-reactive epitopes but, moreover, the enolase of C. albicans also contains additional non-cross-reactive IgE-binding epitopes. Recently, enolase has also been shown to be an allergen in Hevea brasiliensis latex [58, 59]. Comparison of the protein sequences of the enolase of H. brasiliensis with Cla h 6 and Alt a 5, revealed sequence identities between 62 and 60%. Although these identities are less close than those observed among fungal enolases they are high enough to generate cross-reactivity between these allergens [60].

Breitenbach/Simon-Nobbe 60 Consequently, enolase can be called a highly cross-reactive allergen, which is not only a fungal pan-allergen, but also exhibits cross-reactivity with latex enolase.

Epitope Mapping of C. herbarum Enolase

For a more detailed investigation of C. herbarum enolase, epitope mapping was performed. Among the various methods available, we chose an approach based on the polymerase chain reaction. Eight different oligonucleotide primers equally distributed along the entire molecule were designed, whereby four of them were ‘forward’ and the other four were ‘reverse’ oriented. These primers were used to amplify 10 different, partially overlapping peptides of Cla h 6, all of which were fused to the non-IgE-binding glutathione-S-transferase (GST) of E. coli. Investigation of the IgE reactivities of these ten fusion proteins was performed with sera of 10 patients sensitized to C. herbarum. Analysis of the respective immunoblots revealed that 6 of 10 peptides specifically reacted with the sera of those patients, which also reacted with rnfCla h 6. Two peptides only displayed IgE reactivity with few patients and another 2 peptides did not exhibit any IgE reactivity at all. Control experiments showed that GST alone did not bind patient IgE and that none of the peptides unspecifically reacted with the sera of nonatopics. One of the 6 IgE-reactive peptides, namely GST-rCla h6 (120–189), represents the overlapping region of the other 5 larger IgE-reactive fusion peptides, indicating that this peptide harbors at least one immunodominant IgE epitope of C. herbarum enolase. In order to investigate the topology of this peptide in the context of the entire enolase, modeling of the C. herbarum enolase structure was performed [14] based on the crystal structure of S. cerevisiae enolase-1 [61]. Modeling seemed possible as the enolase sequences from C. herbarum and S. cerevisiae are 74% identical. The three-dimensional structure of Cla h 6 modeled in this way is shown in figure 4. The peptide rCla h 6 (120–189) is highlighted in the context of the entire protein. Based on the modeling, it is apparent that rCla h 6 (120–189) exhibits an extended structure spanning the body of the globular protein twice and reaching the surface three times. The marked amino acids depict possible IgE-binding sites (fig. 4). The 69 amino acids long peptide rCla h 6 (120–189) was not only expressed as GST fusion protein but also subcloned as a nonfusion peptide. Comparison of the IgE reactivity of the GST fusion and the nonfusion peptide revealed that the GST fusion part seems to support a native-like three- dimensional structure as the nonfusion peptide itself did not bind patient IgE. For clarifying this observation, we analyzed the solution structure of rCla

C. herbarum and A. alternata Allergens 61 P177

K141

R127

R163

Fig. 4. Modeled three-dimensional structure of C. herbarum enolase. C. herbarum enolase was modeled on the basis of the three-dimensional structure of S. cerevisiae enolase [60, 62]. The C. herbarum enolase monomer is shown in magenta, whereas the peptide rCla h 6 (120–189) is displayed in blue. Those residues which were predicted to interact with patient IgE are marked. For further explanations, see text.

h 6 (120–189) fused to a 6xHis tag by circular dichroism spectroscopy (fig. 5). The results indicate that the nonfusion peptide may adopt a new structure in solution, which results in a loss of IgE reactivity.

Other Allergens

Since the 1980s several groups have undertaken the task of identifying the allergens of A. alternata and C. herbarum by means of SDS-PAGE, Western Blot, crossed radioimmunoelectrophoresis, crossed immunoelectrophoresis, isoelectric focusing, RAST and RAST inhibition assays [63–69]. In the 1990s the knowledge about fungal allergens has increased dramatically by the use of molecular biological techniques. Before giving an overview about our own work, we will briefly summarize the cloning results together with data obtained by protein purification and

Breitenbach/Simon-Nobbe 62 0

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Ϫ10

Ϫ15 185 195 205 215 225 235 245 255 Wavelength (nm)

Fig. 5. Circular dichroism spectroscopy of peptide (120–189) fused to a 6xHis-tag. The circular dichroism spectrum of 6xHis-rCla h 6 (120–189) (protein concentration was 27 ␮M in salt-free water) was recorded on a Jasco spectropolarimeter (J-810) at 185–260 nm. Computer-assisted analysis of the data showed that the peptide is folded with 11% helix, 35% ␤-sheet, 27% loop and 26% random structural elements.

characterization by various groups:

A. alternata By immunological screening of an A. alternata ␭gt11 cDNA library, an IgE-binding fragment was identified, showing sequence homology to hsp70 (70-kD heat shock protein) from C. herbarum. This allergen, termed Alt a 3, is recognized by 5% of the allergic subjects [70]. Alt a 2, another allergen, was also identified by immunological screening of a cDNA library. Analysis of the cDNA did not reveal any homology to a known allergen but to a transposable region and a mouse RNA-dependent eukaryote initiation factor-2 a-kinase. The recombinant protein is recognized by 61% of the A. alternata-allergic patients, rendering Alt a 2 a major allergen [32].

C. herbarum The allergen Ag-54 (Cla h II) was isolated by protein-chemical methods and represents a 25-kD protein with an isoelectric point of 5.0 [29, 71, 72]. This allergen contains a large carbohydrate moiety accounting for 80% of its mass.

C. herbarum and A. alternata Allergens 63 Table 1. List of C. herbarum and A. alternata allergens

Allergen source Systematic and Biological function MW, kD Accession No. References original names

A. alternata Alt-1 31 Yunginger et al. [28] Alt a 1 28 Matthiesen et al. [51] Alt a 29K 29 Curran et al. [48]

Alt a 11563 31 Paris et al. [66] Alt a 1 U82633 Unger et al. [16] Alt a 1 U86752 De Vouge et al. [18, 75] Alt a 2 25 U62442 Bush et al. [32] Alt a 3 heat shock protein 70 U87807 De Vouge et al. [70] U878078 Alt a 4 protein disulfide isomerase 57 X84217 Alt a 5 enolase 45 U82437 Simon-Nobbe et al. [14] Alt a 6 acidic ribosomal protein P2 11 X78222, P42037 Achatz et al. [13] U87806 Alt a 7 YCP4 homolog protein 22 X78225, P42058 Achatz et al. [13] Alt a 10 aldehyde dehydrogenase 53 X78227, P42041 Achatz et al. [13] Alt a 12 acidic ribosomal protein P1 11 X84216, P49148 C. herbarum Cla h 1 13 Aukrust et al. [71] Sward-Nordmo et al. [73] Cla h 2 23 Aukrust et al. [71] Sward-Nordmo et al. [73] Cla h 3 aldehyde dehydrogenase 53 X78228, P40108 Achatz et al. [13] Cla h 4 acidic ribosomal protein P2 11 X78223, P42039 Achatz et al. [13] X77253, P42038 Zhang et al. [31] Cla h 5 YCP4 homolog protein 22 X78224, P42059 Achatz et al. [13] Cla h 6 enolase 46 X78226, P42040 Achatz et al. [13] Cla h 12 acidic ribosomal protein P1 11 X85180, P50344 hsp70 70 X81860 Zhang et al. [31]

Information is taken from the ‘Official list of allergens’ of the IUIS Allergen Nomenclature Subcommittee (ftp://biobase.dk/ pub/who-iuis/allergen.list) and supplemented by additional relevant publications.

On the basis of RAST inhibition experiments and deglycosylation of the allergen it was shown that the protein moiety is responsible for the IgE-binding capacity of Ag-54. This result was stressed by the fact that the carbohydrate moiety alone did not specifically bind patient IgE [73]. Zhang et al. [31] isolated a cDNA clone (Ch3.1) showing high sequence homology to hsp70. This allergen is recognized by 38% of the patients allergic to C. herbarum. Our group has cloned 11 allergens of C. herbarum and A. alternata by immunological screening of ␭-ZAP II expression libraries [13–16]. A complete list of the C. herbarum and A. alternata allergens identified so far including results from other groups [31, 32, 70, 74, 75] is given in table 1.

Breitenbach/Simon-Nobbe 64 However, the 9 allergens cloned from A. alternata and the 6 allergens cloned from C. herbarum are not sufficient to explain the complex reactivity patterns obtained in immunoblots using the corresponding mold extracts developed with IgE from sera of sensitized patients. Therefore, we have to assume that the major part of the allergenic structures produced by the molds are still unknown. In an effort to clone the whole allergen repertoire of C. herbarum, the premade ␭-Zap II cDNA expression library was mass excised in vivo [76] and phagemid DNA containing the inserts was isolated from infected E. coli cells. Agarose-gel-purified cDNA inserts isolated from the phagemids after EcoRI/XhoI restriction were used to construct a unidirec- tional C. herbarum phage surface display library in a modified pJuFo vector [77], as described elsewhere in this volume [19]. The library kept in liquid phase was affinity enriched for phage-displaying allergenic proteins using solid-phase-immobilized IgE of a serum pool from C. herbarum-sensitized individuals [78]. After four rounds of affinity selection, a large population of phagemids displaying IgE-binding molecules potentially covering the whole allergen repertoire present in the subcloned library was obtained. Obviously enrichment of phagemids depends on both the frequency of primary clones present in the original library and the abundance of allergen-specific IgE present in the serum pool used for screening [47]. A manual screening of a limited number of clones by restriction analysis is likely to detect only the most efficiently enriched clones present in the large population. To be able to detect all different clones enriched, more powerful screening formats are required [79]. We have adapted robot technology to screen enriched cDNA libraries displayed on phage surface by high-density array hybridization [47, 79, 80]. 5,376 single clones from the enriched C. herbarum library were robot-picked and arrayed onto 384-well culture plates to obtain working copies [47]. After high- throughput PCR amplification all inserts were high-density spotted onto nitro- cellulose membranes and iteratively hybridized with labeled probes derived from the known allergens. These experiments showed that 5 cDNAs previously identified as coding for C. herbarum allergens were present with different frequencies among the arrayed colonies. For further hybridization rounds, 10 nonhybridizing clones were chosen and sequenced, and inserts used to prepare further hybridization probes by PCR [79]. After 3 rounds of hybridization, more than 80% of the clones were identified, and representative inserts of each hybridization class were sequenced. This work revealed 28 new sequences potentially encoding IgE-binding molecules, among these nuclear transport factor 2, also isolated from A. alternata, thioredoxin and two inserts without any sequence homology to known proteins already shown to bind IgE from C. herbarum-sensitized individuals [M. Weichel, pers. commun.]. The Davos group is now in the process of subcloning all new sequences to produce

C. herbarum and A. alternata Allergens 65 proteins to be tested for their allergenicity and to identify the sequences of the remaining 20% of the arrayed clones. They hope to be able to reconstruct the almost complete allergenic repertoire of C. herbarum in the near future.

Production of Highly Purified Alt a 1 and A. alternata Enolase for Clinical Use

Alt a 1 The cDNA sequence coding for the short (mature) form of the Alt a 1 subunit starting with asp-26 was cloned into the E. coli expression vector pMW172 [41, 42]. The cloning site is devised so that the (partial) cDNA followed in frame just after the vector-encoded start codon. Purification of the expressed protein was performed by inclusion body preparation followed by ammonium sulfate precipitation and DEAE-chromatography, and yielded 99% purity as estimated by SDS-PAGE and IgE immunoblots. In a nonreducing PAGE this material displayed an apparent molecular weight of 29–30 kD indicating a homodimer. In a reducing SDS-PAGE Alt a 1 migrated at 14.6 kD. Patient blots showed that this band strongly reacted with A. alternata-specific serum IgE. Samples of the same batch were tested for the absence of any cytotoxic contaminations in a highly sensitive in vitro test [81]. The recombinant proteins were added to EBV-transformed cytotoxic T cells in a concentration range from 0.001 to 10 ␮g/ml. No change in viability or tritium incorporation was seen in the treated cells. In contrast, cells treated under the same conditions with the toxic allergen Asp f 1 were effectively killed. We conclude that our preparation of Alt a 1 is nontoxic. Based on this and on earlier observations with other allergens purified using the same method, Alt a 1 and Alternaria enolase (see below) were approved by the local ethics committee for clinical testing.

A. alternata Enolase The coding region of A. alternata enolase cDNA was also cloned into pMW172 and transformed in E. coli. Purification of expressed Alt a 5 was done by inclusion body preparation followed by isopropanol and ammonium sulfate precipitations. Finally Sephacryl chromatography was performed ending up with a material which appeared as a single band with essentially no contamination on reducing SDS-PAGE. The immunological reactivity of the protein was determined by IgE immunoblots and did not differ from that of the native enzyme. Furthermore, the enzymatic activity of the protein was determined as described [82]. Although there

Breitenbach/Simon-Nobbe 66 are no literature data on the specific activity of A. alternata enolase, we conclude that based on the values published for yeast enolase-1, a substantial part of the pure recombinant A. alternata enolase isolated from E. coli is enzymatically active. Finally, the preparation was tested for toxicity and approved for clinical testing as described above. A third A. alternata allergen, shown to be a homolog of the yeast protein YCP4, was also purified as an rnf protein using similar methods as above, tested for toxicity and approved for clinical use.

Clinical Study with Recombinant A. alternata Allergens

We performed a clinical study [33] at a local hospital during 1997 in order to assess the diagnostic value of the above-described pure recombinant allergens of A. alternata in comparison with two commercial A. alternata extracts. Seven patients with a positive clinical history for A. alternata sensitization, 5 normal healthy subjects and 5 atopics not sensitized to any mold allergen according to clinical history and previous SPT, were tested. The study was approved by the local ethics committee. All patients and control subjects were first routinely tested as follows: SPT was performed with a standard panel of aeroallergens (including 5 molds) and blood samples were taken for total and specific IgE determination. RAST for IgE antibodies to A. alternata (Pharmacia, Uppsala, Sweden) was performed by using the allergen-specific CAP system with results converted into RAST classes from 0 to 4 according to the manufacturer’s instructions. No antihistamine medication was used by any subject. Six of the 7 patients (but no control subjects) were RAST positive with a RAST class greater than 2. One patient was negative, but this person still showed a positive skin reaction to rnf A. alternata enolase (see below). Both SPT (concentration range 0.1–100 ␮g/ml) and intradermal tests (concentration range 1 ng/ml–1 ␮g/ml) were performed. Positive and negative control solutions were the same as in the routine SPT (physiological saline and 0.1% histamine, respectively). After 15 min the weal size was measured and photographs were taken. The two commercial A. alternata extracts used in the study were Pangramin from ALK (Horsholm, Denmark) and an A. alternata extract from Stallergènes (Fresnes, France). Six of the 7 patients reacted positively with rnfAlt a 1 and 2 of 7 patients reacted positively with rnfAlt a 5 (enolase). The two recombinant allergens together detected all of the 7 patients. None of the patients reacted with the minor allergen, A. alternata YCP4. Very importantly, none of the 10 control subjects showed any unspecific skin reaction with the rnf allergens used,

C. herbarum and A. alternata Allergens 67 showing that these solutions are safe and free of pyrogenic contaminations. The performance of the commercial extracts was less convincing as 2 out of 10 healthy control subjects reacted positively in intradermal tests with Pangramin, indicating that this extract contains unspecific pyrogenic compounds which is not only a problem because of its diagnostic value but also because of safety considerations. The SPT with the atopic patients revealed that the extract from Stallergènes detected 7/7 patients whereas Pangramin detected only 5/7 patients. Therefore we can say that SPT and intradermal testing showed that the two recombinant allergens, Alt a 1 and A. alternata enolase were superior to the commercial A. alternata extracts used with respect to positive and negative predictability in the diagnosis of A. alternata allergy.

Discussion and Outlook

Taken together, all of the facts reported above about the allergens of A. alternata and C. herbarum show that in spite of considerable problems with commercially available extracts of the two molds, we are now in a position to improve the situation through the use of pure rnf allergens of these two molds. It is to be expected that not only diagnosis (as described in the preceding section) but also therapy can be substantially improved. This applies not only to conventional immunotherapy, but also to new forms of therapy (to be discussed elsewhere) which are all based on knowledge of the complete allergen repertoire and on production and exploration of the immunological properties of rnf allergens.

Acknowledgments

We are grateful to Hansjörg Prillinger for his help in identifying the molds and for supplying well-characterized strains of C. herbarum and A. alternata from the strain collection of the Institut für Gärungsgewerbe, Berlin, Germany, and for his help and advice with strain identifications. We are extremely grateful to Reto Crameri for his support and advice over many years and for toxicity testing. Both Reto Crameri and Gernot Achatz helped us with the improved phage display system. We are very much obliged to Markus Susani for supply of purified nonfusion allergens of A. alternata and C. herbarum. We are thankful to all present and previous members of our laboratory and to all our collaborators for their excellent contributions to the research on mold allergens. These are: Hannes Oberkofler, Andrea Unger, Erich Lechenauer, Gerald Probst, Barbara Kessler, Ursula Denk, Fatima Ferreira, Peter Briza, Andreas Kungl, Andrey Kajava and Josef Thalhamer. This work was supported financially by the Austrian Science Fund, grant S8812-MED.

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M. Breitenbach University of Salzburg, Department of Genetics and General Biology, Hellbrunnerstrasse 34, A–5020 Salzburg (Austria) Tel. ϩ43 662 8044 5786, Fax ϩ43 662 8044 144, E-Mail [email protected]

Breitenbach/Simon-Nobbe 72 Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 73–93

Molecular Cloning of Aspergillus fumigatus Allergens and Their Role in Allergic Bronchopulmonary Aspergillosis

Reto Crameri

Swiss Institute of Allergy and Asthma Research, Davos, Switzerland

Allergic reactions to moulds, in particular to Aspergillus fumigatus, are difficult to diagnose for various reasons. Most important, standardized A. fumigatus allergen preparations are not available. Commercial fungal extracts are subject to large batch-to-batch variations and are therefore unsatisfactory for diagnostic applications. Furthermore, moulds of the genus Aspergillus are associated with a wide variety of pulmonary complications, ranging from benign colonization of the lung to life-threatening diseases, such as invasive aspergillosis or allergic bronchopulmonary aspergillosis (ABPA). A. fumigatus is able to produce a wide number of more than 40 IgE-binding molecules. Expression of many of these allergens depends on the growth phase of the fungus making standardization of extracts a difficult task. Moreover, the molecular nature of the IgE-binding molecules is largely unknown and the pathophysiologic role played by each molecule in the clinically distinct pulmonary complications remains to be elucidated. ABPA is a potentially fatal disease resulting from IgE-mediated responses to A. fumigatus grown through noninvasive colonization of the lung in conjunction with deterioration of the lung function. However, since most of the criteria used to diagnose ABPA are not specific for the disease, serologic findings should strongly contribute to confirm or exclude a suspected ABPA based on clinical signs. The reliability of skin tests and serologic methods used to diagnose ABPA mainly depends on the composition of the antigen preparations used. The large differences reported in the incidence of both, sensitization to A. fumigatus and ABPA might partly be explained by the lack of reliable A. fumigatus extracts. The aim of our research is to molecularly clone, produce and evaluate the whole repertoire of IgE-binding molecules produced by A. fumigatus. Large-scale serologic and skin test evaluations of some of the cloned A. fumigatus allergens revealed the existence of disease-specific allergens, opening new perspectives for diagnosis and management of ABPA.

Host Defense Mechanisms and A. fumigatus-Related Diseases

A. fumigatus is a member of the genus Aspergillus, which, according to Raper and Fennell [1] includes 132 species, subdivided into 18 groups. Aspergilli are ubiquitously distributed in our environment and among the most important opportunistic fungal pathogens for humans [2]. Only a few members of the genus are recognized opportunistic pathogens and among these A. fumigatus is the etiological agent found in 80% of the Aspergillus infections in humans [3]. The reasons for the pathogenicity of A. fumigatus, which mainly affects patients suffering from asthma, cystic fibrosis or other forms of immunodeficiency [4, 5], are not fully understood. However, the immune status of the individuals seems to be more important for the development of aspergillosis than the virulence of the mould itself [6]. In fact, patients receiving chemotherapy, undergoing total-body irradiation, organ transplantation or prolonged high- dose corticosteroid treatment are particularly prone to developing Aspergillus infections [7, 8]. As an opportunistic pathogen that only causes disease acci- dentally when host defenses are impaired, A. fumigatus has not developed sophisticated survival strategies, as have better-developed pathogens such as Listeria or Yersinia. Therefore, it is not surprising that, in spite of considerable efforts, no distinct A. fumigatus virulence factors have as yet been identified unequivocally [9, 10]. Studies involving mutants obtained by targeted gene disruption showed that the potential virulence factors catalase, alkaline protease, metalloprotease, hyphal growth factor chsE, and fungal ribotoxin restrictocin do not influence pathogenicity of the fungus in mouse models [11–14]. Several studies showed interactions between A. fumigatus conidia which, due to their small size of 2–3 ␮m, can reach the terminal respiratory tract and matrix proteins, such as fibrinogen, laminin, fibronectin and collagen, suggesting a role of these physical interactions in host tissue adherence [15–18]. Adherence of a fungus to the host epithelial surfaces constitutes a primary and crucial step in the onset of colonization [19, 20]. Several host defense mechanisms have developed in the context of innate immunity and play a predominant role in the clearance of A. fumigatus from the respiratory tract. The first line of defense against fungal colonization is mucociliary clearance, which can be modulated by different secondary metabolites secreted by Aspergillus and other microorganisms. It has been shown that substances secreted by the fungus are able to induce slowing and disorganization of ciliary beating frequency resulting in enhancement of the

Crameri 74 retention time of the conidiae in the airways [21]. Since remaining spores are ingested and killed by monocytes/macrophages [22], clearing the lung from inhaled conidia, an important property for the onset of aspergillosis is the capacity of Aspergillus to interfere with both the phagocytosis and killing capacity of phagocytic cells allowing A. fumigatus spores to survive ingestion by alveolar macrophages [23]. Additionally, neutrophil granulocytes attack hyphae that are too large for ingestion and kill them by oxidative and nonoxidative mechanisms [24]. Each line of defense by itself is able to protect the host against a large conidial inoculum over long periods of time: only if all defense lines are overcome can Aspergillus cause invasive disease [25].

A. fumigatus-Related Diseases

The different diseases related to A. fumigatus which usually remain mutu- ally exclusive can be broadly classified according to their clinical conditions into systemic mycoses, saprophytic colonization and allergic diseases [26]. Systemic (invasive) aspergillosis is considered as an infection affecting immunocompromised subjects suffering from prolonged granulocytopenia or other hematological malignancies [4, 27, 28]. The rate of successful treatment in established invasive aspergillosis is associated with a fatality rate of almost 100%, mainly due to the lack of adequate diagnostic techniques for early detection of the fungal pathogen [4]. Saprophytic bronchopulmonary colonization refers to fungal infestation of the airways without evidence for direct tissue damage [26]. As a special case aspergilloma, also termed mycetoma or fungus ball, is a saprophytic manifestation of an Aspergillus growing in preformed lung cavities of patients with bronchogenic carcinoma, tuberculosis, sarcoidosis or, rarely, ABPA [29]. A. fumigatus-related allergic disorders include allergic rhinitis, allergic sinusitis, allergic asthma and ABPA [26, 30]. A common feature of these atopic diseases is a Th2-type immunologic response, which dictates the production of IgE antibodies against A. fumigatus allergens followed by an increased sensitization of mast cells. A corresponding positive skin test to A. fumigatus extracts is therefore regularly found in subjects suffering from one of these diseases [26, 30]. The symptoms of A. fumigatus-induced allergic rhinitis are indistinguishable from those caused by other allergens [26], and the diagnosis is based on case history, skin reactivity and positive RAST to the appropriate allergens [31]. Allergic fungal sinusitis is a chronic disease that affects multiple sinuses without tissue invasion [32]. Several fungi including A. fumigatus have been implicated in allergic sinusitis. The patients may show skin reactivity, precipitating antibodies and elevated allergen-specific IgG and IgE levels to A. fumigatus extracts [26, 32], immunologic characteristics also found in ABPA [33, and see below].

Aspergillus fumigatus Allergens 75 The symptoms related to mould-associated asthma can be distinguished from those attributable to other allergen-induced asthma by a history of exposure, skin test reactivity or in vitro demonstration of fungus-specific IgE in patients’ sera [34, 35]. The immunologic and inflammatory response in A. fumigatus-related asthma, responsible for the fungus-induced early and late asthmatic reactions, is similar to the responses induced by inhalant allergens derived from different allergenic sources such as the house dust mite [36]. However, for therapeutic reasons, A. fumigatus-related asthma must be clearly distinguished from ABPA which requires steroid therapy to control ongoing lung destruction by allergic inflammation and tissue reaction [37]. The diagnosis of ABPA is complicated by a number of characteristics shared by ABPA and A. fumigatus-related asthma [37] and even more complicated in A. fumigatus- sensitized patients suffering from cystic fibrosis [30].

Diagnosis and Epidemiology of Allergic Bronchopulmonary Aspergillosis

Transient colonization of the airways by A. fumigatus can induce an abnormal host response to the fungus characterized by the presence of large numbers of eosinophils and lymphocytes in the airway walls, increased levels of serum IgE, precipitating antibodies to Aspergillus antigens [38] in conjunction with deterioration in lung function [39]. This clinical entity termed allergic bronchopulmonary aspergillosis (ABPA) was first described by Hinson et al. [40] in 1952 and considered a rarity [41], but is currently diagnosed with much greater frequency [42] as a result of the improvement in serologic and radiological methods of diagnosis [43]. Greenberger et al. [37] and Patterson et al. [44, 45] continued to refine the diagnostic criteria of this complex clinical syndrome. The currently accepted diagnostic criteria for ABPA are reported in table 1. The assessment of peripheral blood eosinophilia, chest roentgeno- graphic infiltrates, central bronchiectasis and elevated total serum IgE poses few problems to the experienced clinician. Unfortunately, these criteria are not sufficient to diagnose the disease [47]. The remaining criteria, immediate skin reactivity to Aspergillus, serum precipitins and elevated serum IgE and IgG to A. fumigatus, although depending on the composition of the extract used [48, 49], should, however, strongly contribute to confirm or exclude ABPA suspected on clinical signs [50]. The lack of standardized A. fumigatus extracts [51] may account partly for the discrepancy in the incidence of ABPA reported to range from 7 to 22% among asthmatic patients sensitized to A. fumigatus [52, 53] and from 0.1 to 12% in patients with cystic fibrosis [54, 55]. The diagnosis of ABPA in patients with cystic fibrosis is further complicated by a number of

Crameri 76 Table 1. Diagnostic criteria of ABPAa

1 Asthma 2 Current or previous pulmonary infiltrate 3 Central bronchiectasis 4 Peripheral blood eosinophilia (1,000/mm3) 5 Immediate skin reactivity to Aspergillusb 6 Serum precipitins to Aspergillusb 7 Elevated specific serum IgE and serum IgG to A. fumigatusb 8 Elevated total serum IgE (Ն1,000 ng/l)

a According to Greenberger and Patterson [37]. b Outcome strongly depending from the quality of the extract used. Not all of these criteria may be present in a patient at a given time because they vary with the activity and stage of the disease [44, 46]. A serologic diagnosis of ABPA can be considered as established if criteria 6, 7 and 8 are positive [45]. If skin tests are negative, ABPA can be excluded [37]. clinical findings shared by the two diseases, including pulmonary infiltrates and centrally located bronchiectasis [54, 55]. The true prevalence of ABPA remains highly speculative: ABPA is not recognized in the international classification of diseases and the diagnostic criteria lack clinical uniformity and standardized tests [56]. This intriguing situation is best described by the prevalence of immediate skin sensitivity to crude A. fumigatus extracts, a criterion absolutely required for diagnosing ABPA [37, 44, 45]. Depending on the study, prevalences varying between 2 and 38% for asthmatic patients and from 35 to 81% for patients suffering from cystic fibrosis were found [56]. These studies highlight the difficulty in determining the prevalence of a condition in which diagnostic criteria are not established. The best estimate of the prevalence of ABPA among asthmatic patients and patients with cystic fibrosis calculated by Novey [56] from the available surveys suggests that ABPA could be present in between 0.25 and 0.8% of all asthma patients in the USA. An incidence rate of ABPA of no more than 11% and closer to 7% in patients suffering from cystic fibrosis is suggested by the same author [56] based on the limited data available. This incidence is high enough to consider ABPA as an important disease entity, which should be excluded in every patient with asthma or cystic fibrosis [37, 45].

Cloning and Molecular Characteristics of A. fumigatus Allergens

Several attempts have been made to characterize and purify relevant A. fumigatus allergens using conventional biochemical methods such as gel filtration

Aspergillus fumigatus Allergens 77 [57], preparative isoelectric focusing [58], ion exchange [59] and affinity chromatography [60] or use of monoclonal antibodies [61]. However, none of these methods provided sufficient quantities of purified allergens for diagnostic applications [26]. Although biochemical purification of Asp f 1, a major allergen of the fungus, allowed determination of the amino acid sequences of tryptic peptides resulting in identification of the protein as a ribotoxin [62], it was also clearly shown that such preparations can be contaminated with other allergens with similar immunochemical properties [63], illustrating the difficulties in obtaining pure native allergens. The sequence information obtained at protein level for this ribotoxin was used to clone the gene coding for Asp f 1 [63], the first fungal allergen cloned. Asp f 1 revealed sequence identity to restrictocin, an 18-kD ribotoxin produced by Aspergillus restrictus and never described as allergen [64] that is >99% identical to the Asp f 1 sequences derived from different clinical isolates of A. fumigatus [65, 66]. All these proteins are related to ribotoxins such as ␣-sarcin [67] and mitogillin [68], ribonucleolytic enzymes strictly restricted to the genus Aspergillus [69]. Therefore, Asp f 1 can be considered a species-specific allergen in contrast to other IgE-binding proteins of A. fumigatus, which are highly cross- reactive with related proteins from phylogenetically distant species [70, 71].

Together with honey bee venom phospholipase A2 [72] and the birch pollen allergen Bet v 1 [73], Asp f 1 is one of the best-investigated allergens at biochemical and clinical level [30] and still the only fungal allergen for which a crystal structure is available [74]. Although screening of cDNA libraries constructed in bacteriophage ␭ with sera of sensitized patients yielded some additional A. fumigatus allergens [75, 76] (table 2), the breakthrough in cloning A. fumigatus allergens [30, 87, 88], and IgE- binding proteins from other complex allergenic sources [89–91], was achieved by screening of cDNA libraries displayed on the surface of filamentous phage. The basic concept of linking phenotype, expressed as gene product displayed on the phage surface, to its genetic information integrated into the phage genome (fig. 1) allows the screening of large libraries for the presence of specific clones using the discriminative power of affinity purification. The selection procedure involves the enrichment of phage binding to an immobilized target [92–94]. Identification of allergens is facilitated by the common property of the molecules to interact with IgE [95] achieved by interaction between gene product displayed on phage surface and solid-phase-bound IgE during consecutive rounds of phage selection and amplification [30]. The procedure allows selective isolation of all allergen- expressing clones present in a cDNA library consisting of millions of single phages (fig. 2). As a consequence of the physical linkage between genotype and phenotype, sequencing the DNA of the integrated section of the phage genome can readily elucidate the amino acid sequence of the displayed allergen. This elegant method of cloning has been used to identify an array of novel genes from cDNA

Crameri 78 Table 2. Characteristics of the cloned A. fumigatus allergens

Allergen Function aa CDSa MW IgE-binding Skin test Accession References kD in vitro, %b reactivityc no.

Asp f 1 ribotoxin 149 C 16.9 66 pos. S889330 63, 77 Asp f 2 fibrinogen binding protein? 268 C 37 ND NT U56938 76 Asp f 3 peroxisomal protein 168 C 18.5 88 pos. U58050 78, 79 Asp f 4 unknown 286 P 30 42 pos. AJ001732 47, 80, 81 Asp f 5 metalloprotease 388 C 42.1 85 pos. Z30424 30, 82 Asp f 6 MnOD 207 C 23 35 pos. U53561 70, 80, 83 Asp f 7 unknown 112 P 11.6 39 pos. AJ223315 30, 82

Asp f 8 P2 ribosomal protein 111 C 11.1 10 pos. AJ224333 71 Asp f 9 unknown 302 P 32.3 66 pos. AJ223327 30, 82 Asp f 10 aspartic protease 325 C 34.4 18 pos. X85092 30, 82 Asp f 11 cyclophylin 171 C 18.8 50 pos. 84 Asp f 12 heat shock protein 90 ? P 65 ND NT U92465 75 Asp f 13 alkaline serine protease 282 C 34 ND NT Z11580 85, 86 Asp f 15 serine protease? 152 C 19.5 29 pos. AJ002026 84 Asp f 16 43 ND NT G3643813 Asp f 17 unknown 197 P 19.4 43 pos. AJ224865 84 Asp f 18 vacuolar serine proteinase C 34 ND NT Y13338 86

a Coding sequences: C = complete, P = partial. b 100 sera from patients with or without ABPA were tested. ND = not determined. c NT = Not tested, pos. = positive.

libraries displayed on phage surface [94, 96], among these also a wide variety of A. fumigatus allergens [30, 88]. Physicochemical and biochemical characteristics of the A. fumigatus allergens cloned thus far are listed in table 2. Many of these allergens do occur as homologous allergens in more than one fungal species and could account for cross-reactivity among moulds [82]. This is clearly the case for Asp f 3, a peroxisomal protein demonstrated to be cross-reactive with two homologous proteins of Candida boidiini [78] and recently isolated as IgE-binding protein also from the yeast Malassezia furfur [90]. Asp f 6 (manganese superoxide

dismutase), Asp f 8 (P2 acidic ribosomal protein) and Asp f 11 (cyclophilin) belong to the class of phylogenetically highly conserved proteins [70, 71, 82]. Interestingly, these proteins share a high degree of sequence identity of more than 50% with the corresponding homologous human proteins [71, 83, 97]. The human and A. fumigatus proteins expressed as recombinant proteins in E. coli are recognized by IgE antibodies from individuals sensitized to the homologous A. fumigatus allergens. Moreover, both the fungal as well as the human proteins

Aspergillus fumigatus Allergens 79 s

cDNA Ju Fo product

g3p (~5/43 kD)

g6p (~5/12 kD)

ssDNA (~6,500 nt) ~900 nm DNA Inserts

g8p (~2,800/5 kD

g7p (~5/3.5 kD)

g9p (~5/3.5 kD) ~6 nm

Fig. 1. Physical linkage between genetic information and gene product. The cDNAs are cloned after the Fos leucine zipper and the protein products are captured and immobilized on the phage surface through interaction and covalent linkage with the modified gIII gene product of filamentous phages fused to the Jun leucine zipper [92–96]. induced proliferation in peripheral blood mononuclear cells and positive skin reactions in A. fumigatus-allergic patients showing specific IgE responses to the recombinant A. fumigatus proteins [71, 83]. These observations clearly demonstrate that autoreactivity to self-antigens may occur in chronic allergic diseases and suggest that the human proteins can act as autoallergens in vivo [71, 83]. The mechanisms responsible for these autoimmune reactions remain to be elucidated; however, the responses against self-antigens in individuals sensitized to the corresponding A. fumigatus allergens indicate an autoantigenic T-cell- mediated pathogenesis in chronic inflammatory responses to the fungus [82]. Recent investigations in patients suffering from severe forms of atopic dermatitis showed frequent IgE reactivity against autoantigens [98], supporting our hypothesis that autoimmune reactivity in atopy is not a rare event [97]. An array of allergenic proteins like Asp f 2, Asp f 4, Asp f 7, Asp f 9 and other recently cloned allergens (table 2) fail to show any homology to known protein sequences and therefore the biochemical function of these gene products remains unknown. However, these allergens can substantially contribute to

Crameri 80 Fig. 2. Selective enrichment of phage displaying allergens. Human serum IgE from sensitized individuals coated to a solid phase surface is used to capture phages displaying IgE-binding molecules. Phages that bind specifically to IgE can be selectively enriched during consecutive rounds of phage growth and adsorption [92–96]. improve the diagnosis of sensitization to A. fumigatus [30]. Many other allergens, including Asp f 1, Asp f 5, Asp f 10, Asp f 13, Asp f 15 and Asp f 18, correspond to enzymes showing a high degree of sequence homology to proteins cloned from different fungal species [82]. In spite of the fact that many IgE- binding proteins cloned from different allergenic sources exhibit enzymatic activity [99], the role of the biochemical function of a protein in the induction of allergic diseases has to be questioned. In fact, engineered enzymatically inactive forms of PLA2 from bee venom [100] and of Phl p 5b from timothy grass [99] did not lose any histamine-releasing activity from basophils of sensitized individuals. In contrast, there is increasing evidence that the three-dimensional structure of the allergens is important for the differential regulation of IgE and IgG4 responses independently of the enzymatic activity of the involved molecules [101, 102]. This evidence is corroborated by the demonstration that structural proteins without enzymatic activity cloned using high-throughput screening of an enriched A. fumigatus cDNA library displayed on phage surface [95, see below] can act as allergens. We assume that the allergens reported in

Aspergillus fumigatus Allergens 81 table 2, together with the new allergens derived from robotic screening [103] should cover the whole allergenic repertoire of A. fumigatus which represents one of the most complex allergenic sources [104–106].

Clinical and Diagnostic Evaluation of Recombinant A. fumigatus Allergens

Although in vitro methods, including enzyme linked immunosorbent assay (ELISA) and Western blot analysis, provide evidence for the IgE-binding ability of a protein, the final demonstration of the clinical relevance of a recombinant allergen is the ability of the preparation to elicit type I skin reactions in allergic individuals [83]. This is especially important for recombinant proteins produced in heterologous expression systems, which may not be correctly folded and therefore unable to interact with IgE [107]. A major concern in using recombinant allergens for the diagnosis of allergic diseases is whether the results obtained in vitro with ELISA or ImmunoCAP® determinations correlate with the ability of the protein to induce skin reactions. The recombinant allergens Asp f 1, Asp f 3, Asp f 4 and Asp f 6 have been tested in large-scale skin studies in patients suffering from asthma or cystic fibrosis and coexisting A. fumigatus-sensitization, and have been demonstrated to be reliable diagnostic reagents [47, 49, 66, 79]. The overall results of these studies that compare skin test reactivity with serum IgE levels in groups of patients suffering from different A. fumigatus-related complications are summarized in table 3. The fact that only individuals with detectable allergen- specific IgE levels in serum reacted against the corresponding allergen in skin challenges is important for the reliability of in vitro tests (fig. 3). Furthermore, the levels of rAsp f 1, 3, 4 and 6-specific IgE in serum significantly correlated with the magnitude of the wheal surfaces in all cases [47, 66, 79], a result confirmed by quantitative skin tests performed with recombinant phospholipase A2 [107, 110]. By using allergen extracts [111] and, in particular, mould extracts [112], discrepancies between results of skin tests and allergen-specific IgE determinations have been reported. Accordingly, based on our studies, the specificity of in vivo and in vitro tests can be substantially improved using recombinant allergens, whereas the sensitivity depends on the frequency of the sera recognizing the single component. All four allergens are suitable for the development of fully automated reagents for a quantitative diagnosis of sensitization. rAsp f 1 [108], rAsp f 3 [79], rAsp f 4 and rAsp f 6 [47] immobilized in the Pharmacia ImmunoCAP® System yielded a highly significant correlation between specific IgE determined by ELISA and the quantitative ImmunoCAP determinations. The excellent

Crameri 82 Table 3. Correlation between intradermal skin test reactivity and absolute levels of allergen-specific IgE in serum of A. fumigatus-sensitized individuals and healthy controls

a Patient group Allergen-specific IgE (kUA/l) against

rAsp f 1 rAsp f 3 rAsp f 4b rAsp f 6b

ABPA with Positive skin test 10.79 14.10 11.30 3.91 Negative skin test 0.34 0.34 0.39 0.74 A. fumigatus-sensitized with Positive skin test 4.97 4.86 0.34 0.34 Negative skin test 0.34 0.34 0.36 0.35 Healthy control individuals All negative in skin test 0.34 0.34 0.36 0.34

a Determined with the Pharmacia CAP system. b These allergens are highly specific for ABPA and all A. fumigatus-sensitized individuals without ABPA score negative in skin challenges [47]. All individuals with allergen-specific serum IgE levels below approximately 2 ␮g/l are negative to intradermal allergen challenges, demonstrating the specificity of the in vitro IgE determinations [47, 79, 108, 109]. correlations between wheal surface and allergen-specific IgE in serum obtained for each single allergen [47, 79, 109] allow an approximate quantification of the amount of allergen-specific IgE needed for a positive skin reaction. The cut-off values for a positive skin test response were found to be approximately 0.35, 0.35, 0.9 and 1.2 kUA/l for rAsp f 1, 3, 4 and 6, respectively, corresponding to an absolute allergen-specific IgE serum concentration in the range of 2 ␮g/l. The statistically significant correlation between serologic data and skin test results suggests the possibility, after adequate cal- ibration of the in vitro systems [108], of relying on serologic data obtained with recombinant allergens to diagnose sensitization.

The Role of A. fumigatus Allergens in Allergic Bronchopulmonary Aspergillosis

The availability of highly pure recombinant allergens from this complex fungal allergenic source allows determination of patient-specific reactivity patterns and therefore evaluation of the involvement of each single molecule in the pathogenesis of the different A. fumigatus-related diseases. Large serologic studies involving patients sensitized to A. fumigatus suffering from

Aspergillus fumigatus Allergens 83 rAsp f 1 rAsp f 3 1,000 10,000 Specific serum IgE (EU/ml) Sensitized/ ABPA Sensitized/ ABPA asthma asthma 1,000 100

100

10 10 Specific serum IgE (EU/ml) 1 1 Ϫϩ ϩ Ϫ Ϫ ϩ ϩ Ϫ Intradermal skin test

Fig. 3. Intradermal skin test with rAsp f 1 and rAsp f 3 and allergen-specific IgE values. rAsp f 1 or rAsp f 3 allergen solutions were injected into the skin of A. fumigatus- sensitized asthma patients with or without ABPA. Results were grouped according to negative (Ϫ) or positive (ϩ) intradermal skin tests and plotted against the allergen-specific IgE values determined by ELISA. The hatched area represents the cut-off value indicating the level of allergen-specific IgE in serum required for a positive skin test.

asthma or cystic fibrosis with or without ABPA revealed the existence of disease-specific allergens [47, 80]. Although it was known that serum IgE of patients suffering from ABPA do recognize protein bands in Western blots performed with fungal extracts not recognized by sensitized individuals without ABPA [34, 46], the biochemical nature of these components remained unde- fined. Molecular cloning of A. fumigatus allergens allowed the biochemical characterization of some of these components as cytoplasmic proteins with a defined function, such as manganese superoxide dismutase (Asp f 6) [83] or

P2 acidic ribosomal protein (Asp f 8) [71] or, as in the case of Asp f 2 [76] and Asp f 4 [30, 80, 81], as allergens with unknown biological functions. These allergens provide a highly reliable, specific and sensitive serologic diagnosis of ABPA (table 4) [80, 81, 113] contributing to the solution of an old diagnostic problem. The rAsp f 4- and rAsp f 6-based serological diagnosis of ABPA has a specificity of 100% and reaches a sensitivity of 90% in asthmatic patients sensitized to A. fumigatus [80], whereas the serological discrimination between A. fumigatus sensitization and ABPA in patients suffering from cystic fibrosis reached 100% [81]. Although sensitization to secreted A. fumigatus allergens, like Asp f 1 or Asp f 5 [30], can easily be explained as a consequence of fungal exposure in the respiratory tract, the specific IgE responses to cytoplasmic allergens in patients with ABPA require further explanation.

Crameri 84 Table 4. Discrimination between ABPA and A. fumigatus sensitization by serology with recombinant A. fumigatus allergens

Patient group rAsp f 1, % rAsp f 3, % rAsp f 4a, % rAsp f 6a, %

ABPA with Allergic asthma (n ϭ 60) 83 88 80 55 Cystic fibrosis (n ϭ 20) 100 100 80 70 A. fumigatus-sensitized with Allergic asthma (n ϭ 40) 45 53 0 0 Cystic fibrosis (n ϭ 20) 60 90 0 0 Control individuals Healthy (n ϭ 20) 0 0 0 0 CF-controlsb (n ϭ 10) 0 0 0 0

a These allergens are highly specific for ABPA and allow serologic discrimination between ABPA and A. fumigatus allergy with a specificity of 100% and a sensitivity of 90% in A. fumigatus-sensitized allergic asthmatics [30, 80]. In A. fumigatus-sensitized patients with cystic fibrosis specificity and sensitivity reach 100% [81]. b Cystic fibrosis patients not sensitized to A. fumigatus [30, 81].

Specific sensitization to nonsecreted proteins in ABPA suggests substantial differences in the pathway of exposure to, and immunologic recognition of, the pathogenic fungus between sensitized patients with and without ABPA. Intracellular allergens are unlikely to be present as free aeroallergens in contrast to secreted allergens which might be present in the environment [114]. This could partly explain the lack of specific IgE raised against these proteins in A. fumigatus-sensitized individuals [80]. Allergic individuals are assumed to become sensitized to proteins secreted shortly after spore germination during the time between deposition in the airway and clearance [77]. In contrast, patients suffering from ABPA have or had the fungus growing in the lung [37, 38] and, as a result of fungal damage related to cellular defense mechanisms, become exposed more strongly to secreted and nonsecreted proteins than individuals suffering from simple allergy. Although it is known that infection by pathogens or parasites can induce differential immune responses in infected individuals [115], it was not yet observed that a single pathogen can elicit differential IgE responses in clinically related diseases. These observations open new perspectives for a serologic discrimination between clinically distinct IgE-mediated allergic diseases related to A. fumigatus. The availability of the pure substances will aid development of highly specific reagents such as monoclonal antibodies suitable for the in vivo

Aspergillus fumigatus Allergens 85 localization of the substances during the time course of infection and thus contributing to further elucidate the pathophysiologic mechanisms involved in the development of ABPA.

Utilizing the Allergenic Repertoire of A. fumigatus Identified with Advanced Technologies

Allergen cloning by phage surface display technology has substantially reduced the time required for the isolation of cDNAs encoding IgE-binding proteins to a short period of a few weeks [30, 88]. This approach has been successfully applied to cDNA libraries constructed using mRNAs derived from different complex allergenic sources [88–93]. The subsequent cloning of IgE-binding molecules from these sources resulted in the production of whole panels of fungal allergens [103–106]. However, single clones enriched from phage surface display libraries derived from complex allergenic systems are expected to be multiply represented in the large phagemid population selected, as a direct consequence of amplification and enrichment of IgE-binding molecules occurring during each round of phage selection and amplification [94]. Therefore, identification of clones encoding products derived from rare mRNA species among billions of phages obtained after several rounds of selection and amplification remains a major problem. To enable fast and cost-effective characterization of all clones of an enriched cDNA library, high-throughput screening technology is required. The combination of selective enrichment and automated robot technology for picking and high-density spotting of clones onto filter membranes has proven to be very efficient for the isolation of virtually all allergen-specific cDNA clones present in enriched libraries [103]. Thereby, all clones representing a specific gene present in a library are identified by hybridization with labeled cDNA probes derived from inserts encoding genes of interest. Negative clones can be regrouped on new filters and used for further rounds of hybridization with a different set of DNA probes. This technology permits a fast identification of all different clones present in the enriched library and drastically reduces the manual work involved. Using three rounds of hybridization, we were able to identify 71 different cDNAs encoding allergens of an A. fumigatus cDNA library displayed on the phage surface, enriched by selection with serum IgE of individuals sensitized to the mould [Kodzius et al., in preparation]. This allergen panel is likely to represent the whole IgE-binding repertoire of the pathogenic fungus and will allow to further investigate the immune responses occurring in the clinically distinct A. fumigatus-related allergic diseases at the molecular level. Moreover, the consequent application of functional enrichment and robotic-based screening will allow to rapidly generate information and reagents to facilitate progress in the whole field of life science.

Crameri 86 Conclusions

The development of phage surface display technology for cDNA cloning allowed rapid progress in the identification and characterization of allergenic molecules produced by A. fumigatus. This technique, corroborated by robotic- based high-throughput screening technology, allowed identification at the molecular level of basically the whole repertoire of IgE-binding molecules produced by this fungal pathogen. It must be pointed out, that A. fumigatus represents one of the most complicated inhalative sources of allergens. The availability of the cDNAs encoding more than 70 A. fumigatus allergens will contribute to generate rapid progress in understanding the pathophysiologic role played by each single molecule in the clinically distinct allergic diseases related to A. fumigatus. All recombinant allergens tested so far are able to elicit strong type I skin reactions in sensitized individuals and, moreover, are applicable to fully automated diagnostic systems suitable for routine diagnosis. The results show that recombinant allergens are clearly superior to fungal extracts for diagnostic purposes as deduced from the significant correlations obtained between skin test outcome and serology. Large-scale serological studies showed that specific IgE responses to at least 4 allergens (Asp f 2, Asp f 4, Asp f 6 and Asp f 8) are highly specific for sera of patients suffering from ABPA. These disease-specific allergens allow a serological discrimination between ABPA and A. fumigatus-sensitization with 100% specificity and 90% sensitivity in asthmatic patients, whereas sensitivity and specificity in patients suffering from cystic fibrosis can practically reach 100%. Such discrimination is not possible using conventional fungal extracts, highlighting the potential of recombinant allergens for diagnostic uses. Production, characterization and evaluation of the whole IgE-binding repertoire of A. fumigatus obtained by phage display and high-throughput screening technology are likely to allow further improvement of the differential diagnosis of A. fumigatus-related allergic diseases. The wide application of these advanced technologies will have implications far beyond allergen cloning and characterization.

Acknowledgments

I am grateful to Prof. Dr. K. Blaser and Prof. Dr. H. Lehrach for continuous support and encouragement. I am particularly indebted to the clinics in Davos for the intensive clinical support and to all my coworkers and collaborators for practical support which made progress in this field of research possible at all. This work was supported by the Swiss National Science Foundation (grants 31.50515.97 and 31.063381.00) and by the Ciba-Geigy Jubiläumsstiftung.

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Aspergillus fumigatus Allergens 91 92 Crameri R, Suter M: Display of biologically active proteins on the surface of filamentous phages: A cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production. Gene 1993;137:69–75. 93 Crameri R, Jaussi R, Menz G, Blaser K: Display of expression products of cDNA libraries on phage surfaces. A versatile screening system for selective isolation of genes by specific gene- product/ligand interaction. Eur J Biochem 1994;226:53–58. 94 Crameri R: pJuFo: A phage surface display system for cloning genes based on protein-ligand interaction; in Schaefer B (ed): Gene Cloning and Analysis: Current Innovations. Wymondham, Horizon Press, 1997, pp 29–42. 95 Crameri R, Hemmann S, Blaser K: pJuFo: A phagemid for display of cDNA libraries on phage surface suitable for selective isolation of clones expressing allergens. Adv Exp Med 1996;409:103–110. 96 Suter M, Foti M, Ackermann M, Crameri R: A cDNA cloning system based on filamentous phage: Selection and enrichment of functional gene products by protein/ligand interactions made possible by linkage of recognition and replication functions; in Kay B, Winter KJ, McCafferty J (eds): Phage Display of Peptides and Proteins. San Diego, Academic Press, 1996, pp 187–205. 97 Appenzeller U, Mayer C, Menz G, Blaser K, Crameri R: IgE-mediated reactions to autoantigens in allergic diseases. Int Arch Allergy Immunol 1999;118:193–196. 98 Natter S, Seiberler S, Hufnagl P, Binder BR, Hirsch AM, Ring J, Abeck D, Schmidt T, Valent P, Valenta R: Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients. FASEB J 1998;12:1599–1569. 99 Bufe A: The biological function of allergens: relevant for the induction of allergic diseases? Int Arch Allegy Immunol 1998;117:215–219. 100 Förster E, Dudler T, Gmachl M, Aberer W, Urbanek R, Suter M: Natural and recombinant enzy-

matically active or inactive bee venom phospholipase A2 has the same potency to release histamine from basophils in patients with hymenoptera allergy. J Allergy Clin Immunol 1995;95: 1229–1235. 101 Wymann D, Akdis CA, Blesken T, Akdis M, Crameri R, Blaser K: Enzymatic activity of soluble

phospholipase A2 does not affect specific IgE, IgG4 and cytokine responses in bee sting allergy. Clin Exp Allergy 1998;28:839–849. 102 Akdis CA, Blesken T, Wymann D, Akdis M, Blaser K: Differential regulation of human T-cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants. Eur J Immunol 1998;28:914–925. 103 Crameri R, Walter G: Selective enrichment and high-throughput screening of phage surface- displayed cDNA libraries from complex allergenic systems. Comb Chem High Throughput Screen 1999;2:63–72. 104 Crameri R, Kodzius R, Konthur Z, Lehrach H, Blaser K, Walter G: Tapping allergen repertoires by advanced cloning technologies. Int Arch Allergy Immunol 2001;124:43–47. 105 Crameri R, Kodzius R: The powerful combination of phage surface display of cDNA libraries and high throughput screening. Comb Chem High Throughput Screen 2001;4:145–155. 106 Crameri R: High throughput screening: A rapid way to recombinant allergens. Allergy 2001;56 (suppl 67):30–34. 107 Müller UR, Dudler T, Schneider T, Crameri R, Fischer H, Skrbic D, Maibach R, Blaser K, Suter

M: Type I skin reactivity to native and recombinant phospholipase A2 from honeybee venom is similar. J Allergy Clin Immunol 1995;96:395–402. 108 Crameri R, Lidholm J, Grönlund H, Stüber D, Blaser K, Menz G: Automated specific IgE assay with recombinant allergens: evaluation of the recombinant Aspergillus fumigatus allergen I in the Pharmacia CAP system. Clin Exp Allergy 1996:26;1411–1419. 109 Moser M, Crameri R, Brust E, Suter M, Menz G: Diagnostic value of recombinant Aspergillus fumigatus allergen I/a for skin testing and serology. J Allergy Clin Immunol 1994;93:1–11. 110 Müller U, Fricker M, Wymann D, Blaser K, Crameri R: Increased specificity of diagnostic

tests with recombinant major bee venom allergen phospholipase A2. Clin Exp Allergy 1997;27: 915–920. 111 Van der Zee JS, de Groot H, van Swieten P, Jansen HM, Aalberse RC: Discrepancies between the skin test and IgE antibody assays: Study of histamine release, complement activation in vitro, and occurrence of allergen specific IgE. J Allergy Clin Immunol 1988;82:270–281.

Crameri 92 112 Vijay HM, Perlmutter L, Bernstein IL: Possible role of IgG4 in discordant correlations between intracutaneous skin tests and RAST. Int Arch Allergy Appl Immunol 1978;56:517–522. 113 Kurup VP, Banerjee B, Hemmann S, Greenberger PA, Blaser K, Crameri R: Selected recombinant Aspergillus fumigatus allergens bind specifically to IgE in ABPA. Clin Exp Allergy 2000;30: 988–993. 114 Swanson MC, Agarwal MK, Reed CD: An immunochemical approach to indoor aeroallergen quantitation with a new volumetric air sampler: Studies with mite, roach, cat, mouse and guinea pig antigens. J Allergy Clin Immunol 1985;76:724–729. 115 Reiner SL, Locksley RM: The regulation of immunity to Leishmania major. Annu Rev Immunol 1995;13:151–177.

Prof. Dr. Reto Crameri, PhD Head Molecular Allergology, Swiss Institute of Allergy and Asthma Research, Obere Strasse 22, CH–7270 Davos (Switzerland) Tel. ϩ41 81 410 08 48, Fax ϩ41 81 410 08 40, E-Mail [email protected]

Aspergillus fumigatus Allergens 93 Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 94–113

Defense Mechanisms of the Airways against Aspergillus fumigatus: Role in Invasive Aspergillosis

H.F. Kauffman, J.F.C. Tomee

Laboratory for Allergology and Pulmonology, Department of Allergology, University Hospital, Groningen, The Netherlands

Fungal infections, including those by Aspergillus fumigatus, have become more prevalent in recent years. Intensified medical treatment causing iatrogenic immunosuppression (e.g. in organ transplantation and cancer therapy) may explain the increase. Also in patients with acquired immunosuppression, as with the acquired immuno deficiency (AIDS) pandemic, fungal infections are increasingly found. When fungi interact with the human host two factors determine the outcome. Characteristics of the fungus facilitating its invasiveness and survival in the human host (collectively described as the ‘fungal virulence factors’) and the ability of the host to cope with the fungal threat (the host defense strategies). Especially in the case of pathogenic fungi such as A. fumigatus the fungus may overwhelm the host defense system by causing immunosuppression in various ways and damaging the local barrier function of the respiratory epithelium [1]. In doing so, the fungus may cause derailment of host defenses leading to a self- perpetuating cycle of inflammation which will further facilitate fungal invasion [1, 2]. The pathogenicity of A. fumigatus in the animal and human host is remarkable and several factors may explain its pathogenicity. The human airways are continuously exposed to large numbers of fungal spores that enter the lower airways. Nevertheless, human airways have a remarkable capacity to eliminate most of the inhaled fungal spores and only a small number of fungi are known to cause airway diseases, often under conditions of altered immune activity (e.g. in atopy or immunosuppression). The general strategy of the airways to resist fungal infection is aimed at elimination of the offending fungus by ciliary clearing of the airways, defensive properties of the resident cells in the airways and phagocytic cell activity. Although the genus Aspergillus encompasses over 350 species, most infections are caused by A. fumigatus, Aspergillus niger, Aspergillus flavus and Aspergillus terreus. Especially, A. fumigatus is known as a cause of multiple airway diseases ranging from harmless saprophytic colonization, local inflammatory disease in immunocompetent individuals to life-threatening invasive disease in immunocompromised patients. In this review we will focus on airway diseases caused by A. fumigatus in both immunocompetent and immunocompromised patients with emphasis on the different defense strategies of the airways against this fungus.

Overview of Manifestations of Aspergillosis

An intriguing aspect of the biological behavior of A. fumigatus is its ability to colonize the human bronchopulmonary system under various circumstances. Aspergillus species are ubiquitous in the environment and are inevitably inhaled into the airways. Following the inhalation of Aspergillus conidia or mycelial fragments, colonization of the airways may result. Colonization does not necessarily result in disease, yet, contrary to the seemingly innocuous ‘nonpathogenic saprophytic colonization’ in healthy individuals, acute and often fulminant invasive disease may occur in patients who are vulnerable to infection due to underlying lung diseases or immunosuppression. In addition to its ability to colonize the human respiratory tract, Aspergillus has a significant potential to act as a powerful allergenic source. Aspergillus conidia (and mycelial fragments) in the bronchial tree may result in the development of an allergic state in individuals with a predisposition to develop allergic sensitization (atopy), as in patients with Aspergillus-related asthma and allergic bronchopulmonary aspergillosis (ABPA). Over the past decades various different classifications of pulmonary aspergillosis have been proposed. Probably one of the best known and widely accepted of all has been presented by Bardana [3]. In this system pulmonary aspergillosis is classified into four groups which will be discussed here in more detail.

Nonpathogenic Saprophytic Colonization

Aspergillus species may cause saprophytic infestation of the human respiratory tract without causing manifest tissue damage. Mostly, this form of saprophytic colonization goes without disease and therefore this condition is not an infection as such. Generally, saprophytic colonization remains untreated; however, in patients with enhanced susceptibility to fungal infection, notably after organ transplantation, leukemia, or cancer chemotherapy, antifungal treatment is often warranted.

Defense Mechanisms against Fungi 95 Saprophytic colonization is found with increased incidence in patients with underlying pulmonary diseases, such as in advanced stages of chronic obstructive pulmonary disease, chronic asthma requiring administration of adrenal corticosteroids, primary ciliary dyskinesia syndrome and cystic fibrosis [4–7]. A. fumigatus is the predominant species cultured from the respiratory tract although other Aspergillus species may also be found occasionally.

Aspergilloma

Aspergilloma (mycetoma or fungus ball) usually is regarded as a saprophytic growth of a fungus in a preformed and mostly poorly drained lung space, typically in the upper lung fields. Pulmonary aspergilloma is mostly caused by A. fumigatus although other Aspergillus species may also be associated with aspergilloma formation. Aspergilloma may be found in patients with a history of a disease, mostly tuberculosis or sarcoidosis, where cavities or large bronchiectatic cysts have formed. Formation of aspergilloma may follow or may precede other pulmonary illnesses including ABPA. The average size of a pulmonary aspergilloma is 3–5 cm in diameter and may be visualized by conventional roentgenography or (com- puted) tomography of the thorax as a typical air-crescent sign (Monod’s sign) surrounding a dense structure (i.e., the fungus ball). Diagnosis of pulmonary aspergilloma requires this typical chest picture together with either a positive culture of a specimen obtained from the cavitary lesion or positive IgG antibody titers against A. fumigatus measured by an enzyme-linked immunosorbent assay (ELISA) or serum precipitin. The pulmonary aspergilloma is composed of cellular debris, fibrin, tangled fungal hyphae, mucus and a few inflammatory cells. The occurrence of pulmonary aspergilloma in the general population is unknown, but has been estimated to be 0.01–0.02%. The natural history of pulmonary aspergilloma is highly variable. Pulmonary aspergilloma may exist for a long period without any adverse effects. Spontaneous lysis has been reported in up to 10% of the cases. The most common symptom is recurrent hemoptysis, which may cause fatal asphyxia. Treatment of active disease usually consists of resection of the fungus ball or antifungal therapy.

Hypersensitivity-Induced Aspergillosis

Aspergillus Asthma Exposure to Aspergillus allergens in patients with atopy may lead to allergic reactions in the upper (rhinitis, sinusitis) or lower airways (Aspergillus asthma, ABPA). In the lower airways Aspergillus asthma is caused by a type I allergic

Kauffman/Tomee 96 reaction upon exposure to Aspergillus conidia or hyphae. Sensitization to Aspergillus is found mostly in combination with sensitization to other inhalant allergens, such as house dust mite, tree pollen and grass pollen.

Allergic Bronchopulmonary Aspergillosis ABPA is regarded as a complication of asthma, and occurs in approximately 1% of asthmatic patients. In addition to fever and malaise, the acute phase presents as an acute, easily reversible asthmatic syndrome with dyspnea and transient pulmonary infiltrations of eosinophils, neutrophils and lymphocytes, which may be effectively treated with corticosteroids. This condition usually progresses to a corticosteroid dependent and more intractable asthmatic state with transient pulmonary infiltrates (pulmonary eosinophilia) and a high sensitization to Aspergillus. From there the usual progression of the disease is to fibrosis and bronchiectasis. Most patients come to medical attention before the age of 35 years. The disease is a common complication of cystic fibrosis. Eight diagnostic criteria for ABPA have been proposed by Rosenberg et al. [8] and Patterson et al. [9] and these include: (1) episodic bronchial obstruction (asthma); (2) peripheral blood eosinophilia (Ͼ1,000/mm3); (3) elevated total IgE level in serum; (4) specific IgE and IgG to Aspergillus antigen in serum; (5) immediate (type I) skin reactivity to Aspergillus antigen; (6) precipitating antibodies to Aspergillus; (7) transient pulmonary infiltrates, and (8) central bronchiectasis. Sputum often contains A. fumigatus and sometimes may grow Aspergillus. Proximal bronchiectasis is a characteristic bronchographic finding of patients with ABPA, and atelectasis and cavitations can sometimes be found [10, 11]. ABPA is immunologically characterized by type I, type III and type IV hypersensitivity reactions. The type I immune reactions mediated by IgE antibody result in bronchospasm, eosinophilia and immediate skin reactivity, all of which can be experimentally induced by cutaneous or bronchial provocation with A. fumigatus extracts. The severe pulmonary eosinophilic infiltrate is possibly mediated by an extended or late-type reaction induced by factors from mast cell degranulation and activation of epithelial cells [2]. The development of the bronchiectatic lesions may be the consequence of both the excretion of proteolytic enzymes by A. fumigatus and a type III (Arthus’ or immune-complex- mediated) reaction mediated by circulating or precipitating antibody complexes. Strongly elevated titers of IgG and IgA antibodies have been described in the peripheral blood and in bronchoalveolar fluid [12, 13]. Type IV delayed hypersensitivity reaction may be involved as evidenced by in vitro lymphocyte transformation responses to Aspergillus antigens [8], and may cause granulomas and contribute to mononuclear infiltration. Although the airway damage, resulting in proximal bronchiectasis, may be mediated by various of the aforementioned immunologic mechanisms, at least

Defense Mechanisms against Fungi 97 some of the airway destruction may be explained by microinvasive processes in the airway wall. Studies have suggested that patients with ABPA display evidence for ‘limited fungal invasion’ of the lung parenchyma [14–17]. Riley et al. [18] demonstrated viable fungi within the parenchyma of a patient with ABPA. A lung biopsy specimen obtained from a child with cystic fibrosis and ABPA indicated a marked disruption of elastin layers in bronchioles, whereas hyphae of A. fumigatus were observed in the parenchyma [19]. Some have suggested that this microinvasive activity may be mediated by the release of fungal products, such as proteases, in the area of the airway wall [2, 15, 20–23].

Hypersensitivity Pneumonitis Hypersensitivity pneumonitis (or extrinsic allergic alveolitis) is an inflammatory interstitial lung disease possibly resulting from hypersensitivity type III and type IV reactions following persistent or intense exposure to antigens of bacterial, animal, or fungal origin or to reactive chemical sources. Hypersensitivity pneumonitis occurs primarily in nonatopic individuals and more frequently in men than in women and children. The clinical symptoms occur typically within 4–8 h following the exposure to the offending agent with resultant systemic and respiratory manifestations often with fever. Histologically, the disease is characterized by a diffuse mononuclear cell infiltration of alveolar wall, alveoli, terminal bronchioles, and neighbouring interstitium leading to radiological abnormalities. The physiological correlates are restriction of lung volumes and impaired oxygenation. Damage to the peripheral airways and surrounding parenchyma results in lung restriction; however, overt lung obstruction, probably as a result of lesions located in the bronchioles, is present in some patients. Diagnosis is often made on the basis of a history of environmental or occupational exposure, the presence of elevated IgG antibodies against the environmental antigen as determined by ELISA or precipitating antibodies to the suspected antigen, the presence of suppressor cytotoxic lympho- cytosis in bronchoalveolar lavage fluid, and granulomatous alveolitis in lung biopsy specimens may confirm the diagnosis. The most frequent and commonly known is ‘farmer’s lung disease’ resulting from inhalation of large quantities of thermophilic actinomycetes (Thermoactinomycetes vulgaris, Micropolyspora faeni). Various reports have related hypersensitivity pneumonitis to intense exposure to Aspergilli as well. Aspergillus clavatus, present in mouldy barley on brewery floors, may cause ‘malt-worker’s lung’ [24]. Aspergillus umbrosus, A. fumigatus and A. terreus, present in mouldy oats, corn or hay, may cause ‘farmer’s lung disease’[25–27]. A. fumigatus has been identified as the causative organism in ‘greenhouse lung’ [28] and ‘tobacco worker’s lung’ [29]. Over the past decades the immunopathogenesis and cellular mechanisms of hypersensitivity pneumonitis have been ascribed to cell-mediated immune mechanisms,

Kauffman/Tomee 98 resulting in perpetual lung injury and chronic inflammation characterized by fibrotic and granulomatous processes in the lung interstitium.

Invasive Pulmonary Aspergillosis

The most severe and life-threatening pulmonary disease caused by Aspergillus is invasive aspergillosis. The term invasive aspergillosis generally denotes histopathologically demonstrated fungal invasion into tissue, preferably with confirmation by culturing the organism. The incidence of invasive fungal infections has increased dramatically in the last decades due to the growing number of immunocompromised patients who survive longer than in the past, the widespread use of immunosuppressive drugs, a large aging population with increased numbers of malignancies, and the AIDS pandemic. Generally, as holds true for bacterial and viral infections, immunosuppression is the major condition that increases the susceptibility to fungal infections, including aspergillosis. Immunosuppression may result from various causes, including immunosuppressive and antibacterial therapy or chemotherapy. Underlying diseases leading to T cell depletion as in patients infected with human immunodeficiency virus and the use of invasive procedures, such as catheters and prosthetic devices, are recognized as major risk factors for developing invasive aspergillosis. Prolonged neutropenia is the leading cause of invasive aspergillosis. The risk has been calculated to increase from 1% per day after the first 3 weeks of neutropenia to 4.5% per day after 5 weeks [30]. The important role of neutrophils in the protection against Aspergillus is also illustrated by the increased incidence of invasive aspergillosis observed in patients with a genetic defect in neutrophil function as in chronic granulomatous disease.

Host defense against Aspergillus

The defense system of the airways includes both the innate, nonadaptive defense at the level of the mucociliary system and the adaptive immune response. Special attention will be given to the innate defense system as an important first-line defense against inhaled microorganisms.

Innate Defense Strategies of Airways against Fungi

The innate nonadaptive mucosal defense system of the airways of healthy individuals is a highly efficient primary defense for the elimination of

Defense Mechanisms against Fungi 99 microorganisms. Some parts of this primary defense are not influenced by immunosuppressive therapy and function as the only barrier against invading fungi under severe immunosuppressed conditions. The defense against A. fumigatus by the mucosal airway system is diverse. Cell-mediated defense plays a crucial role in the host defense against most fungal pathogens, including Aspergillus. Alveolar macrophages, polymorphonuclear cells and defense effector proteins produced by resident cells cooperate in the control and elimination of the fungus in the airways [2]. Different functional levels may be distinguished (fig. 1). By junctional contacts the epithelial cells of the airway mucosa function as a physical barrier that prevents the penetration and passage of microorganisms. In addition, excretory cells present in the epithelial cell layer produce glycocon- jugates, defensive proteins and enzymes that together constitute a protective environment. The mucus layer (or epithelial lining fluid) of the airways facili- tates the elimination of inhaled particles, including fungal spores. Mucociliary clearance is mediated by coordinated ciliary movement from specialized epithelial cells that transport the epithelial lining fluid together with impacted material, e.g. fungal spores, out of the airways into the pharynx (fig. 1). The clearance rate of the epithelial lining fluid is dependent on the ciliary beating frequency that can be modulated by multiple factors and mediators, e.g. prostaglandins, that are released during infectious and asthmatic inflammatory responses. Pseudomonas aeruginosa, Haemophilus influenzae and Aspergillus may release factors that inhibit the beating frequency [31], facilitating the survival of these microorganisms in the airways. The mucosal airway defense is mediated by defense effector proteins that have a direct antimicrobial activity which is not dependent on the cellular defense system (fig. 1) [32, 33]. Antibacterial and antifungal proteins are constitutively produced by airway epithelial cells. Although the regulation of these defensive proteins is largely unknown, the production was shown to be enhanced by bacterial stimulation [32–35]. These defensive proteins mainly belong to the family of the cationic defensins, most of them being salt-sensitive, and contribute to the innate defense in vertebrate and invertebrate organisms. Both ␣-defensins derived from neutrophils and the human ␤-defensin-1 from epithelial cells, have strong bactericidal activity [32]. Other members of the ␤-defensin family, tracheal antimicrobial peptide and lingual antimicrobial peptide have been shown to be inducible proteins in epithelial cells of the mucosal surface [36]. Antileukoprotease, also known as secretory leukoprotease inhibitor, has been studied with respect to its capacity to inhibit serine proteases, but aside from being an antiprotease, it was also demonstrated to possess strong antifungal activity [37, 38]. Recombinant antileukoprotease expressed pronounced fungicidal and fungistatic activity towards metabolically active A. fumigatus conidia and C. albicans yeast cells,

Kauffman/Tomee 100 Phagocytic cells Antifungal/bacterial Alveolar macrophage Monocyte Clearance proteins Opsonization Neutrophil Transport Lysozyme Sp-A Phagocytosis Oxygen radicals system Killing SLPI/ALP Sp-D Nitric oxide • Ciliary ␤-Defensins MBP ␣-Defensins beating TAP Complement IL-1␤, TNF-␣ frequency LAP sIgA • Mucin LL-37

Fungal spores

Proteases

-ELF Cytokines Neutrophils -Epithelial cell layer IL-8 Lymphocytes -Basal membrane IL-6 MCP-1

Inflammation

Fig. 1. Innate defense of the airway mucosa in the immune competent host. The first barrier of the airway mucosa is the epithelial cell layer. On top of the epithelial cells, a mucus layer (epithelial lining fluid, ELF) contains various proteins that are produced by both columnar and basal epithelial cells, and by specific excretory cells. The rate of transportation of the mucus layer by coordinated cilia beating to the pharynx determines the rate of clearance of particles that are deposited on the mucus layer (first block, left upper corner). Within the mucus layer several antifungal proteins are present with killing potency for bacteria and fungi (second block, upper left). These components include lysozyme, secretory leukoprotease inhibitor (SLPI) or antileukoprotease (ALP), ␤-defensins, tracheal antimicrobial peptide (TAP), lingual antimicrobial peptide (LAP), and LL-37 as antimicrobial peptides. Block three shows glycoproteins that bind to microorganisms (opsonization) which will prevent binding to the epithelial cells and will give help to phagocytic leukocytes. Some of these components (the family of C-type lectins and collectins) are the surfactant proteins A and D (Sp-A, Sp-D), the mannan-binding protein/lectin (MBP) and components of the complement system. Secretory IgA also belongs to the first barrier defense and is produced as an adaptive response in contrast to the nonadaptive innate recognition. The second part of the defense is the phagocytosis and killing by phagocytic cells that can recognize the microorganism by innate receptors of the Toll-like receptor family. The phagocytic cells will kill the fungi with the help of antifungal proteins (defensins) and the production of oxygen radicals and nitric oxide (block upper right). During the activation and killing process phagocytes will produce IL-1␤ and tumor necrosis factor-␣ (TNF-␣). These proinflammatory cytokines will stimulate the epithelial cells in enhancing the production of defense proteins (see second and third block) and will stimulate the epithelial cells to produce various chemokines (IL-8, RANTES, MCP-1, IL-6). These chemokines will attract neutrophils, monocytes and lymphocytes to give additional help in the defense against the fungi (lower part of the figure).

Defense Mechanisms against Fungi 101 whereas metabolically quiescent A. fumigatus conidia were totally resistant. Antileukoprotease is composed of two highly homologous domains. The COOH- terminal domain contains the proteinase inhibitor site that also blocks the serine proteinase activity of A. fumigatus. Until recently, the function of the second NH2 terminal domain has been largely unknown. However, the fungicidal activity of recombinant antileukoprotease molecule was found to be localized primarily in the

NH2 terminal domain. This may be explained by the more basic properties of the NH2 terminal domain. On a molar base, the fungicidal activity of recombinant antileukoprotease was comparable with the fungicidal activity of a mixture of human neutrophil ␤-defensins (HNP1-3) and also with that of lysozyme. Currently, more of these proteins are detected in the epithelial lining fluid that may play an important role in the defense against infectious agents such as bacteria, fungi and viruses [32–36, 38]. The mechanisms by which these cationic proteins, especially defensins, kill microorganisms are not fully understood. The bactericidal activity of defensins were found to be associated with pore formation in the outer membrane of bacteria. Comparable mechanisms may also pertain to the fungicidal activity. In fact, binding of cationic antimicrobial proteins to the fungal cell wall chitin, a prerequisite for pore formation, has been demonstrated. In addition to the cationic defensive proteins, products such as serum-derived transferrin and polymorphonuclear cell-derived lactoferrin inhibit growth of microorganisms by strongly binding to iron. C-type lectins, glycolipids and glycoproteins are involved in the innate defense of the airways by the binding to surface structures of fungi (fig. 1; opsonization) [39]. These glycoproteins will impede the attachment of microorganisms to the epithelial surface, preventing access to the host. Components of this defense are the collectins, e.g. surfactants (Sp-A, Sp-D) that together with complement and mannose-binding protein/lectin act through the recognition of ‘patterns of molecular structures’ present on the surface of large groups of microorganisms [40, 41]. Complement components are deposited on the surface of microorganisms in an inflammatory milieu. These microorganism-bound opsonins may then bind to the cell surface of macrophages and enhance phagocytosis and killing. The importance of complement, particularly the opsonic component C3, has been documented in the defense against different fungi [42, 43]. Yet, the role of complement (and antibody) in the defense against Aspergillus is far less clear, and contrasting data have been reported [44–48]. Kurup [48] found that prior opsonization of A. fumigatus conidia with normal rabbit serum, rabbit anti- A. fumigatus serum, complement, or lung lavage fluid, did not markedly enhance attachment, internalization or killing of the conidia by rabbit alveolar macrophages, suggesting that opsonization of Aspergillus is not required for binding to rabbit macrophages. Terminal components of the complement system

Kauffman/Tomee 102 may also cause lysis of microbial cell membranes. Fungi, however, are resistant to lysis by these terminal components, even in the presence of specific antibody, presumably as a result of their thick cell wall. Therefore, as opposed to bacteria and viruses, fungi are not killed following incubation in serum. Although the role of complement in the human host defense against Aspergillus is not clear, some studies indicate that activation of complement by Aspergillus depends on the presence of complement receptors on the fungal surface. C3 deposition via activation of the alternative complement pathway was shown for A. fumigatus conidia, especially during germination of the spores. Like resting conidia, hyphae are poor activators of complement [49] and are damaged by neutrophils in vitro in the absence of complement [50]. In summary, the activation of the alternative pathway seems to be the main mechanism of the activation of the complement system by the conidia. However, its role in vivo in the defense against A. fumigatus needs additional studies. In contrast to the nonadaptive defense system discussed so far, secretory IgA (sIgA) is produced as the result of a specific adaptive immune response. Binding of sIgA to the surface of fungi will prevent the binding of the spores to the epithelial surface. Furthermore, it has been shown that sIgA bound to immobilized antigen is important for the activation of certain cell types, e.g. eosinophilic leukocytes [51, 52]. sIgA against A. fumigatus in human bronchoalveolar lavage fluid has been shown to be locally produced and is enhanced in bronchoalveolar fluid in patients with ABPA [13]. Phagocytosis and killing by phagocytic cells such as alveolar macrophages and polymorphonuclear cells in the epithelial lining fluid constitute a crucial role in the innate defense of the airways (fig. 1). Macrophages selectively protect against conidia and their importance in mediating the first line of host defense against inhaled Aspergillus has been clearly demonstrated. In murine models macrophages destroyed inhaled conidia and prevented lethal infection in vivo even in neutropenic or athymic mice. Phagocytic cells have receptors that recognize surface structures of microorganisms, the so-called ‘pattern recognition receptors’ [53]. For example, studies by Turner [41] have shown that fungal mannan is recognized by the mannan-binding protein present on phagocytic cells. The efficiency of phagocytosis and subsequent killing is improved by opsonization with sIgA and the various glycoproteins (described above) that are present in the epithelial lining fluid. Those conidia that have escaped the first line of cellular defense may germinate and grow as the hyphal form (fig. 1). Aspergillus hyphae have a long, dichotomously branched, filamentous shape that cannot be readily phagocytosed by a single cell. Protection against the hyphal form is mediated by polymorphonuclear cells and monocytes (fig. 1). Adhesion to hyphae and release of the reactive oxygen intermediates and cationic peptides by these cells mediating the

Defense Mechanisms against Fungi 103 second line of the innate defense are considered key mechanisms in causing hyphal damage. The strong clinical association of severe neutropenia and pathological dysfunction of polymorphonuclear cells with the high prevalence of invasive aspergillosis illustrates the importance of these cells in host defense. Following phagocytosis, macrophages are able to kill intracellular microorganisms by several mechanisms. Normally, phagocytosis leads to the production of reactive oxygen species (referred to as the ‘respiratory burst’), such as nitric oxide, hydrogen peroxide, superoxide anion, and hydroxyl radi- cal, that mediate the oxidative killing of microorganisms. In some pathological situations such as in patients with chronic granulomatous disease, the macrophages, monocytes and neutrophils fail to produce reactive oxygen species in response to phagocytosis. Aspergillus infections are common in these patients, suggesting that reactive oxygen species are required to kill the fungus in vivo. After binding and phagocytosis, alveolar macrophages and monocytes release pro-inflammatory cytokines, e.g. IL-1, interferon-␥ and tumor necrosis factor-␣ (fig. 1). These cytokines activate resident cells (epithelial cells, fibro- blasts) to produce chemokines such as IL-8, granulocyte-monocyte-colony- stimulating factor, RANTES (regulated on activation normal T cell expressed and secreted) that will result in a second wave of cell recruitment (mononuclear and polymorphonuclear cells). In recent years the epithelial cell has been recognized as a central player for regulating the natural and acquired immune system of the host at mucosal surfaces. In a study with epithelial cell lines from human airways it was shown that fungal serine protease activity present in culture filtrates from A. fumigatus induced the production of interleukin IL-8 and IL-6 and monocyte chemotactic protein-1 and caused cell detachment in a dose-dependent fashion [54, 55]. Chymostatin, antipain, phenylmethylsulfonyl fluoride and heat treatment completely inhibited fungal protease activity, cytokine production and cell detachment, whereas antileukoprotease partially inhibited these activities [55]. These observations have shown that by causing cell detachment, fungal proteases may decrease the physical barrier function of the epithelium. However, by eliciting a cytokine response, the epithelium may signal the mucosal inflammatory response resulting in the recruitment of phagocytes against A. fumigatus. In a more recent paper it was also shown that, in contrast to other fungi, proteases from A. fumigatus at higher concentrations are able to inhibit chemokine production by epithelial cells. It was proposed that this silencing of epithelial cells may play an additional role in the pathogenicity of A. fumigatus by preventing detection by infiltrating phagocytes [56]. Furthermore, it was shown that spores of A. fumigatus can directly bind to epithelial surface cultured in vitro [57]. The role of this binding to epithelial

Kauffman/Tomee 104 cells in the defense system is as yet unclear. In one study it was shown that attachment of spores to the epithelial surface is followed by phagocytosis by the epithelial cell, suggesting a defensive role [57], whereas in another study binding of spores to the epithelial surface was followed by germination and penetration by hyphae into the epithelial cell [58]. Until now the significance of the binding of fungal spores to the epithelium for a defensive or invasive role is not clear, and further studies are needed. However, it is beyond doubt that binding of microorganisms to host tissues is a prerequisite for survival of the organism in the human airways in protecting it from mucociliary clearing. The innate defense strategies described above likely are the major defense mechanism of the central airways. These mechanisms are highly efficient in eliminating fungi from the airways and may at least in part explain why immunocompromised patients are only in part affected by life-threatening invasive aspergillosis. The significance of the innate defense is also supported by the observation that cultures of sputum samples of patients with aspergillosis, e.g. ABPA, often remain negative even if fungal hyphae can be detected in the sputum samples by cytology, indicating the efficient killing of fungi in the airways.

The Adaptive Immunological Response

During growth of Aspergillus in lung tissue, antigen presentation by antigen- presenting cells may result from either phagocytosis of intact spores, fungal particles or soluble antigens that are released from the growing tip of the fungus. Antigen presentation will activate B and T lymphocytes, resulting in both cellular and humoral immune responses, while highly specific killer T cell response may develop. Depending on the immunologic status of the patient, a Th1- or Th2- like response may predominate. In immunocompetent patients the immunologic response to Aspergillus is dependent on the immune status of the patient. In nonatopic patients the presentation of antigens of Aspergillus to the immune system will result in a Th1-type response with strong IgG and IgA antibody formation and recruitment of polymorphonuclear cells. This is seen in patients with aspergilloma and patients with cystic fibrosis with Aspergillus infections. In patients with cystic fibrosis, infections with A. fumigatus regularly will develop into ABPA, with concurrent more severe loss of airway function. Although it is generally assumed that antibody formation will facilitate the elimination of the fungus, the proteases liberated from polymorphonuclear cells may cause severe damage of the airway tissue. In atopic patients the Th2-type response is characterized by IL-4 and IL-5 cytokine production. IL-4 dictates B cells to produce IgE antibodies against

Defense Mechanisms against Fungi 105 Aspergillus antigens, followed by an increased sensitization of mast cells. Furthermore, IL-5 is responsible for the recruitment of eosinophils from the bone marrow to the airways. As was described above, aspergillosis in atopic patients may develop into Aspergillus-associated asthma and in the more complicated manifestation of ABPA. An important difference between the two manifestations is the binding and growth of Aspergillus hyphae on and between epithelial cells in ABPA, which has never been demonstrated for Aspergillus asthma. The continuous presence of the fungus at the epithelial surface will result in abundant liberation of antigen, followed by strong antibody responses (IgG, IgA and IgE) in the blood and at local sites. These antibodies can be detected in the bronchoalveolar lavage fluid [12, 13]. Furthermore, extremely high titers of total IgE may be found, which is characteristic for exacerbations of ABPA [12, 59]. It was described above that proteases from A. fumigatus are able to cause epithelial cell detachment and induce the production of chemokines. Destruction of the epithelial cell barrier is rapidly followed by repair mechanisms, resulting in the influx of serum proteins and extracellular matrix proteins to the lumen site of the epithelium [60]. Both spores and mycelium of A. fumigatus have surface structures that are able to interact with extracellular matrix molecules and serum factors, such as fibrinogen, collagen I and IV, laminin and complement C3 [47, 61–65]. Therefore, damage and repair mechanisms of the airway mucosa may facilitate the binding of Aspergillus to the damaged sites of the airways (fig. 2). Binding of Aspergillus to the epithelial surface may be responsible for local chemokine production by proteolytic attack, resulting in a rapid recruitment of phagocytic leukocytes. It has been argued that in patients with asthma this local inflammatory response of epithelial cells [2] may result in the development of ABPA with massive infiltration of eosinophils [19]. These eosinophils may cause additional damage to the epithelial cell layer by release of their toxic granular proteins. This will cause additional binding of Aspergillus, which will further facilitate the survival of the fungus in the tissue. In this way Aspergillus will modulate a defensive immunological response in atopic asthmatic patients into a perpetuating cycle of damage to the airways, promoting its continuous growth in the airways. Although information is lacking on the further progress of damage of the airways of patients with ABPA, the continuous findings of bronchiectatic lesions, fibrosis and haemoptysis during exacerbations of the disease, together with the presence of antibodies against proteases of A. fumigatus, suggest that next to toxic components eventually released by Aspergillus also the proteolytic components may play an important role in the inflammatory responses found in patients with ABPA. In summary: it may be concluded that in immunocompetent patients A. fumigatus generates a strong perpetuating cycle of inflammatory responses

Kauffman/Tomee 106 Antifungal/bacterial Clearance proteins Opsonization Phagocytic cells Phagocytosis Transport Lysozyme Oxygen radicals Sp-A Killing system SLPI/ALP Nitric oxide Sp-D ␤-Defensins ␣-Defensins • Ciliary MBP TAP beating Complement LAP IL-1␤, TNF-␣ frequency sIgA • Mucin LL-37

?? A. fumigatus

Proteases? Binding to MMP

-ELF -Epithelial cell layer Migration Signaling -Basal membrane ? of fungus by cytokines Blood vessel

Transport to other organs

Fig. 2. Innate defense against fungi in the immunocompromised patient. Abbreviations as in figure 1. In the immunocompromised patient the phagocytic activity and production of the first wave of proinflammatory cytokines (IL-1␤, TNF-␣) is largely or totally diminished (right upper corner). In contrast to the cellular response, the innate barrier functions against fungi shown in the upper left corner (blocks 1–3) remains active and will prevent infections of the airways against the majority of bacteria and fungi that are inhaled. A. fumigatus has the capacity to impair this innate response at several points and will be able to cause damage to the epithelial surface by means of different proteases that are excreted. Damage will result in exposure of mucosal extracellular matrix proteins (MMP), a target of binding for Aspergillus spores and mycelium. Binding to the mucosal site is the first and often fatal event that may start invasive aspergillosis. Whether epithelial cells are still able to respond to the challenge by Aspergillus proteases is very likely but unknown. However, the cells that have to respond to these signals are largely absent (lower left part). The way by which Aspergillus is able to penetrate the lung tissue and spread (via the blood circulation) to various organs of the body is largely unknown as well.

that induces additional damage to the airways. This self-damaging mechanism will facilitate the local invasion by the fungus. Although proteases of A. fumigatus appear to play a role in different manifestations of aspergillosis in immunocompetent patients, their role in

Defense Mechanisms against Fungi 107 immunocompromised patients is much less clear and still a matter of debate. Arguments pro and contra have been reviewed recently [66, 67]. In patients with strong immunosuppression and neutropenia, activation of epithelial cells by proteases may still be possible, but this will not result in a self-damaging inflammatory response as has been described for ABPA or cystic fibrosis patients with Aspergillus infections. Recently we were able to isolate two strains of A. fumigatus from a patient who died from invasive growth of Aspergillus in the lung tissue. Both strains were unable to grow on a collagen-containing medium, suggesting that these two strains of A. fumigatus were not easily producing the elastin- and collagen-degrading serine proteases [Kauffman, unpubl. obs.]. The production of these collagen-degrading proteases has been proposed as virulence factor for invasive strains of A. fumigatus.

Effect of Immunosuppressing Agents on the Innate and Adaptive Defense Mechanisms

During immunosuppressed conditions the cellular defensive role of T cells is most vulnerable (fig. 2). Immunosuppressive therapy following organ transplantation is aimed at suppressing cellular systems that are involved in the rejection of the transplanted organ. This will result in the loss of the cytotoxic T cell function, which is most vulnerable to immunosuppression. Generally this will result in a partial loss of the cellular defense, while the humoral response to Aspergillus still may be functional. Strong antibody responses against A. fumigatus are found under immune-suppressive therapy after lung transplantation [68]. Also, observation in bronchoalveolar fluid after lung transplantation still shows normal numbers of alveolar macrophages and polymorphonuclear cells [Kauffman, unpubl. obs.]. Tuned therapy regimens try to strike a balance between acute rejection of the transplanted organ and the threat of viral, bacterial or fungal infections. However, after strong immunosuppression, as is used after e.g. bone-marrow transplantation, both cellular and humoral responses may be lost (fig. 2). As described before, these immunocompromised patients are vulnerable to severe and life-threatening invasive aspergillosis. In contrast to immunosuppression of the adaptive immune response, the effect of immunosuppressive agents on the innate defense responses is very limited. However, the limited information available suggests that the effect of corticosteroids and cyclosporin A may be different on resident cells of the airways compared with the effects on immune cells. It has been shown that incubation of epithelial cells with corticosteroids increases both the mRNA expression and the protein release of the secretory leukocyte protease inhibitor [69]. As described above, SLPI has bactericidal and fungicidal activities, suggesting that under certain

Kauffman/Tomee 108 immunosuppressive conditions some components of the innate defense may be enhanced. In contrast, Agerberth et al. [33] found lower antibacterial levels in a single patient that was treated with corticosteroids before bronchoalveolar lavage fluid collection, suggesting that the production of antibacterial components by airway epithelial cells was inhibited at the corticosteroid concentration used in this study. Studies with other immunosuppressants, e.g. cyclosporin A, FK506, showed increased production of cytokines by resident tissue cells (mesangial cells, epithelial cells), indicating that the effect of immunosuppressive agents on resident tissue cells may be different as compared with their effect on immune cells [70–72]. Similarly, we have recently shown that the immunosuppressive drug cyclosporin A induces an enhanced production of cytokines (IL-6 and IL-8) by human airway-derived epithelial cells [73]. Furthermore, the effect of corticosteroids showed bell-shaped dose responses for these cytokines, with a first phase of activation of the cytokine response at concentrations that are used under in vivo conditions after lung transplantation, followed by an inhibitory response at higher concentrations [73]. These observations suggest that the influence of immunosuppressive agents on the local innate defensive mechanism may be different (stimulatory) from the effect (inhibitory) on the adaptive immune response. Further studies are necessary in order to elucidate the modulating role of the different agents used for immunosuppression on the nonadaptive primary defensive system and the adaptive immunological response and the significance for fungal infections.

Concluding Remarks

The pathology of Aspergillus-related disease is broad and depends on fungal-related factors (the fungal virulence factors) and host-related factors (anatomical abnormalities, atopy, impaired local or systemic immunity). Aspergillus species are ubiquitous in the environment and are inevitably inhaled into the airways. In healthy individuals A. fumigatus has low pathogenicity and inhalation of A. fumigatus conidia may cause innocuous colonization of the airways. However, opportunistic infections may occur in susceptible patients who have breaches in their defense systems. Defense against A. fumigatus is diverse and includes cellular and non- cellular components. Cell-mediated defense by alveolar macrophages, polymorphonuclear cells and monocytes plays a central role in the host defense against Aspergillus both in the innate and acquired defense mechanisms. Soluble antimicrobial and fungicidal proteins produced by epithelial cells may cause direct killing of inhaled fungal spores. In addition, opsonizing soluble products may facilitate phagocytosis and killing of Aspergillus by alveolar

Defense Mechanisms against Fungi 109 macrophages and polymorphonuclear leukocytes and may contribute to its elimination by inhibiting attachment of the fungus to host tissues. Epithelial cells also play a crucial role in the defense against Aspergillus by the release of proinflammatory cytokines, recruiting cells of the host defense to the site of fungal penetration. It has been argued that the strategies of A. fumigatus may depend on the immunological status of the patient. Acting as a powerful allergen source, exposure to A. fumigatus may result in the development of an allergic state in individuals with a predisposition to develop allergic sensitization (atopy). In these patients proteases may facilitate the pathogenic presence of Aspergillus, by inducing an inflammatory (eosinophilic) response causing damage to the epithelial cell layer. In immunocompromised patients inflammatory self-damaging by proteases of fungal origin is absent. In these patients the crucial cell-mediated defense is lost and the residual defense is mainly composed of the innate defense factors derived from epithelial cells. It is discussed that immunosuppression mainly acts on the crucial cellular defense of the airways. However, the effect of the immunosuppressive agents on the innate response capacities of resident cells, such as epithelial cells or mucus-producing cells, is hardly known. Immunosuppressive agents may influence such cells differently as compared with their influence on immune cells.

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H.F. Kauffman, PhD., Assoc. Prof. University Hospital Groningen, Clinic for Internal Medicine, Department of Allergology, Laboratory of Allergology and Pulmonology, Hanzeplein 1, NL–9713 GZ Groningen (The Netherlands) Tel. ϩ31 50 3613703, Fax ϩ31 50 3121576, E-Mail [email protected]

Defense Mechanisms against Fungi 113 Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 114–128

Secreted Proteinases and Other Virulence Mechanisms of Candida albicans

Michel Monoda, Margarete Borg-von Zepelinb

a Service de Dermatologie (DHURDV), Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, and b Department of Bacteriology, University Clinics, University of Göttingen, Germany

Candida albicans is a major source of different types of fungal infections in immunocompromised patients [1]. Chronic superficial C. albicans infections of the skin or the mucosae are commonly seen in patients with cell-mediated immunological disorders, e.g. AIDS patients, whereas disseminated infections due to C. albicans and related Candida spp. predominantly occur in patients with severe defects of their phagocytic system, e.g. neutropenic patients, in immunosuppressed patients following organ transplants or in cancer patients undergoing aggressive cytostatic therapies (for a review, see Lortholary and Dupont [2]). Furthermore, the widespread use of broad-spectrum antibiotics has caused an increase in mucosal Candida infections, such as thrush, infections of the intestinal tract and vaginitis in normal hosts. In fact, Candida spp. are ubiquitous human commensals, mainly residing in the gastrointestinal tract without causing any harm. Since they are not highly virulent microorganisms, Candida infections only occur under favorable conditions in the host, which allow the fungus to do more than just colonize mucosae. Candida infections have emerged as a significant medical problem at the end of the twentieth century concomitantly with AIDS and immunosuppressive therapies. C. albicans and related Candida spp. are prototrophic, which contributes to establish virulence. Uracyl or adenine auxotrophy diminishes the virulence of C. albicans [3, 4]. Efficient iron assimilation is also essential for C. albicans virulence [5]. Since prototrophy is also found in many other yeasts which are not necessarily pathogenic, it is conceivable that C. albicans must possess traits which make this species more adapted than others to overcome the mucosal barriers protecting the host from infections and deep mycosis. The aim of this chapter is to briefly review different traits of virulence of C. albicans and to discuss the role of the secreted aspartic proteinases (Sap) in more detail.

Site-Directed Mutagenesis and Genomics to Investigate Virulence Factors

Numerous putative virulence factors have been investigated using genetic tools and animal models by observing the specific effects of gene disruption and growth of the organism in vitro and in vivo. Many knockouts of genes result in strains that show lower lethality for mice than their parent strains without affecting growth in vitro in a rich medium. The construction of mutants of C. albicans in an isogenic background is technically difficult due to the permanent diploid status of this asexual yeast. The ‘ura-blaster’ system [6], which allows the sequential disruption of targeted alleles, has been applied in many investigations using a C. albicans with a ura3/ura3 background [7]. Strains constructed by the ura-blaster technique have the URA3 gene of C. albicans inserted into the targeted gene of interest. It is of critical importance that the orotidine 5-decarboxylase enzyme, encoded by the URA3 gene inserted into the targeted gene retains sufficient activity, since both uracyl auxotrophy and mutations in the pyrimidine biosynthetic pathway have previously been shown to diminish the virulence of C. albicans. However, retrospectively, the URA3 activity within the disruption cassette was shown (1) to vary and (2) to be lower than in the parental strain [8]. Therefore, the results of some virulence studies in animal models has to be interpreted with precaution. It is possible now, to replace URA3 with a dominant selection marker conferring resistance to mycophenolic acid that does not influence virulence [9, 10]. This marker in combination with the FLP recombinase allows sequential disruption of both alleles of a gene to generate site-targeted mutants from wild-type strains [11]. The relevance of putative virulence attributes can be based on comparisons of C. albicans with apathogenic yeasts like Saccharomyces cerevisiae and other less pathogenic species. Shotgun sequencing of the C. albicans genome was completed with the sequencing of 10.4 haploid genome equivalents [12]. Roughly 80% of the genes found are closely homologous to the genes of S. cerevisiae. Interestingly, many genes shown to be involved in the virulence of C. albicans belong to the remaining 20% of the genes which have no equivalents in the S. cerevisiae genome. Furthermore, virulence factors also appear to be organized in large protein families specific for C. albicans. This applies especially to the families of secreted aspartic proteinases and cell surface agglutinin-like proteins. As discussed below, numerous genes necessary to

C. albicans Virulence Factors 115 C. albicans for establishing infection were shown to be involved in dimorphism and adherence, which have to be considered as essential virulence traits, or encode secreted hydrolases. However, disruption of genes involved in other functions (e.g. nutritional factors and efflux pumps) for which a homolog exists in many yeasts can also render C. albicans less virulent [3–5, 13].

Dimorphism

In response to certain environmental conditions, C. albicans can switch from a yeast-like organism to pseudohyphal or hyphal forms. Hyphae grow by apical tip extension, and division with true septa occurs in a fission-like manner. In contrast, pseudohyphae arise by budding and are chains of distinct cells where the mother and daughter cells fail to separate after division [14]. The ability to switch between yeast cells and long hyphae is considered an essential virulence trait of C. albicans [15]. During mucosal and deep infections, budding yeasts transform to filaments (fig. 1). Evidence that hyphae are essential for tissue invasion was demonstrated using mutants unable to switch to the filamentous form in serum and were avirulent in a mouse model [16]. The yeast and filamentous forms of C. albicans (fig. 1) are interconvertible and differ in many adhesion and hydrolytic factors themselves associated with virulence. The formation of hyphae is induced by many signals, including N-starvation and/or C-starvation, pH and embedded/microaerophilic conditions [for a review, see Ernst 17].

Switching System of C. albicans

Also linked with virulence is a second form of cellular transformation in which cells alternate at high frequency (10–2–10–3) between different phenotypes, each expressing a unique combination of characteristics [18–20]. Switching involves the coordinated regulation of many unlinked genes and can affect many characters such as colony morphology, cell shape, cell size, cell wall morphology, aspartic proteinase secretion, adherence, virulence in animal models and susceptibility to antifungal drugs. The different phenotypes are strain specific. Switching in C. albicans is ordinarily observed through changes in colony morphology. The particular strain WO-1, that switches from a white to an opaque form at frequencies of 10–2–10–4 per cell cycle, has been the object of intensive research [19, 20]. Recently, phenotypic switching events in this strain have been shown to be regulated by the transcriptional factor Efg1p [21].

Monod/Zepelin 116 a b

c d

Fig. 1. Yeast and filamentous forms of C. albicans. a C. albicans grown in Sabouraud liquid medium. b Direct mycogical examination of a vaginitis sample. c C. albicans colonizing human nonkeratinized buccal epithelium. A tissue sample (diameter 2 mm) was infected with 2 ϫ 105 blastospores of C. albicans CBS 2730. After 4 h of incubation, the epithelial surface was invaded by germ tubes. d C. albicans invading endothelial cells. Cultured endothelial cells from dog vessels were infected with C. albicans CBS 2730 blas- toconidia (105 cells/ml). After 4 h of incubation, target cells were invaded by C. albicans filaments. a, b Fluorescence micrographs of C. albicans stained with blankophor. c, d Scanning electron micrographs.

Opaque cells produce the secreted aspartic proteinases Sap1p, Sap2p and Sap3p in vitro, while the white form of the same strain cultured under identical conditions produces Sap2p only [22–24]. In an intravenous infection model, the white phenotype was more virulent than the opaque phenotype. In contrast, in a murine cutaneous candidiasis model, the opaque phenotype was more infectious [25]. In addition to yeast-hyphal dimorphism, phenotypic switching can provide a means for rapid adaptation of C. albicans to a changing environment in different anatomical locations.

C. albicans Virulence Factors 117 Adherence

Infection with C. albicans generally involves adherence and colonization of superficial tissues. Cell wall mannoproteins of C. albicans are pivotal for these functions [for a review, see Calderone and Braun 26]. Glycosylation occurs through either N-linkage to asparagine or O-linkage to serine or threo- nine residues via mannosyltransferases. Blocking the adhesion of C. albicans to epithelial cells in vitro could be achieved by either mannose supplementation, pretreating yeasts with the mannose-specific lectin concanavalin A [27], exposing C. albicans to tunicamycin [28], a drug which inhibits mannoprotein synthesis, or by the addition of an anti-Candida antibody with specificity for mannan [29]. Moreover, cell surface defects of C. albicans strains involving mannoproteins are correlated with a reduction in adherence [30], and null mutants of MNT1, which encodes for a mannosyltransferase involved in O-glycosylation, showed reduced adherence to buccal epithelial cells [31]. The virulence of these mutants was strongly attenuated in both systemic and vaginal animal models. The well-characterized agglutinin-like sequence (Als) proteins are highly glycosylated cell surface proteins with both N- and O-linked carbohydrates [32–34]. They are encoded by the ALS gene family, which contains at least 9 members [35] with similarities to ␣-agglutinin (Ag␣1p) of S. cerevisiae, a cell surface glycoprotein involved in cell-to-cell interactions during mating [36]. The C-terminus of each predicted Als protein encodes a consensus sequence for the addition of a glycosylphosphatidylinositol (GPI) anchor. Heterologous expression of ALS genes in the nonadherent S. cerevisiae confers the ability to adhere to extracellular matrix glycoproteins [37] and to cultured human cells [38]. Als proteins, like other cell wall proteins, such as Hpw1p and Saps (see below), are activated specifically during hyphal development. Immunohistological staining demonstrates the production of Als proteins on the cell wall of C. albicans in infected tissues of mice with disseminated candidiasis [39]. C. albicans possesses specific molecules with limited homology to the vertebrate integrins able to bind to extracellular matrix proteins, such as collagen, fibronectin and laminin, but also ligands of host cells and iC3b of the complement system [for reviews, see Calderone and Braun 26 and Hostetter 40]. In particular, a gene encoding a protein similar to mammalian leukocyte integrins, INT1, has been cloned from C. albicans [41]. The INT1 gene product shows homology to the ␣M chain of the human iC3b receptor CR3, and is not present in other yeast species. INT1 expression in S. cerevisiae was sufficient to mediate adherence of this yeast which is normally nonadherent to epithelial cells. Furthermore, disruption of INT1 in C. albicans suppressed hyphal growth, adhesion to epithelial cells and virulence in mice.

Monod/Zepelin 118 Lectin activity is expressed by C. albicans [42] but so far, only one gene, EPA1 (for epithelial adhesin 1), encoding a specific lectin of the related species Candida glabrata was cloned [43]. From its predicted amino acid sequence, this lectin protein appears to be GPI anchored. Deletion of this gene reduced adherence of C. glabrata to epithelial cells in vitro by 95%. Heterologous expression of the EPA1 gene in the nonadherent S. cerevisiae also confers the ability to adhere to cultured cells. Stable attachments between germ tubes and mammalian cells were found with C. albicans [44]. A surface protein in the fungus with similarities to mammalian small prolin-rich protein, encoded by the gene HWP1 (hyphal wall protein) serves as a substrate for epithelial transglutaminase enzymes that can covalently cross-link Hpw1p to epithelial proteins. This binding mechanism involving covalent cross-linkage of the fungus to host cell surface proteins appears to be unique for the adherence of microorganisms. In summary, C. albicans possesses a broad panel of molecules involved in adherence with different binding mechanisms which can function independently. Many of these molecules, such as members of the Als family, Int1p, Hwp1p, appear to be specific for C. albicans and are not found in other yeasts. Secreted aspartic proteinases, which are also a particularity of C. albicans are additionally involved in adherence. Their multiple roles in virulence will be discussed below.

Secreted Hydrolases

Many pathogenic microbes possess constitutive and inducible hydrolytic enzymes that help them invading host tissues by destroying, altering, or damaging membrane integrity leading to dysfunction or disruption of host structures. C. albicans produces a variety of secreted hydrolases which are implicated in pathogenesis.

Phospholipases

Extracellular phospholipases have been implicated as virulence factors in many microorganisms, such as Listeria monocytogenes [45], Toxoplasma gondii [46, 47], and Entamoeba histolytica [48]. The secretion of phospholipases by C. albicans was first detected by growing the yeast on solid media containing egg yolk and CaCl2 and visualizing a halo of precipitation of phos- pholipid digestion products with calcium around the colonies [49]. Phospholipase activity was shown to be correlated with adherence to buccal

C. albicans Virulence Factors 119 epithelial cells and pathogenicity in mice [50]. C. albicans genes PLB1 and PLB2, encoding phospholipases, were cloned [51, 52]. Results obtained from PLB1-deficient mutants constructed using the ura-blaster methods were shown to contribute to lethality of mice infected by C. albicans. Phospholipase Plb1p is not involved in adherence, but contributes to virulence by facilitating increased penetration and damage to host cells; this enzyme may also enhance the ability of C. albicans to cross the gastrointestinal barrier and to enter blood vessels.

Secreted Aspartic Proteinases

Among various potential virulence factors, Saps have been intensively investigated. High titers of anti-Sap antibodies are observed in sera of patients with candidiasis [53] and the presence of Sap antigens has been demonstrated on the surface of fungal elements colonizing mucosa, penetrating tissues during disseminated infection and evading macrophages after phagocytosis of Candida cells [54–56]. Evidence for the role of Sap in virulence has also been derived from experiments with the specific inhibitor pepstatin A that is able to block adherence and invasion of C. albicans [57–60]. Ten genes encoding Saps have been cloned from C. albicans to date [61, 62, our own unpublished results]. Sap1p, Sap2p and Sap3p as well as Sap4p, Sap5p and Sap6p form two subgroups each containing closely related enzymes, whereas Sap7p and Sap8p form distinct branches in clustering trees [61, 62]. The SAP9 and SAP10 deduced amino acid sequences show a putative GPI anchor at the C-terminus of the translation products. The Candida Saps, are synthesized as precursors in a preproprotein form. The prepeptide, or signal peptide, of 16–18 amino acid (aa) residues is necessary for entering the secretory pathway by transporting the protein across the membrane of the endoplasmic reticulum. With the exception of Sap7p, which has a putative 195 aa propeptide, the Candida Saps have a relatively short propeptide (32–58 aa) which contains one to four Lys-Arg sequences, one of which is immediately before the N-terminus of the mature form of the enzyme. The fact that C. albicans inhabits diverse host niches leads to the question whether different Saps are expressed by C. albicans in reaction to specific environmental conditions. This question has been addressed by a number of laboratories (1) by Northern blotting analysis [63], (2) by using RT-PCR performed on in vitro and in vivo samples [64–66], (3) by using a gene expression reporter system [67], (4) by constructing mutants bearing mutations in either individual or multiple SAP genes [65, 68, 69] and (5) by determining which of these proteinases are involved during yeast adherence to mucosae and tissue invasion by

Monod/Zepelin 120 using immunological methods [70]. The availability of pure recombinant Sap1p to Sap6p produced in Pichia pastoris helped to determine the specificity of monoclonal and polyclonal antibodies raised against peptides or active individual Sap.

Aspartic Proteinases in the Adherence Process

Using RT-PCR techniques, the expression of different SAP genes was detected in samples of human oral candidiasis [63, 65]. In an in vitro model of oral candidiasis using reconstituted human epithelium, the different proteinase genes were sequentially expressed [64]. SAP1 and SAP3, then SAP6 and finally SAP2 and SAP8 expression was observed at the time when increasing damage to reconstituted human epithelium occurred [64], and the lesions caused by ⌬sap1,3 double mutants were shown to be strongly attenuated [65]. In contrast, no attenuation of the lesions in reconstituted human epithelium was observed using a ⌬sap4,5,6 mutant [64]. In an experimental rat vaginitis model, the expression of SAP1 and SAP2 was shown by Northern blot analysis [63]. In particular Sap2p seemed to have a potential importance in vaginal infection as deduced from infection assays using proteinase-deficient mutant strains in a rat vaginitis model [71]. Altogether these results show the importance of the SAP1- SAP3 gene subfamily in mucosal adherence. Since the introduction of highly active antiretroviral therapy using HIV aspartic proteinase inhibitors and nucleoside analogs, oropharyngeal candidiasis, the most common fungal disease in patients suffering from HIV infection, is less often observed even in the presence of viral resistance to treatment [72–74]. The availability of purified recombinant Sap produced in P. pastoris allowed testing the effects of four different HIV aspartic proteinase inhibitors, ritonavir, saquinavir, indinavir and nelfinavir on C. albicans Sap activity. Interestingly, Sap1p, Sap2p and Sap3p, apparently involved in Candida adherence, were those inhibited by HIV proteinase inhibitors [75]. The influence of these HIV proteinase inhibitors on the adherence of various Candida strains to epithelial cells was correlated to the direct effect of the HIV proteinase inhibitors on the different Saps [75, 76] (fig. 2), leading to the hypothesis that the frequency of oropharyngeal candidiasis might be decreased still more by inhibition of Saps involved in C. albicans adherence. In another study, indinavir and ritonavir were shown to have a good anti-C. albicans effect in a rat vaginitis model [77]. Globally, these results suggest that the dramatically lower rates of oropharyngeal candidiasis due to C. albicans in individuals receiving HIV aspartic proteinase inhibitors reflect not only an improvement in the immune system as measured by an increase in CD4 counts but that at least some of these

C. albicans Virulence Factors 121 a

Fig. 2. Fluorescence micrographs of C. albicans SC5314 adhering on epithelial Vero cells in the absence a and in the presence b of the HIV proteinase inhibitor ritonavir at a concentration of 200 ␮m.

HIV proteinase inhibitors may display a specific anti-Sap activity leading to a reduced number of C. albicans yeasts on epithelial cells. Therefore, development of specific aspartic proteinase inhibitors might be useful in the treatment of mucosal candidiasis. The precise function of Candida Saps in the adherence process is not known, but two hypotheses can be advanced: (1) the Candida Saps could act as ligands to surface proteins of the specific host cells, which do

Monod/Zepelin 122 not necessarily require their enzymic activity. (2) The Candida cells use Saps as active enzymes to affect target structures of their host cells leading to conformational changes of surface proteins or ligands of epithelial cells allowing a better adherence of the yeasts.

Aspartic Proteinases in Deep-Seated Candidiasis

Deep-seated, or systemic candidiasis is a secondary disease of patients suffering from an impairment of their immunological defense system due to unrelated underlying diseases, such as hematological disorders or different forms of cancer as well as organ transplantation, which often involve the cellular immune system. Specifically, a lack of granulocytes appears to be important in the pathogenesis of candidiasis. Deep-seated candidiasis is considered an endogenous infection. After colonizing the mucosal surfaces, Candida yeasts penetrate the mucosal barrier, and to the blood system. The second defense line formed by macrophages and granulocytes constitutes a major obstacle for the establishment of systemic Candida infections. Engulfed Candida have the ability to resist phagocytes [78] similarly to L. monocytogenes [79]. After ingestion of yeast cells by isolated peritoneal macrophages, Sap4p, Sap5p and Sap6p have been shown to be expressed by C. albicans [69]. However, the three closely related isoenzymes Sap4p, Sap5p and Sap6p could never be recovered up to now from C. albicans in vitro cultures although SAP4, SAP5 and SAP6 mRNAs were detected during hyphal cell growth stimulated by serum at neutral pH [80, 81]. SAP4, SAP5 and SAP6 appear to be expressed by one or several yet unknown macrophage intracellular factors via the transcription factor CaTec1p which specifically recognizes repeated CATTC(A/C) sequences in the promoters [82]. Results of the viability tests of wild-type strain C. albicans SC5314 and of the ⌬sap4,5,6 mutant DSY459 in macrophages suggest that Sap4p–Sap6p have a role as pathogenicity factors [70]. Other results support the importance of Sap4p–Sap6p in deep-seated candidiasis. (1) The members or at least one individual member of the Sap4p–Sap6p subfamily were shown to contribute to virulence in mice peritonitis [83]. (2) Mice and guinea pigs infected with DSY459 survived significantly longer than animals infected with the parental nonmutated strain Sc5314 [69]. Sap4p–Sap6p isoenzymes, which are optimally active at pH 5.0, could act as cytolysins, as described for Trypanosoma cruzi, Listeria and Shigella [84]. Filamentous growth C. albicans has a destructive action on macrophages [16]. However, as previously reported [54], Sap4p–Sap6p could additionally be involved in the direct destruction of intracellular components of the macrophages by affecting some key enzymes of the macrophage oxidative

C. albicans Virulence Factors 123 metabolism which is important for optimal microbicidal activity [for a review, see Miller and Britigan 85]. Immunohistological staining also allowed to demonstrate the presence of Sap proteins on the cell wall of C. albicans in infected tissues of patients and mice with disseminated candidiasis [56]. Therefore, Saps could also play an important role in tissue invasion by the fungus. Using a gene expression reporter system, SAP2 expression appears to be strongly induced after dissemination into deep organs [67]. In conclusion, the ability of C. albicans to adhere to mucosae in the oral and vaginal tracts, to invade deep organs and to resist to phagocytic cells apparently requires the use of combinations of different specific proteinases suitable to each particular condition during the infection. Expansions of genes to form a gene family could reflect selection during evolution to allow a better adaptation of the organisms to different environmental conditions.

Acknowledgment

We thank Dr. Mary Holdom for a critical review of the manuscript and assistance with the English.

References

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Dr. Michel Monod, Service de Dermatologie, Laboratoire de Mycologie, BT422, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne (Switzerland) Tel. ϩ41 21 314 0376, Fax ϩ41 21 314 0378, E-Mail [email protected]

Monod/Zepelin 128 Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 129–166

Cutaneous Mycology

Thomas Hawranek

Department of Dermatology, Landeskliniken Salzburg, Salzburg, Austria

In 1839 it was shown for the first time that fungi can cause disease. Schoenlein and Gruby discovered what was later called Trichophyton schoenleinii, the causative agent of scalp infection or favus [1]. But for many years, fungi somehow lived in the shadow of bacterial diseases: their importance grew during the last decades as a consequence of worldwide travel as well as the increased use of immunosuppressive drugs and the appearance of AIDS. Opportunistic infections caused by unknown fungi or fungi initially thought to be nonpathogenic were increasingly recognized. Dermatology is involved not only because the majority of fungal infections affect the skin or the subcutaneous tissues, but also because systemic infections may present with cutaneous lesions and therefore lead to the diagnosis of systemic disease. Many of the subcutaneous and systemic mycoses were first described by dermatologists. Excellent reference books [1–7], comprehensive reviews [8–12] and web sites are available on the theme. A good starting point for excursions into the world of mycology online is, for instance http://fungusweb.utmb.edu/mycology/. This short excursus on cutaneous mycology cannot and will not give an extensive overview on this vast field, but tries to expound some clinical aspects of fungal-induced dermatological diseases. Whether they display pleasant activities, like the production of cheese or the fermentation of alcohol, or do harm by destroying crops or by attacking man, fungi are closely linked to our lives. Fungi may affect man in three ways: by causing allergy (cutaneous, respiratory and other forms), by their toxins (whether by ingestion of poisonous fungi or by production of mycotoxins) or by invasion. The molecular phylogeny and systematics of the fungi will be discussed elsewhere in this book [13]. A simple division of fungi with respect to clinical aspects is that into molds and yeasts, the former characterized by the formation of septate and nonseptate hyphae, the latter by an unicellular life cycle with reproduction mainly by budding. The so-called dimorphic fungi, such as Histoplasma or Coccidioides, may switch forms depending on environmental conditions, such as temperature, grow as molds at room temperature and as yeasts at 37 °C or in human tissue. The immense number of names given not only to the whole diversity of up to 250,000 species, but often also to one and the same of the approximately 200 pathogens, has led to some confusion, partly brought into order by an interna- tionally accepted nomenclature. Confusion also arose from giving different names to one and the same disease, depending on which of the many different pathogenic agents was demonstrated in a special case. Identification of a particular fungal pathogen is essential in many respects; it will have an important role in therapeutic decision-making as in otomycosis, and with the arrival of the new antifungals it has become important to differentiate between yeast and dermatophyte infection as well as between dermatophytes themselves, as they may not respond similarly. Identification of the pathogen may also lead to the source of an infection and therefore enable initiation of measures against spread. A useful classification of fungal infections groups them according to the site of the infection combined with the immune reaction of the host (table 1).

Pathogenesis of Cutaneous Fungal Infection

As the mammalian physiological barriers protect against microbial invasion, fungal diseases in man are rather uncommon considering the wide distribution of these microorganisms. In addition, nonimmunological and immunological defenses hinder the virulence of fungi [14]. All humans have contact with dermatophytes during their lifetime, but only a few develop clinical symptoms, fungal infection therefore usually depends on a breakdown of one of these defense lines. Dermatophyte growth is restricted to the stratum corneum, the keratinized hair shaft and the keratinized parts of the nail. From there, the disease is transmitted by direct contact or via fomites in the form of parasitic arthroconidia, the infective agents of the fungi. These are extremely resistant to environmental rigors surviving up to 20 months in scales or hair, which therefore are the usual source of infection. As there is not much space between the layers of the stratum corneum, arthroconidia, which are formed by fragmentation of hyphae, are the ideal form of growth. This fragmentation is promoted by high carbon dioxide tension, humidity, a temperature of ideally 37 °C, a physiological pH, certain metabolites and still unidentified cutaneous factors. Transversal growth between the cell layers is facilitated by keratinases produced by fungi as well as hydrolytic enzymes (e.g. lipases, proteases, phosphatases) and finds its clinical expression in the peripheral expansion of the skin lesion.

Hawranek 130 Table 1. Clinical classification of the most common fungal infections and their pathogens

Superficial mycoses Characterization: cosmetically apparent infections without invasion of living tissue or immune response of the host 1. Pityriasis versicolor (M. furfur) 2. White piedra (T. ovoides, T. asakii, T. asteroides and others) 3. Black piedra (P. hortai) 4. Tinea nigra (H. werneckii) Cutaneous mycoses Characterization: superficial infection of skin, hair and nails; usually no invasion of living tissue, defense reaction of the host 1. Dermatophytosis (ringworm, tinea) (multiple dermatophytes) 2. Candidiasis (C. albicans and others) Subcutaneous mycoses Characterization: chronic infection of skin and subcutaneous tissue after traumatic implantation of the geophilic agent. 1. Sporotrichosis (S. schenckii) 2. Chromoblastomycosis (Fonsecaea pedrosoi, Cladophialophora carrionii and others) 3. Mycetoma (Madura foot) (Madurella mycetomi, Pseudoallescheria boydii, Acremonium, Exophiala and others) 4. Subcutaneous zygomycosis (entomophthoromycosis and mucormycosis) 5. Lobomycosis (L. loboi) 6. Rhinosporidiosis (Rhinosporidium seeberi) Systemic (deep) mycoses by pathogenic fungi Characterization: the immunocompetent host is infected by the dimorphic fungi as they change their morphological form; patients usually get infected by inhalation of conidia; restricted to certain geographic areas 1. Coccidioidomycosis (C. immitis) 2. Paracoccidioidomycosis (P. brasiliensis) 3. Histoplasmosis (H. capsulatum) 4. Blastomycosis (B. dermatitidis) Systemic (deep) mycoses by opportunistic fungi Characterization: the immunocompromised host is overcome by fungi of actually low virulence 1. Candidiasis (C. albicans and others) 2. Cryptococcosis (C. neoformans) 3. Systemic zygomycosis (mucormycosis) (Rhizopus arrhizus, Mucor, Absidia and others) 4. Aspergillosis (Aspergillus spp.) 5. Pseudallescheriasis (P. boydii) Rare mycoses 1. Pheohyphomycosis (E. jeanselmei, Cladophialophora, Exophiala and others) 2. Hyalohyphomycosis (Acremonium spp., Penicillium spp., Beauveria spp. and many others) 3. Protothecosis (Prototheca spp.)

Cutaneous Mycology 131 Essential steps in dermatophyte infection are an initial close contact between arthroconidia and corneocytes of the stratum corneum, adherence by physical or chemical binding, rapid germination (usually within 4–6 h) to evade elimination by continuous epithelial desquamation and finally penetration of the stratum corneum. Defense mechanisms of the host include elimination by epidermal turnover (decreased infection in psoriasis, increased in keratoderma), unsaturated fatty acids of the sebum (explaining relative resistance of the adult scalp against tinea capitis as well as clearing of this disease during puberty), vitamin-K-like substances in sweat, unsaturated transferrin in human serum (binding of the iron needed for fungal growth) and a body temperature of about 37 °C. Cell-mediated immunity often leads to spontaneous resolution of infection, whereas a decrease in T-helper cells and an increase in T-suppressor cells tend to lead to chronicity. Antibodies seem to have no effect on the course of the infection. Neutrophils and monocytes play an important role in the early phases of invasion. Other factors associated with fungal infections are very young and very old age, endocrine disorders like diabetes, Cushing’s disease, sex, genetic and racial factors, hot and humid climates, malnutrition as well as lack of knowledge about the prevalence and carrier status and absence of control measures [15].

Clinical Mycology

Superficial Mycoses

The so-called superficial mycoses are characterized by invasion restricted to the stratum corneum and thus are usually not associated with a remarkable inflammatory response of the host.

Pityriasis versicolor This cosmetically disturbing condition, first described in 1846, is caused by the lipophilic yeast Malassezia furfur, the hypha-producing, invasive form of Pityrosporum ovale (mainly on the scalp) sive orbiculare (mainly trunk), physiological saprophytes of the human skin from where they may be cultured in up to 100% of the cases. Whether or not these two latter forms represent different species or two forms of the same organism is still discussed. Certain predisposing factors (humidity, heat, oily skin, hyperhidrosis as well as hereditary disposition and immunodeficiency) lead to the distinct clinical lesions presenting as asymptomatic, rarely itching macules of hypopigmentation (in summer affected areas fail to tan) and hyperpigmentation (winter) on the upper trunk, neck and shoulders, slightly scaling when scraped. The disease is uncommon in

Hawranek 132 childhood. Recently reported entities are systemic disease by Malassezia – fungemia described mostly in neonates receiving parenteral lipids through central catheters as well as a form of onychomycosis in AIDS patients. Usually, the clinical picture in combination with the detection of short curved hyphae and ovoid budding yeast cells (the so-called ‘spaghetti and meat- balls’) by light microscopy is sufficient for diagnosis. Often inspection under Wood’s light (a lamp emitting UV light at a wavelength of above 365 nm) may demonstrate weak yellow fluorescence, which however disappears after bathing [16]. Occasionally, however, it may be necessary to culture the fungi on agar with sterile olive oil. In most cases, topical treatment with selenium sulfide or a number of other agents including imidazoles and terbinafine extended also to areas not yet visually affected will be successful, repigmentation taking up to several months. A single dose (400 mg) of ketoconazole or fluconazole as well as itraconazole 200 mg daily for 5–7 days has been equally effective. Before treatment, it is advisable to point out to the patient that relapse after successful therapy is the rule, but may be avoided by prophylactic measures such as topical therapy weekly (low compliance [17]) or ketoconazole 400 mg once a month. Pityrosporum folliculitis, a distinct clinical picture, is characterized by scattered itching acneiform, small follicular papules, sometimes pustules, on the back and shoulders of young patients. In contrast to acne, it is often aggravated by the use of local antibiotics and sun exposure. Hyphae are usually absent by light microscopic examination of pustular material. Pityrosporum yeasts take part in the pathogenesis of seborrheic dermatitis and have also been associated with certain facial eczemas in young atopic women.

White Piedra Trichosporon asakii, Trichosporon inkin, and Trichosporon mucoides are regularly isolated from clinical specimens. In addition, Trichosporon ovoides (Trichosporon beigelii) was isolated as causative agent of capital white piedra. These inhabitants of soil, lakes and plants in subtropical and temperate climates including Europe, North America and Japan produce small and soft white to cream-colored nodules on hair shafts of a usually restricted area which may be easily stripped off. Breakage or splitting of hair is common. The condition is not contagious. Cultures should be grown without the use of cycloheximide. Treatment includes shaving of all affected hair and/or topical clotrimazole, oral ketoconazole being an alternative possibility. In immunocompromised patients T. asakii may lead to a serious systemic infection called trichosporonosis.

Black Piedra This infection by Piedraia hortai presents with tightly adherent dark nodules on hair shafts causing breakage. Light microscopy and culture consolidate

Cutaneous Mycology 133 the diagnosis. Shaving is the treatment of choice; oral terbinafine 250 mg daily for several weeks may be an alternative.

Tinea nigra This harmless condition, caused by Hortaea (Phaeoannelomyces) werneckii, is acquired by direct inoculation from various sources via minor trauma and most commonly involves one hand. This dematiaceous fungus is endemic in maritime regions of tropical and subtropical climates and occasionally affects travelers on vacation, producing mostly asymptomatic, sometimes itchy, slowly extending brown to black macules after incubation periods of supposedly weeks to decades. Misinterpretation as melanoma may lead to unnecessary excision. After consolidation of diagnosis by light microscopy and culture, treatment with topical keratolytic agents, 10% thiabendazole or topical imidazoles has been effective.

Cutaneous Mycoses

Dermatophytosis (Ringworm, Tinea) The dermatophytes, a group of filamentous fungi invading the epidermal stratum corneum and keratinized skin appendages as hair and nails in up to 20% of the population [18], may be divided according to their natural habitats: anthropophilic species are spread from human to human, zoophilic species par- asitize animals, and geophilic species live on soil as saprophytes. The latter two are also capable of causing human disease. Spread of zoophilic and geophilic species from human to human is uncommon. Zoophilic species usually cause a more severe clinical variant producing suppurative lesions. Trichophyton rubrum and Trichophyton mentagrophytes var. interdigitale are the two most commonly isolated species in Europe. Dermatophytes are discriminated by macroscopical and microscopical features (table 2) described in the section on laboratory diagnosis.

Special Clinical Patterns

Tinea corporis This dermatophyte infection confined to the trunk and extremities takes a subacute to chronic course (weeks to years) and produces the typical and rather well-known lesion called ringworm (fig. 1), a usually round, often irregular scaly lesion with a significantly more inflamed, raised border containing a

Hawranek 134 Table 2. Characteristics of some dermatophytes

Species Geography Remarks Culture Microscopy

Anthropophilic dermatophytes T. rubrum cosmopolitan most common and widely distributed downy or fluffy white ‘snowball’ Macroconidia long, cylindrical, thin dermatophyte of man; mostly feet and appearance; reverse: dark red, walled, usually absent; microconidia nails infected; Wood’s light negative sometimes melanoid; granular rare also, clavate to pyriform, and dysgonic varieties situated along the sides of hyphae T. mentagrophytes cosmopolitan mostly tinea pedis and corporis; no tinea flat, downy, white in the beginning, single-standing pear-shaped conidia var. interdigitale capitis, but positive hair penetration then becomes powdery and cream- along the hyphae, typical spiral hyphae test; positive urease test. colored, sometimes velvety occasionally, sometimes macroconidia; chlamydospores M. audouinii cosmopolitan used to be the most common cause of downy white surface to grayish- pectinate (‘comb-like’) and racquet epidemics of tinea capitis in Europe white ‘mouse-fur’, reverse salmon (rackett) hyphae, thick-walled and N. America, Ectothrix; Wood’s to peach-pink, sometimes rusty; chlamydospores; rare micro- and light positive slow growth (bizarre) macroconidia M. ferrugineum E. Europe, epidemics of tinea capitis in children, waxy, convoluted, slowly growing no macro- or microconidia, but hyphae Asia, C. Afr., ectothrix invasion of hair; Wood’s thallus, color cream to yellow of irregular shape with prominent septae S. America light positive to red (‘bamboo hyphae’) T. schoenleinii Eurasia, causes favus, a chronic and scarring gray to gray-brown, cream-colored, no macro- nor microconidia, but hyphae Africa form of tinea capitis with typical orange-brown; waxy and folded, then tend to build antler-like branches at the scutulae, Wood’s light positive becoming flat; grows well at 37 °C end, sometimes resembling chandeliers T. violaceum cosmopolitan finely scaling lesions, typical ‘black dot’ violet, seldom white, glabrous or branched, distorted, thick hyphae; tinea capitis; endothrix hair invasion waxy, heaped and folded, slowly no macro- or microconidia growing colonies; growth stimulated by thiamine; reverse deep purple T. soudanense N. C. Africa endothrix tinea capitis; Wood’s light slowly growing, yellow-orange typical is the right angled, reflexive negative (apricot) cultures with flat or folding, branching of hyphae; rarely velvety surface; often broad fringed pear-shaped microconidia, margin (‘eyelashes’); reverse apricot no macroconidia; chlamydospores

Cutaneous Mycology 135 Table 2. Continued

Species Geography Remarks Culture Microscopy

T. tonsurans cosmopolitan fine scaling lesions mainly on the scalp; cream to buff or yellow-brown irregular, branched (‘tree-like’) endothrix invasion; Wood’s light powdery or velvety, sometimes hyphae with numerous negative folded colonies; reverse yellow- microconidia of different brown or red-brown morphology (‘balloon-like’, ‘matchstick-like’ etc.); macroconidia rare T. megninii Europe, onychomycosis and tinea corporis white suede-like colonies turning to clavate microconidia and long Africa mostly in the upper exposed parts pink or violet with time; wide radial pencil-shaped macroconidia of the body of older men for unknown grooves; reverse light wine-red reasons Epidermophyton cosmopolitan common cause for skin lesions as well typical olive-green to khaki color, macroconidia borne directly from floccosum as onychomycosis; epidemics among slowly growing colonies with hyphae, often in clusters, of broadly persons using the same shower pleomorphic tufts in older cultures; clavate, ‘beaver tail’ appearance with and facilities reverse yellowish-brown 0–4 cells; no microconidia Zoophilic dermatophytes M. canis cosmopolitan cats (!) and dogs; tinea frequently white wooly surface, reverse shows numerous spindle-shaped, rough and seen in children; ectothrix; Wood’s the typical deep yellow, rarely thick-walled macroconidia, few light positive orange or brownish microconidia T. verrucosum cosmopolitan cattle; highly inflammatory infections very slow growth (ϭ small macro- and microconidia rarely seen; of exposed areas, especially the colonies) of white or buff, flat or on thiamine sometimes few, but typical beard and scalp; ectothrix, but raised and folded colonies; ‘rat tail’ or ‘string bean’ shaped Wood’s light negative; grow better reverse white or buff macroconidia; chlamydospores in at 37 °C (!) and with thiamine chains (‘string of pearls’); terminal vesicles with yeast extract T. mentagrophytes UK, NZ hedgehogs and their mites; in man white, flat, powdery colonies, the many large clavate microconidia on the var. erinacei mostly exposed areas of the skin; reverse shows a typical bright sides of the hyphae, macroconidia endothrix invasion in tinea capitis; yellow smooth, thin-walled and of different size Wood’s light negative

Hawranek 136 T. mentagrophytes cosmopolitan ‘mouse favus’; in man rarely scutulae; white and downy first, then slender microconidia along the hyphae, var. quinckeanum seldom endo- or ectothrix invasion; becomes powdery and heaped and becoming broad and pear-shaped Wood’s light negative folded; reverse yellow-brown with age; macroconidia rarely seen M. equinum Europe, horses; rare; ectothrix; Wood’s light suede-like to fine powdery, white 2 to 4-celled spindle-shaped, irregular N. Am., positive to pale salmon, radial folds, reverse thick-walled macroconidia, rare Austr. buff to salmon microconidia M. nanum cosmopolitan pigs; hair invasion possible but Wood’s white to cream-colored powdery, typically egg-shaped, rough-walled light negative reverse brown-orange macroconidia with 2 cells M. persicolor Eur., Austr., voles and bats; rare cause of tinea in white to pink powdery to downy pear- to club-shaped microconidia, Africa, N. Am man surface, reverse red-brown; rapid single-standing or in clusters; rare growth club-shaped macroconidia T. gallinae cosmopolitan fowl; rare cause of tinea in man; white to pink, velvety to downy, multicellular, sometimes curved ectothrix hair invasion; Wood’s light reverse orange-pink to thick-walled macroconidia with a negative strawberry-red blunt tip, microconidia T. equinum cosmopolitan horses; rare in man; ectothrix hair white to buff, velvety to downy solitary pear-shaped to elongated invasion; Wood’s light negative surface, often with yellow fringe; microconidia along the hyphae, reverse yellow, then red brown; macroconidia rare and of clavate shape most require niacin for rapid growth of different size Geophilic dermatophytes M. gypseum cosmopolitan soil; solitary lesions of skin and scalp cinnamon-colored, powdery surface, ellipsoidal cucumber-like, multicelled, (ectothrix but Wood’s light negative) often with a fluffy white taft, thin-walled macroconidia, clavate reverse yellow-brown microconidia M. cookei cosmopolitan geophilic, but reported in dogs, powdery to granular, brownish, ellipsoidal cucumber-like macroconidia, rodents, and man reddish, yellowish, reverse deep but mostly thick-walled; numerous purple-red microconidia

Cutaneous Mycology 137 majority of ‘active’ fungi (fig. 2) and often producing prominent hair follicles. Multiple lesions are not uncommon. Facial involvement is characterized by possible flares in sunlight. Management: Besides measures as boiling or disinfecting clothes, topical antimycotic therapy is the treatment of choice. Ointments should be applied several centimeters beyond the border of the lesion and treatment should be continued over at least 1 week after clearance of the lesions. However, these recommendations are seldom followed, resulting in chronic or recurrent disease (apart from the problem of resistance). In these cases, and when multilocular lesions or an infection of extensive spread have to be treated, systemic antifungals usually for 1–2 weeks are necessary, terbinafine usually giving the best results in dermatophytosis.

Tinea inguinalis (Tinea cruris) This very contagious condition (usually spread via contaminated towels, saunas) presents with bilateral and itchy scaling in the groin (fig. 2), usually including the pubic region and the upper region of the thighs and sometimes the perineum. The frequently occurring candidiasis of this region usually may be easily differentiated by the typical peripheral satellite lesions, pustules and/or papules outside the border of the main lesion. The bacterial erythrasma usually is associated with fine wrinkling and has a typical red-brownish color; Wood’s light may be helpful for diagnosis. Management: Treatment rules are very similar to those of tinea corporis (local therapy should be continued for at least another week after clearance of the lesions), prophylactic measures may include boiling of underwear, usage of different towels for the affected region, examination of the feet to see whether tinea pedis may be the source of autoinoculation, avoidance of occlusive and synthetic garments and possibly weight loss.

Tinea capitis This dermatophyte infection of the scalp and hair of sometimes quite strik- ing appearance is reported to have had tremendous influence on the lives of people in old times: subjects with tinea capitis were allowed to keep their heads covered in the presence of the monarch, and sometimes were not allowed to emigrate to the United States [19]. Although such drastic measures are historical anecdotes, tinea capitis nevertheless may lead to some social discomfort even today. Usually not a disease of adults, this sporadic infection sometimes causes epidemics in schools. Its clinical appearance varies from mildly scaling lesions over alopecia (patchy hair loss) to highly inflamed, suppurative (kerion) variants, the latter usually caused by zoophilic species as the leading pathogen in

Hawranek 138 Fig. 1. Ringworm. Typical round lesion on the calf with inflamed, raised border.

Fig. 2. Tinea ‘cruris’. Note the typical scaling on the ‘active’ border of the lesion containing the majority of ‘active’ fungi.

Western Europe. Itching is no obligate symptom, hair loss usually is reversible except in some cases of kerion. Lymphadenitis is a common complication even in mild disease [20]. ‘Gray patch’ tinea capitis caused by Microsporum (canis and audouinii) is differentiated from the other forms by some authors. There are three forms of hair invasion: ectothrix (M. canis, M. audouinii, Microsporum ferrugineum, Trichophyton verrucosum), endothrix (Trichophyton tonsurans, Trichophyton violaceum, Trichophyton soudanense) and favic (Trichophyton schoenleinii). Favus is a rarely seen condition in small endemic areas of Northern Africa, the Middle East and the USA. It presents with typical dish-like crusts (scutulae) and scarring alopecia and may also affect adults. Wood’s light: This lamp emitting UV light at a wavelength of above 365 nm is helpful in the diagnosis of tinea capitis (bright greenish yellow), erythrasma,

Cutaneous Mycology 139 a bacterial skin disease (brick red) and pityriasis versicolor (yellow) if observed in a darkened room. Dermatophytes regularly inducing fluorescence are M. canis and M. canis var. distortum, M. audouinii, M. ferrugineum and T. schoenleinii, whereas Microsporum nanum and Microsporum gypseum do so only occasionally. Very recent infection may not yet show this quality, and fluorescence may remain even after cessation of viability. Management: tinea capitis is the typical example for the importance of an accurate identification of the pathogen, as proceedings are different for anthropophilic dermatophytes transmitted from child to child and for zoophilic species usually originating from one common source. If more than one child is affected in the first case, the whole class as well as the family members should be examined with brush samples (see Collection of Material for Laboratory Diagnosis) done simultaneously. Active disease should be treated systemically (see below); the carrier status (i.e. asymptomatic patient ϩ positive culture) demands only local therapy with ketoconazole shampoo (or selenium sulfide) for about 3 weeks. Initially, adjuvant measures may include hair cut, protection (dressing or cap) and sterilization of important fomites (e.g. combs, brushes, toys, etc.) even if the organisms may be found on a great variety of locations and there are no uniform recommendations for the method [21]. Cultures should be repeated after 1 month of treatment and again before discontinuation of therapy. Whether or not infected children should be kept away from kindergarden or school remains a moot point, the usual recom- mendation is to wait until light microscopy is negative [10], others [4] tend to let children return after 1 week’s topical treatment with ketoconazole shampoo (or selenium sulfide). These precautions are not necessary with zoophilic infections, here the source (housepets) as well as every infected person should be treated systemically. Antifungal agents used in tinea capitis: griseofulvin 10–20 mg/kg body weight for at least 6–8 weeks up to several months (still the ‘gold standard’) or – if resistance to griseofulvin occurs – terbinafine 62.5 mg daily from 10 to 20 kg, 125 mg up to 40 kg and 250 mg above 40 kg body weight for 4 weeks or itraconazole suspension 5 mg/kg daily for 2–4 weeks or fluconazole 2–8 mg/kg daily for 4–6 weeks. Monitoring of hepatic, renal and hematopoietic function may be indicated [22]. The role of griseofulvin as the gold standard of treatment may be questioned in the near future because of a growing risk/benefit ratio [23] compared to the new drugs. Although valid studies concerning treatment with those new antifungals are still lacking (e.g. terbinafine for T. tonsurans, itraconazole for M. canis) and appropriate dosing and duration of therapy, it is very likely that these drugs prove to be of excellent efficacy, offering pulse therapy and short- course therapy in addition [24].

Hawranek 140 Fig. 3. Sycosis barbae. This infection caused by T. verrucosum in a milker is an impressive clinical example of the aggressive behavior of zoophilic dermatophytes compared with anthropophilic species.

Bacterial superinfection (Staphylococcus aureus, gram-negative bacteria) rarely demands antibiotic treatment; scarring may be prevented by prednisone 1 mg/kg daily for 1–2 weeks in severe cases.

Tinea barbae Mainly adults handling animals (farm, zoo) or straw contaminated by infected mice are affected by this highly inflammatory, usually pustular variant (sycosis barbae, fig. 3), caused in most cases by T. verrucosum.

Tinea pedis (‘Athlete’s Foot’) Up to 70% of the ‘western’ population is reported to suffer from this harmless, but stubborn disease unheard of in certain aboriginal tribes, a fact which has been attributed to the avoidance of occlusive footwear (‘tinea pedis follows in the footsteps of shoe-wearing nations’ [25]). So tinea pedis is rare in Southeast Asia, the original endemic area of T. rubrum where the worldwide epidemic is said to have originated from. Partly due to the lack of sebum, which contains fungistatic lipids, even cultures of clinically normal toewebs give positive results in up to 21% of the samples. Transmission usually occurs by walking barefoot on contaminated floors where the fungi are able to survive in skin scales for many months. The infection commonly starts with scaling in the third or fourth interdigital space (interdigital form). The clinical picture may be mild with erythematosquamous lesions sometimes covering the whole sole and extending upwards (squamous form, ‘moccasin-type’) almost always caused by T. rubrum or a more severe blistering disease (vesicobullous form) generally attributable to T. mentagrophytes var. interdigitale. Especially in chronic courses with interdigital maceration (‘dermatophytosis simplex’) concomitant bacterial

Cutaneous Mycology 141 infection (e.g. Pseudomonas aeruginosa leading to greenish discoloration, Proteus, Staphylococcus aureus) is very common (‘dermatophytosis complex’), most bacteria being resistant to penicillin and its derivates. In mild disease sensitivity of cultures has been reported to reach about 85%. The more symptoms, bacterial superinfection and inflammation, the more difficult it is to isolate the pathogenic fungus in cultures, lowering sensitivity by approximately 50%. Management: Therapy is very similar to that of tinea corporis. Concomitant onychomycosis bears the constant danger of reinfection, therefore a cure of this disease should be aimed at. Chronic disease tends to require systemic therapy (e.g. itraconazole or terbinafine for 2 weeks) with clinical recovery some time after cessation of therapy. Additional measures include daily bathing of feet with meticulous drying and daily change of absorbent socks (100% cotton) which should be boiled or washed with special preparations. Nonocclusive shoes should be preferred, changed and left to dry over 3 days, prepared daily with antifungal footpowder inside. Relapses may be avoided by long-term topical antifungals twice weekly at least [26]. Unfortunately, recurrences must be regarded as a rule.

Tinea manuum Infection of typically only one hand usually occurs simultaneously with (bilateral) infection of the feet (or groins), often involving fingernails (in fact onychomycosis may be a source of reinfection, thus suggesting treatment of onychomycosis). Clinical appearance may be characterized by fine, nonsymp- tomatic, so-called ‘dyshidrotic’ scaling or by hyperkeratotic, chronic lesions. Up to 90% of this disease is caused by T. rubrum. Management: Treatment is very much the same as in tinea corporis, but usually requires about 2–6 weeks of systemic medication; topical treatment should be continued for several weeks after clearance of the lesions.

Onychomycosis Fungal nail infections (fig. 4) are a very common disease with an incidence of approximately 5:1,000. The most common pathogens are T. rubrum followed by T. mentagrophytes var. interdigitale, invading the nail from the distal and lateral ends leading to onycholysis (i.e. separation of the nail from the nailbed), discoloration, thickening and dystrophy. Isolation of the pathogen by culture may prove difficult even in samples positive on light microscopy. An extensive survey of onychomycosis is given in Hay et al. [27]. For onychomycosis caused by molds, see below. Management: Because of the characteristic physical features of the nail, pharmacokinetics play a major role in therapy [28]. Topical therapy (ciclopirox and amorolfin lacquer), more successful with thin nails and if the lunula is

Hawranek 142 Fig. 4. Onychomycosis. Another typical manifestation of dermatophyte infection, starting from the distal border of the nail. spared, may be tried if Ͻ 60% [29] of the nail is affected, another possibility is atraumatic removal of the nail by bifonazole/urea followed by prolonged topical bifonazole. In extensive distal disease, proximal onychomycosis or failure of local therapy, systemic therapy with itraconazole (400 mg/day for 1 week every month for 3–4 months or terbinafine 250 mg daily for 3 and more months or fluconazole) is indicated. In lateral nail mycosis, poor penetration even of the new antifungals may lead to recurrences [30]. Treatment of concomitant tinea pedis is essential. Long-term management with periodical use of topical therapy for tinea pedis and nails should prevent relapses.

Skin and Nail Infections by Molds Inflammation of the proximal nailfold (often misdiagnosed as bacterial infection) strongly suggests onychomycosis by molds (except Acremonium), especially when associated with the proximal subungual form of fungal nail infection [31]. As most molds do not grow on agars containing cycloheximide, this group of pathogens usually is missed on routine agars. This is why many of these infections were first described only recently. The significance of finding a mold in culture has to be seen with caution [32] as usually they represent a contamination. Cases of onychomycosis caused by molds have been estimated at about 5% [33], prevalence rates ranging from 1.45 to 17.6%. Scytalidium Species. Scytalidium dimidiatum (formerly Hendersonula toruloidea) and its nonpigmented variant, Scytalidium hyalinum, found in the tropics, the USA and the Mediterranean (although a recently published article [31] states that Scytalidium spp. have never been isolated in Italy) are very common in endemic areas, but may be acquired by tourists as well. Clinically, they lead to a scaly tinea pedis et manuum, sometimes including nails starting

Cutaneous Mycology 143 from the distal and lateral ends [34] without considerable thickening of the nail. Scytalidium spp. were never found to cause tinea in areas of the skin with a thin keratin layer. Diagnosis often is made by light microscopic demonstration of irregular hyphae. Colonies mostly grow rapidly. If Whitfield’s ointment is not effective, therapy presents a problem, even if there are anecdotal reports of success with the newer antifungals. Onychomycosis Caused by Scopulariopsis brevicaulis. S. brevicaulis is the most common cause of onychomycosis by molds, presenting with a cinnamon discoloration of the toenails. Direct examination reveals the distinctive, lemon- shaped conidia taking up Parker’s stain quickly. On agar with cycloheximide the partially resistant fungus grows slowly. Therapy is problematic, local therapy may be tried after removal. Onychomycosis Caused by Other Molds. Aspergillus spp., Fusarium spp., Acremonium spp. (all three mostly held responsible for ‘superficial white onychomycosis’), Onychocola canadiensis, Curvularia lunata and many others have been described to cause secondary onychomycosis, often in elderly patients with dystrophic nails due to ischemia. According to a recent study [31], local factors do not play a significant role in fungal invasion. Therapeutic efforts in these cases usually are fruitless, although the same study mentions cure rates of 42.5% for S. brevicaulis, 20% for Acremonium and 29.4% for Fusarium. The best results were obtained by the use of topical therapy (8% ciclopirox nail laquer or topical terbinafine after nail avulsion) whereas systemic therapy with itraconazole or terbinafine was often ineffective. Aspergillus infections were cured in 100% of cases with systemic or topical therapy.

Tinea incognito This condition is characterized by an unusually aggressive behavior of dermatophytes invading the upper dermis thus causing granulomatous folliculitis. The main cause for this is seen in prolonged and inappropriate topical steroid therapy for fungal disease, resulting in loss of itching, scaling and the typical pronounced border of the lesion, producing prominent papules and pustules. Other triggers that have been mentioned [35] are trauma (shaving), long- standing occlusion and the use of local steroid/antifungal/antiviral/antibacterial combination therapy.

‘Superficial’ Candidiasis The first step in the genesis of this common infection of mucous membranes (of the mouth, gastrointestinal tract and vagina where they live as physiological commensals) and the skin seems to be a change in host resistance, the origin of which is detectable in most cases. Predisposing factors include age (very young and very old), moisture, reduced general conditions (e.g. malignancies),

Hawranek 144 hormonal influences (diabetes and other endocrine disorders, pregnancy), immunosuppression (e.g. steroids, whole-body irradiation, immunosuppressive drugs), destruction of the physiological bacterial flora by prolonged use of antibiotics and mechanical factors, whereas for vaginal candidiasis such factors have yet to be found. Candida albicans, is by far the most important pathogen; other pathogens include Candida krusei, Candida glabrata, Candida tropicalis. The typical sign of the invasive phase of Candida is the production of hyphae. Management: Three groups of antifungals are available: for the treatment of candidiasis the polyenes (amphotericin, nystatin, natastatin), the imidazoles (clotrimazole, miconazole, econazole, ketoconazole, fluconazole, itraconazole) and flucytosine, the latter – which is used only for systemic candidiasis in combination with amphotericin B – being relatively susceptible to the development of resistance. Amphotericin B in its recently developed lipid-associated form (reduced renal toxicity) is used intravenously because of the minimal gastrointestinal uptake after oral administration. Of the imidazoles, besides their use as topicals, mainly fluconazole (100–400 mg daily) and itraconazole (100–200 mg daily, now in soluble form with better absorption) are used, as ketoconazole was shown to be of considerable hepatotoxicity. With fluconazole, the problem of primary (mainly C. glabrata, C. krusei and some strains of C. albicans) and secondary (maintenance therapy in AIDS patients) resistance is emerging. Specific antimycotic treatment should be combined with general measures to modify predisposing factors, e.g. drying, hygienic procedures with dentures, treatment of underlying diseases.

Oral Candidiasis (Thrush) Management: Suspensions, tablets or lozenges of nystatin, amphotericin or miconazole several times a day for 10–14 days are usually sufficient in acute cases in children and immunocompetent adult patients. Chronic and unresponsive courses, AIDS patients and chronic mucocutaneous candidiasis usually demand the use of systemic imidazoles, intermittently if possible, because of the risk of development of resistance with continuous therapy [36]. Once resistance has occurred, it can usually be overcome by increasing the dose of fluconazole (stepwise up to 800 mg daily); however, success is seldom persist- ing as response to higher doses often is transitory. Alternative azoles (itraconazole, ketoconazole) show modest cross resistance, itraconazole (as a new liquid formulation in cyclodextrin) being very effective for treatment of primary disease at a dose of 200 mg daily. Unfortunately, this efficacy obviously cannot be reached for fluconazole-resistant strains in the long term, so one is left with toxic amphotericin B at 0.3–0.5 mg/kg intravenously per day initially (omission once to three times per week). In angular cheilitis cure depends on treatment of the oral disease and is usually accelerated by additional topical treatment.

Cutaneous Mycology 145 Fig. 5. Oral candidiasis (thrush). Note the typical white, easily removable patches of pseudomembranous candidiasis.

Pseudomembranous Candidiasis. This condition is characterized by white to gray patches (fig. 5) which are easily removed leaving inflamed epithelium, often accompanied by angular cheilitis (perlèche). In patients without known predisposing factors, this disease, especially its chronic form, is highly suspi- cious of HIV infection, often extending to the pharyngeal and esophageal areas, usually causing retrosternal pain on swallowing. Erythematous (Atrophic) Candidiasis. No pseudomembranes; the mucosal surface is inflamed, often associated with local discomfort. In its chronic variant, bacteria probably play a pathogenic role too, so the use of antiseptics in addition to antifungal treatment is essential. Candida Leukoplakia (Chronic Plaque-Like or Hyperplastic Candidiasis). Here the plaques, most commonly on the cheeks and on the tongue, are not easily removable and may clear with prolonged antimycotic therapy. This condition is difficult to differentiate from other types of leukoplakia.

Genital Candidiasis Candida Balanitis. This infection preferably occurs in the uncircumcised population. In its mild variant, papules develop to pustules or vesicles with minimal inflammation and discomfort; in its severe form, these symptoms aggravate and become persistent, often extending to the prepuce. Vaginal Candidiasis. This condition, usually accompanied by itch and discharge, may appear in its acute form or take a chronic relapsing course presenting a burden for both patient and doctor. No satisfying explanation has been found for chronic recurrent vaginal candidiasis; an interesting neuro- psycho-endocrinological mechanism has been advanced [37], supported by observations of an association with signs of depression [38] and atopy [39].

Hawranek 146 Management: As for treatment, single-dose oral fluconazole 150 mg or itraconazole 600 mg or single-dosed vaginal antimycotic suppositories (clotrimazole, econazole, isoconazole, nystatin) in combination with antimycotic topical therapy is generally sufficient; sometimes prolonged therapy for up to 14 days may be necessary. If failure of therapy or relapse occurs, systemic treatment with fluconazole or itraconazole seems to be useful. As C. glabrata and C. krusei usually are not eradicated by these drugs, high-dose treatment with fluconazole for 3 weeks is recommended for the former, local therapy for the latter. Treatment of the asymptomatic sexual partner is no longer regarded as mandatory [29], whereas examination and appropriate treatment still seems useful [40] in cases of continuing recurrences. The question of chronic relapsing vaginal candidiasis is left open in the recently established guidelines of the European Dermatology Forum [29]; fluconazole 150 mg once a month after menstruation or once a week as a possible maintenance therapy after initial eradication [41] seems to be worth trying in certain cases, however leading to relapse in half of the patients after stopping. Another approach is itraconazole 400 mg monthly [42] which is able to reduce the relapse rate but fails after cessation of therapy. Scrotal and Perianal Candidiasis. These manifestations usually accompany genital disease, but may also develop independently. On the scrotum candidiasis often presents as erythema.

Candida Paronychia and Onychomycosis Interdigital candidiasis and candida paronychia are connected with frequent immersion of the hands in water leading to painful swelling of the nailfolds. Discharge of pus is often associated with bacterial coinfection. The invasion may also spread to the nails causing onycholysis, even if this seems to be a rare occurrence in temperate climates [43]. Other causes of candida onychomycosis are Cushing’s disease and Raynaud’s disease; it also appears in connection with chronic mucocutaneous candidiasis. In the course of diagnostic procedures, a dermatophyte isolated in culture is considered the cause of onychomycosis, whereas C. albicans is envisaged as a possible pathogen. Other Candida spp. may be contaminants requiring reculture [44]. Management: In paronychia, prolonged local therapy with polyene or imi- dazole lotions combined with drying and improvement of circulation are helpful, chronic cases often demand the additional use of topical steroids. For onychomycosis, and perhaps also for paronychia, systemic imidazoles are promising.

Congenital Candidiasis Generalized cutaneous candidiasis of newborns, mostly of mothers who suffered from vaginal candidiasis prior to delivery, often is associated with

Cutaneous Mycology 147 prematurity or intrauterine contraceptive devices. It usually starts on the face and chest and becomes generalized over the next days. Sometimes pulmonary involvement has to be differentiated from Candida sepsis, which seldom involves the skin. Management: Topical antimycotic therapy is sufficient for skin infection, whereas systemic infection demands systemic therapy with amphotericin B and/or fluconazole.

Chronic Mucocutaneous Candidiasis Rare syndrome of unknown origin (possibly based on the inability to develop effective cell-mediated immune responses against Candida [45]) with chronic oral (pseudomembranes), cutaneous (crusted plaques called Candida granuloma) and nail (dystrophy) candidiasis, mostly in children (usually with extensive immunologic defects), but also in adults. This condition should be differentiated from severe candidiasis among plenty of other infections due to a known underlying compromised immune system (it seems important not to forget HIV infection in adults presenting with chronic candidiasis), whereas in patients with chronic mucocutaneous candidiasis (CMC), the candidiasis of skin and mucous membranes represents the dominant clinical feature. Despite the existence of overlaps, differentiation into five clinical forms has proven helpful: a mild autosomal recessive form, a more severe autosomal dominant variant, which perhaps includes a third form (diffuse CMC) with the most severe course (often bronchiectasis). The fourth variant is associated with endocrinopathy, mostly familial polyendocrinopathy syndrome, candidiasis sometimes preceding the endocrine disorder by 10 years, at the moment including a recently described form associated with hypothyroidism which may be transmitted in an autosomal dominant way. The last subtype, late-onset CMC, may be associated with thymoma. Association with vitiligo, pernicious anemia, ovarian failure and lupus erythematosus has been reported. Patients usually present other infections (e.g. warts). No underlying disease is found in most cases, although investigations (endocrine screening tests, immune parameters, chest X-ray to exclude thymoma) are recommended as well as follow-up investigations. Prognosis is variable, spontaneous remissions occur. Management: Prolonged and repeated systemic therapy with fluconazole, itraconazole or ketoconazole – always with the risk of development of resistance – is crucial, treatment of an underlying endocrine disorder has no effect on candidiasis. Genetic counseling is indicated.

Other Forms of Candidiasis Interdigital candidiasis mainly affects persons with continuous water contact and is characterized by macerated skin with erosion in the toe or finger

Hawranek 148 web spaces. Candida intertrigo (flexural candidiasis) is generally easily recognized by the satellite lesions, which are also typical for candida-induced nappy (or napkin, diaper) candidiasis, usually a secondary infection of irritant eczema. Gastrointestinal involvement should be considered. Management: For intertrigo, local antimycotic therapy has to be combined with drying of this region (e.g. linen strips, use of an antimycotic paste), local therapy with potassium permanganate deals with this aspect and is effective against concomitant bacteria as well. In the treatment of diaper candidiasis these points of view are important as well (e.g. frequent changes of napkins); steroids should be avoided if possible.

Subcutaneous Mycoses

This group of mycoses is characterized by traumatic inoculation of geophilic fungi and is seen mainly in tropical and subtropical climates.

Sporotrichosis Geography: Europe (rare), Americas, Australia, Japan, South Africa. As the dimorphic fungus Sporothrix schenckii is a geophilic species found in soil and on plants, persons handling those are predisposed to acquire this chronic infection (‘drunken rose gardener syndrome’) which primarily affects the cutaneous and subcutaneous tissues (‘fixed type’) as well as the adjacent lymphatics (‘lymphangitic type’), forming nodule after nodule along the draining vessels. Prillinger et al. [46] have shown that S. schenckii isolates from soil are genotypically different and belong to Sporothrix albicans. Sometimes, the infection spreads to joints, bones and muscles as well as to the lungs, central nervous system or genitourinary tract (‘disseminated type’). Diagnosis usually is made by culture; histopathology (as a dimorphic fungus Sporothrix develops the yeast form in tissue, often surrounded by eosinophilic asteroid bodies) is less important because of the small number of fungal elements found in biopsies [47]. Therapy with itraconazole 100 mg daily for weeks according to clinical response is often successful; potassium iodide used to be the therapy of choice. Heat therapy (42 °C for 30 min twice a day) may be tried in early and solitary skin lesions, as S. schenckii does not grow above 38.5 °C [48].

Chromoblastomycosis Geography: South-East Asia, Africa, Central and South America. This chronic infection by dematiaceous fungi typically presents with verrucous, slowly growing lesions on the lower legs and hands. Diagnosis is made by light microscopy, culture and histopathological demonstration of

Cutaneous Mycology 149 thick-walled and pigmented ‘sclerotic bodies’ in scrapings or biopsies. Wide excision of an early lesion is considered the simplest successful therapy [49]. Itraconazole 100 mg daily may be the therapy of choice, sometimes in combination with flucytosine. Other possibilities include amphotericin B and flucytosine, flucytosine alone, thiabendazole and local heat.

Mycetoma (Madura Foot) Geography: Middle East, India, Africa, northern parts of South America. This chronic subcutaneous infection (mycetoma after Greek mykes for fungus and oma for tumor, meaning tumor produced by fungi [50]) may be caused by fungi (eumycetoma) or by bacteria, namely Actinomycetales (Actinomycetoma) which resemble fungi macro- and microscopically but have the characteristic features of bacteria on a cellular level [51]. After much controversy about nomenclature [52] these are the commonly used terms today. The typical clinical picture is a swollen foot (rarely hand) with abscesses, which painfully rupture and give birth to red, black and white grains. Black grains give a first hint that the infection most likely is fungal. Immediate direct examination of a grain with KOH is a reliable method to distinguish between mycotic (hyphae visible) and bacterial mycetoma, a decision of some importance as actinomycetoma often heals under antibiotic therapy, whereas eumycetomas frequently require surgery. Reasons for this common failure of antimycotic therapy probably are adaptive changes by the organisms, like cell-wall thickening or pigmentation [53]. Ketoconazole 200–400 mg daily, griseofulvin or itraconazole may be tried. X-ray of the feet often reveals lytic bone lesions. Histopathology consoli- dates the diagnosis.

Subcutaneous Zygomycosis Zygomycetes rarely lead to infections in tropical countries, causing systemic (see systemic mucormycosis) or a localized, disfiguring disease. Therapeutic efforts include surgery, potassium iodide and azoles.

Entomophthoromycosis Basidiobolus haptosporus and Conidiobolus coronatus, an insect pathogen, both lead to painless, wooden, slowly extending swellings, the first mainly on the limbs, whereas the second is typically restricted to the nasal sub- mucosa and the face, where the swellings usually assume bizarre dimensions.

Localized Mucormycosis The same agents as in systemic mucormycosis may lead to a subcutaneous, localized variant of mucormycosis, presenting with plaques, pustules, abscesses, ulcerations and finally skin necrosis.

Hawranek 150 Lobomycosis Reports of this chronic granulomatous skin infection in man mostly come from the Brazilian Amazon valley, where 21% of the cases reported in the medical world literature occur in an Indian tribe obviously highly predisposed to this disease [54]. The bottle-nosed dolphin is another host. Affected are people living in tropical rain forests; the infection is sometimes attributed to contact with dolphins. Attempts to culture the pathogenic fungus Loboa loboi never succeeded. Clinically, keloidal or smooth skin nodules appear on legs and trunk to a various extent. Histopathology reveals a dense infiltrate of ovoid or lemon-shaped organisms. Excision may be successful.

Rhinosporidiosis The majority of cases of this chronic granulomatous disease of the mucosal membranes have been reported from the southern parts of India and from Sri Lanka, others come from equatorial Africa, Europe and the Americas. Clinical findings show inflamed masses with white structures in the nasal cavities leading do nasal bleeding and obstruction. Other affected areas include the sinuses, the (palpebral) conjunctiva, and the oral, penile and vaginal mucosa. Rare skin lesions may stretch out from the conjunctiva; a disseminated cutaneous form has been reported [55]. Incubation and routes of infection remain unknown, minor trauma has been incriminated as a trigger. Diagnosis is confirmed by the presence of spherules staining with mucicarmine in biopsies. By continuing divisions, the spherules grow until about 2,000 sporangiospores are contained in the sporangium, leading to rupture. Attempts to culture the fungus have not been successful. Regarding therapy, electrosurgery, cryotherapy, ketoconazole and sulfones have been tried.

Systemic (Deep) Mycoses Caused by Pathogenic Fungi

Systemic mycoses are caused by dimorphic fungi (growing as molds at 26 °C and as yeasts at 37 °C, i.e. during invasion of the human body) and are usually acquired by inhalation of infectious conidia, thus leading to subclinical or flu-like infections or to primary acute illness [56]. Exposure is often demonstrated by positive skin testing only. Skin lesions may develop at a later stage of dissemination and often lead to diagnosis of the systemic infection, as they are particularly well suited for inspection and further microscopic examination, which usually provides the diagnosis. Coincidence of both erythema nodosum (as a marker of positive outcome) and/or erythema multiforme has been reported in acute disease. Very rarely traumatic implantation of the pathogen (e.g. laboratory accidents) causes local painless nodules or ulcers with regional

Cutaneous Mycology 151 lymphadenopathy (primary cutaneous disease), healing without treatment after a few months. The following short overview tries to present some clinical features of skin involvement in these deep mycoses.

Coccidioidomycosis The soil inhabitant Coccidioides immitis is endemic in semiarid areas of the South-West of the USA (‘San Joaquin Valley fever’), Central and South America. Its conidia are dispersed by earth-moving activities or simply the dust of unpaved streets, causing pulmonary infection by inhalation, usually asymptomatic or appearing as a mild upper respiratory infection. A small percentage of infected patients develop chronic pulmonary disease, and in about only 1% dissemination produces rather nondistinct (papules, pustules, plaques, ulcers, nodules, abcesses) as well as typical solitary large, warty skin lesions. Facial involvement seemed to be connected to meningeal disease in a retrospective study [57]. Serology may be helpful in the diagnosis, although there may be negative results even in disseminated disease. Effective drugs include amphotericin B, ketoconazole, fluconazole and itraconazole.

Paracoccidioidomycosis Habitat of Paracoccidioides brasiliensis is the soil of semitropical areas along rivers and agricultural areas in Latin America. In children, acute dissemination causes severe illness, often associated with pustules or subcutaneous abscesses. In adults (mainly males) – usually many years after an asymptomatic primary pulmonary infection – chronic disseminated disease leads to mucocutaneous lesions (ulcerating papules and pustules, subcutaneous cold abscesses, sometimes scrofuloderma-like manifestations), especially around nose or mouth in about half of the patients. Itraconazole is the drug of choice.

Histoplasmosis Two forms may be differentiated by pathogen, geographical distribution and clinical features. The most common form of histoplasmosis is caused by Histoplasma capsulatum, worldwide inhabitant of bird roosts and the guano of bat caves (‘typical disease of speleologists’). Depending on the amount of inhaled conidia and fitness of the immune system of the host, most infected adults remain asymptomatic, children often presenting with a more severe course. Cutaneous lesions appear in about 5% (up to 25% in immunocompromised patients) and again present as unspecific papules, pustules, cutaneous and subcutaneous nodules, plaques, granulomas, nodules, exfoliative erythro- derma, eczematous changes and lesions similar to molluscum contagiosum, the most characteristic manifestation being painful oropharyngeal mucocutaneous

Hawranek 152 ulcers. Again, amphotericin and azole compounds (especially itraconazole) are used in treatment, which should be initiated as quickly as possible. Histoplasma capsulatum var. duboisii causes African histoplasmosis, skin manifestations being the prominent sign in this variant. Three patterns may be differentiated clinically: superficial (ulcerating papules or nodules, infiltrated plaques, ‘target phenomenon’), subcutaneous granulomas (first hot and tender, then cold, rupture) and osteomyelitic lesions, the latter often demanding surgical procedures combined with amphotericin B and ketoconazole.

Blastomycosis Blastomyces dermatitidis is highly endemic in river banks of most eastern states of North America, and there are worldwide reports of this infection including Africa, India and Europe, where its rare appearance is most probably related to contact with fomites. Skin lesions are a very common sign of chronic disease and appear as either pustules developing to ulcers or papules growing to verrucous lesions over months. African blastomycosis is characterized by bone infection and draining sinuses, traumatic implantation presenting as a chancroid-like lesion. Treatment: amphotericin B is used for severe acute disease, otherwise ketoconazole, fluconazole or itraconazole have been effective.

Systemic (Deep) Mycoses Caused by Opportunistic Fungi

Patients with solid organ transplants tend to suffer from oral candidiasis and cryptococcosis, whereas neutropenic patients (chemotherapy because of cancer or bone marrow transplantation) are susceptible to aspergillosis, systemic candidiasis or mucormycosis. AIDS patients – as a general sign of their defective immune system – present with more severe courses of infectious diseases in general, including mycoses like candidiasis, dermatophytosis or seborrhoic dermatitis. The opportunistic pathogens rarely disseminate to the skin, even if skin manifestations of cryptococcosis are not that uncommon since the appearance of the AIDS pandemic. Diagnosis may be consolidated by biopsy.

Candidiasis Oral candidiasis – besides other predisposing factors – is a common disease in the immunocompromised patient and should remind clinicians of the possibility of HIV infection. It is often associated with esophageal manifestations. Relapsing or chronic persistent vulvovaginal candidiasis has been reported in up to 50% of HIV-infected women, therefore some authors recommend HIV testing in this condition too [58]. Resistance to antifungal drugs is a common problem

Cutaneous Mycology 153 with candidiasis and other fungal infections [36]. In contrast to these two conditions, systemic candidiasis is not a disease of AIDS but of neutropenic cancer patients, intensive care patients and intravenous drug abusers [59]. Fever and muscle tenderness may accompany the appearance of macules changing to papules (with pale centers) and nodules, abscesses and purpuric lesions, follicular pustules being a typical sign of septicemia in heroin abusers. Janeway lesions may be seen in candida endocarditis though rarely. Chronic disseminated (hepatosplenic) disease is characterized by abdominal pain. If skin lesions occur, which is the case in a minority of septicemic patients only, evidence of yeast cells in skin biopsies allows a rapid diagnosis. As blood cultures are not very sensitive, serodiagnosis in this setting offers some diagnostic help.

Cryptococcosis The typical habitat of Cryptococcus neoformans (appearing in two varieties) is soil with pigeon droppings. Despite this fact, pigeon breeders do not show any occupational predisposition to the disease [60]. Infection usually occurs via the respiratory tract, leading to systemic disease (mainly CNS manifestations) in patients with severe underlying disease, systemic corticoid therapy and is especially common in AIDS patients in whom the symptoms of meningitis will often be muted (e.g. absence of a stiff neck because of minor inflammatory reaction of the meninges). Skin lesions present as nodules, ulcers or molluscum-contagiosum-like lesions and occur in up to 10% of the cases, genital ulcerations may be found in up to 40% of the cases. Primary cutaneous cryptococcosis is rare, but is regularly reported in HIV-positive and -negative patients. Diagnosis may be made by light microscopy with Indian ink revealing the encapsulated yeast and confirmed by growth of brown cultures on melanin- precursor-containing agars and by urease production. A rapid latex agglutination test and ELISA are available. Aggressive treatment with amphotericin B and/or flucytosine may be followed by maintenance therapy with fluconazole or itraconazole (which are sufficient in the primary cutaneous form). Studies on triple antimycotic therapy in HIV patients have been published [61].

Systemic Zygomycosis There is still some controversy on terminology depending on whether precedence is given to mycological or clinical aspects. Systemic zygomycosis is a very rare and potentially fatal disease of neutropenic, diabetic, nephro- pathic and burn patients, the latter being especially prone to skin manifestations (i.e. necrosis). Pathogens include Rhizopus spp., Absidia corymbifera, Rhizomucor pusillus, Mucor spp. and Cunninghamella bertholletiae. The most common clinical manifestation is the rhinocerebral form (often presenting as an erythematous induration with central purple-black discoloration), cutaneous

Hawranek 154 lesions may be primary (rapid change in morphology from papules and pustules to necrosis) or secondary in the course of hematogenous dissemination (reddish to purplish subcutaneous nodules). Vectors for transmission in primary cutaneous zygomycosis may include cloth tape securing endotracheal tubes [63] or wooden tongue depressors [64]. Diagnosis by autopsy or biopsy, treatment with lipid-associated amphotericin B.

Aspergillosis Besides causing allergies and mycotoxicosis, Aspergillus (named by the Italian priest Micheli in 1729 after a church instrument called aspergillum used to sprinkle holy water during religious ceremonies [65]) species (Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus and others) typically colonize preformed cavities (aspergilloma) or cause granulomatous disease of inner organs (e.g. bronchopulmonary aspergillosis). On rare occa- sions, they initiate systemic and even fatal disease (invasive aspergillosis). Infection occurs by inhalation or inoculation of spores from soil, water and decaying plants. The most common manifestations are the pulmonary form and sinusitis. Cutaneous embolization leads to erythematous, indurated plaques, hemorrhagic bullae and ulcers; other lesions are described as abscesses and of molluscum-like appearance [66]. Primary cutaneous aspergillosis has been reported with the application of contaminated dressings to preexisting wounds, presenting as ulcerating nodules sometimes leading to hematogenous dissemination. Rare cases have been reported in neonates [67] and in HIV patients [68]. Diagnosis is obtained by biopsy and culture; blood cultures are rarely positive. The prognosis is poor in spite of therapy with amphotericin B and/or flucytosine. Itraconazole has been tried with good results. Prevention (laminar flow, air filtration, amphotericin B nasal spray and aerosol) may be successful.

Pseudallescheriasis Pseudallescheria boydii is a geophilic fungus causing mycetoma and may be a rare cause of pulmonary infections (especially in immunocompromised patients), infections of the CNS and inner organs as well as ear and eye infections. There has been a recent report on an unusual case of cutaneous pseudallescheriasis with pustules and erythema resistant to azole treatment [69].

Rare Mycoses

Hyphomycosis Many species of the vast group of ubiquitous hyphomycetes (i.e. mycelial fungi) show the same tissue morphology and host response in the very rare

Cutaneous Mycology 155 human cases. Whereas pathogens with distinctive features are assigned to separate entities, many of these fungi are categorized into two main artificial groups, the pheohyphomycetes (containing brown pigment) and the hyphomycetes (with colorless walls), each group containing examples of completely unrelated fungi.

Pheohyphomycosis According to definition, these so-called dematiaceous fungi contain melanin in their cell walls which sometimes (e.g. S. schenckii) may be made visible only by specific melanin stains [70]. Regarding the frequency of infections, the major pathogens of more than 100 species involved are Bipolaris spicifera and Exophiala jeanselmei, others include Curvularia spp. Examples of distinct clinical entities are mycetoma, sporotrichosis, chromoblastomycosis, black piedra and tinea nigra, ranging from superficial to systemic disease. Subcutaneous infection often presents with a solitary acral abscess attached to the skin, but without connection to the underlying tissue. Fistulas as well as dissemination may complicate the course in immunocompromised individuals, initial lesions often presenting as firm subcutaneous, indurated lesions. Besides surgical excision in subcutaneous disease, therapeutic attempts with mostly disappointing results included amphotericin B, flucytosine, miconazole, ketoconazole and itraconazole, the latter with promising effectiveness.

Hyalohyphomycosis Species included are Penicillium, Beauveria, Acremonium, Fusarium and Scopulariopsis, with an everexpanding list of organisms involved in human disease.

Protothecosis The achloric algae Prototheca wickerhamii and Prototheca zopfi, although not belonging to the fungi, produce yeast-like colonies on Sabouraud’s agar and are often included with the fungi [54]. The usually verrucous lesions most commonly are observed in immunocompromised patients as an opportunistic infection, and extracutaneous manifestations of the disease are not rare. The histopathologically typical structure is the so-called morula, daughter cells within a theca resembling a soccer ball. Therapy with excision or with combinations of an oral tetracycline with topical amphotericin B may be successful.

Penicillium marneffei Infection This dimorphic fungus, which does not fit into the definition of hyalohyphomycosis, causes disease in bamboo rats of Southeast Asia. Its relation to human disseminated mycosis in immunocompromised as well as healthy persons

Hawranek 156 is not clear; most cases have been reported from Thailand where it is one of the most common AIDS-associated diseases. The skin lesions, which occur in about 50% of the patients, present as papules, ulcers or resemble molluscum contagiosum, and thus have to be distinguished from cryoptococcosis and histoplasmosis by biopsy. Blood cultures are positive in about three fourths of the patients. Because of the high mortality, treatment is necessary. Drugs of choice are itraconazole or ketoconazole for mild to moderate cases and amphotericin B for severe cases. Maintenance therapy may be considered with the azoles [66].

Laboratory Diagnosis

For reasons of limited space and the general focus on clinical aspects in this review, not too much emphasis is laid on technical details, for these, see a reference book [3] or review article [71].

Collection of Material

Before sampling, the lesions should be cleansed with 70% alcohol if covered with ointment. For skin scales, specimens should be gathered with a sterile blunt scalpel from the edge of a lesion (or the roof of a blister); the best transport medium is folded black paper. Nail specimens should be collected from under the edge of the nail with a raspatory (as proximal as possible), debris in paronychia may be gained by the gentle use of a dental probe. Hair (roots) can easily be epilated with an epilation forceps. If T. verrucosum is suspected, it may be necessary to remove many hairs. For the diagnosis of tinea capitis a brush is pressed on the agar after brushing the hair ten times at least, a screening method most valuable in school epidemics. Specimens from mucous membranes are sampled by swabs and should be examined immediately, as yeasts do not survive for long outside a transport medium.

Direct Examination (Potassium Hydroxide Test)

The specimen is immersed in 10% potassium hydroxide dissolving the keratin but leaving fungal elements. The microscopic picture may be enhanced by staining with an equal amount of Parker’s ‘Quink’. Nail material usually needs about 10 min (about 1 h has recently been recommended [29]) to get soft enough to be flattened (the thinner the sample, the easier the detection of fungus; gentle warming may help), hair starts to disintegrate after a few

Cutaneous Mycology 157 minutes. Fungal structures are first searched for at low magnification (ϫ100), then examined at higher magnification (ϫ400). This test does not allow the diagnosis of a specific dermatophyte; a negative result does not rule out mycosis. The superficial mycoses, especially pityriasis versicolor, and – to a lesser degree – S. brevicaulis may be satisfactorily diagnosed by direct examination alone. The fluorochromes, Calcofluor White and Congo red bind to chitin and cellulose, while acridine orange binds to nucleic acids of the fungi. These dyes may be helpful when there is a lack of fungal elements (blastospores, hyphae or pseudohyphae) in the specimen, as even few elements are visible under the fluorescence microscope. By using specific antibodies conjugated with the fluorochromes, it should be possible to identify specific fungi.

Culture and Additional Tests

The specimen is inoculated onto the culture medium, usually Sabouraud’s dextrose agar without and with added antibiotics, e.g. actidione for elimination of contaminating molds and chloramphenicol for suppression of bacterial growth. If infection with the mold S. brevicaulis or yeasts is suspected, actidion should not be added. One culture should be incubated at about 26–28 °C for the propagation of dermatophytes, the other at 37 °C for yeasts. Most dermatophytes need approximately 1–2 weeks for growth; yeasts grow within 1–2 days (slimy little colonies very similar to bacterial cultures). If there is no growth after 4 weeks, cultures may be reported as negative. The cultures are examined for macroscopic features such as color, surface and growth rate (table 2). An easy and good method of preparation for microscopic study is to bring some material, peferably from the margin of the culture, on a glass slide by touching the culture with a sticky tape and mounting it on the slide after putting a drop of lactophenol cotton blue on it. The mount may then be examined looking for unique morphologic features (table 2). As a general rule, macroconidia of Microsporum spp. have rough, thick walls, whereas those of Trichophyton and Epidemophyton spp. are smooth-walled and clavate or broadly clavate. Microconidia of Microsporum spp. are typically pear-shaped and single-standing along the hyphae, whereas the microconidia of Trichophyton spp. are pear-shaped to spherical, numerous and tend to build clusters. They are absent in Epidermophyton spp. Distinctive details of conidia (fig. 6) are best searched for by performing slide cultures. A little piece of agar is inoculated with the fungus on four sides and incubated at 25–30 °C in a moist atmosphere between a mount and a cover slip until sufficient growth becomes apparent, a method also used to set up permanent preparations.

Hawranek 158 Fig. 6. Slide culture. A definitive diagnosis is usually made by distinctive microscopic features, such as the typical spiral hyphae of T. mentagrophytes var. interdigitale.

If sporulation is insufficient, the same procedure may be repeated with cornmeal agar or potato agar, which is sometimes successful. If this fails too, a number of additional tests exist for the identification of dermatophytes, including urea hydrolysis, in vitro hair perforation test, growth on polished rice grains, temperature enhancement or tolerance tests as well as numerous tests for the detection of special nutritional requirements, e.g. thiamine, niacin.

Histopathology, Serology, Identification of Yeasts and Molds

Histopathology (hematoxylin-eosin stain, periodic acid-Schiff stain, Grocott’s methenamine-silver stain) may be helpful in the diagnosis of dermatophytosis and plays an important role in the diagnosis of subcutaneous mycoses. Serology: The detection of specific antibodies does not play any role in the diagnosis of superficial or cutaneous mycoses, but has its place in systemic candidiasis, aspergillosis and especially cryptococcosis, histoplasmosis and coccidioidomycosis. Identification of yeasts: As dermatophytes, yeast colonies are examined for macroscopic and microscopic criteria. C. albicans, the most prominent fungus of this group, may be identified by the presence of terminal chlamydospores on rice agar and of germ tubes after 2 h of incubation in serum at 37 °C (‘germ tube test’). Besides classical tests for the specific identification of yeasts [3], several kits working with biochemical reactions are commercially available. Identification of molds: As macroscopic and microscopic examination of colonies is the only way of identifying molds at the moment, special attention

Cutaneous Mycology 159 Table 3. Aspects of antifungal drug therapy

Substance Appl. Some characteristics Some side effects Some interactions

Imidazoles Miconazole top., i.v. ind.: broad spectrum (excl. aspergillus) i.v.: tachycardia, seizures, anemia; i.v.: oral antidiabetics↑, antifungal agent; no therapy of choice top.: irritation, contact dermatitis coumarin↑, phenytoin↑ Econazole top. ind.: most cutaneous mycoses irritation, contact dermatitis – Ketoconazole top., o. ind.: most cutaneous mycoses, some hepatotoxicity, inhibition of insulin-sparing effects, systemic mycoses; because of its steroidogenesis and other cyclosporin↑, coumarin↑, potential hepatotoxicity has been endocrinologic side effects, disulfiram-like reactions replaced widely by the newer impotence with alcohol; cave antifungal agents terfenadine and astemizole (cardiac arrhythmia) Triazoles Itraconazole o. ind.: most cutaneous, subcutaneous nausea; hepatotoxicity (rare); may ↑ levels of oral and systemic infections; pulse regimen hypokalemia, edema, antidiabetics, coumarin, possible; fungostatic, lipophilic drug gynecomastia, impotence with digoxin, phenytoin, with extensive tissue distribution; higher doses; embryotoxicity cyclosporin should be taken with meals Fluconazole o., i.v. ind.: most cutaneous and systemic gastrointestinal side effects may ↑ levels of oral (candidiasis, cryptococcosis) mycoses (common), hepatotoxicity (rare), antidiabetics, coumarin, exfoliative dermatitis, thrombo-, phenytoin, cyclosporin leukopenia Allylamines Naftifine top. ind.: cutaneous dermatophyte and yeast local irritation, contact dermatitis – infections; fungicidal and antiinflammatory

Hawranek 160 Terbinafine o., top. ind.: very effective for dermatophyte gastrointestinal side effects, loss no influence on clearance infections given orally, low effectivity of taste, hepatitis, erectile of cytochrome

against yeasts (only fungistatic against dysfunction, Stevens-Johnson P450-dependent drugs, but C. albicans) and molds; fungicidal (!), syndrome is affected by inhibitors few relapses or inducers of this system Polyenes Amphotericin B i.v., top. ind.: Candida, Aspergillus and most i.v.: chills (50%), fever (33%), avoid other nephrotoxic systemic mycoses, presumed fungal renal toxicity (up to 20%), drugs (cyclosporine, infections in febrile neutropenic hypokalemia, hypomagnesemia, aminoglycosides etc.); patients; first systemic antifungal drug anemia hypokalemia may be (1960); less toxicity with new lipid deteriorated by steroids complex and liposomal formulations; pre- and post-infusion hydration advisable Nystatin o., top. ind.: Candida o.: occasional gastrointestinal side – effects with high doses Natamycin top. ind.: Candida, dermatophytes irritation – Miscellaneous Griseofulvin o. ind.: dermatophytosis in children (and gastrointestinal symptoms, coumarin levels ↑, adults), still considered as the ‘gold headaches, arthralgias, cyclosporin ↓; standard’ for tinea capitis; not effective photosensitivity, aggravation of barbiturates lead to against yeasts; good safety profile; to lupus erythematosus, allergic griseofulvin ↓; possible be taken with fat-containing food, reactions (up to 7%), side effects interference with improved absorption by micronization; of the nervous system anticonceptive pills; blood count and liver function tests disulfiram-like reactions possibly unnecessary in healthy with alcohol children

Cutaneous Mycology 161 Table 3. Continued

Substance Appl. Some characteristics Some side effects Some interactions

Amorolfine top. ind.: most cutaneous mycoses; nail local irritation (burning, itch etc.) – lacquer for onychomycosis Tolnaftate top. ind.: dermatophytosis (not T. rubrum) local irritation, contact dermatitis – Cyclopiroxolamine top. ind.: most cutaneous mycoses local irritation – Flucytosin i.v., o. ind.: systemic mycoses, given in bone marrow toxicity, avoid other nephrotoxic combination with amphotericin B; nephrotoxicity, hepatotoxicity drugs; cytarabin as resistance when used as monotherapy antagonist

Appl = Mode of application; i.v. = intravenously; o. = orally; top. = topically; ind.: indication.

Hawranek 162 has to be paid to the texture of the colony as well as to the type of conidia and especially the way these conidia are borne on the conidiophores. This may be achieved by a sticky tapemount, but the best method to preserve special conidial arrangements without the conidia becoming detached is slide culture (see above).

Principles of Therapy

Fungi in the stratum corneum may be attacked by keratolysis (ϭ elimination of the pathogen including the site of infection), e.g. by Whitfield’s ointment or topicals containing salicylic acid, or by broad-spectrum antiseptics (mostly dyes like gentian violet or sulfide-containing creams). The third approach involves specific antifungal substances administered topically or systemically. Table 3 gives some characteristic information on antifungal agents, for detailed information see review articles [72–74]. Four groups may be differentiated, all characterized by different modes of action: the allylamines and azoles blocking ergosterol formation in the cell wall in different ways, the polyenes destroying the cell membranes, griseofulvin blocking the intracellular microtubules and flucytosine blocking DNA and RNA synthesis.

References1

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Hawranek 164 36 Graybill JR: The clinical impact of antifungal drug resistance in patients with AIDS; in Aly R, Beutner KR, Maibach H (ed): Cutaneous infection and therapy. New York, Dekker, 1997, pp 233–243. 37 Hawranek U: Vaginalmykosen – Eine rein somatische Erkrankung?, PhD thesis, Salzburg, 1998. 38 Irving G, Miller D, Robinson A, Reynolds S, Copas AJ: Psychological factors associated with recurrent vaginal candidiasis: A preliminary study. Sex Transm Infect 1998;74:334–338. 39 Moraes PS: Recurrent vaginal candidiasis and allergic rhinitis: A common association. Ann Allergy Asthma Immunol 1998;81:165–169. 40 Drake LA, Dinehart SM, Farmer ER, Goltz RW, Graham GF, Hordinsky MK, Lewis CW, Pariser DM, Skouge JW, Webster SB, Whitaker DC, Butler B, Lowery BJ: Guidelines of care for superficial mycotic infections of the skin: Mucocutaneous candidiasis. J Am Acad Dermatol 1996; 34:110–115. 41 Sobel JD: Controversial aspects in the management of vulvovaginal candidiasis. J Am Acad Dermatol 1994;31:S10–S13. 42 Spinillo A, Colonna L, Piazzi G, Baltaro F, Monaco A, Ferrari A: Managing recurrent vulvovaginal candidiasis. Intermittent prevention with itraconazole. J Reprod Med 1997;42:83–87. 43 Midgley G, Moore MK: Nail infections. Dermatol Clin 1996;14:41–49. 44 Drake LA, Dinehart SM, Farmer ER, Goltz RW, Graham GF, Hordinsky MK, Lewis CW, Pariser DM, Skouge JW, Webster SB, Whitaker DC, Butler B, Lowery BJ: Guidelines of care for superficial mycotic infections of the skin: Onychomycosis. J Am Acad Dermatol 1996;34: 116–121. 45 Kirkpatrick CH: Chronic mucocutaneous candidiasis, J Am Acad Dermatol 1994;31:S14–S17. 46 Prillinger H, Messner R, König H, Bauer R, Lopandic K, Molnár O, Dangel P, Weigang F, Kirisits T, Nakase T, Sigler L: Yeasts associated with termites: A phenotypic and genotypic characterization and use of coevolution for dating evolutionary radiations in Asco- and Basidiomycetes. System Appl Microbiol 1996;19:265–283. 47 Davis BA: Sporotrichosis. Dermatol Clin 1996;14:69–76. 48 Belknap BS: Sporotrichosis. Dermatol Clin 1989;7:193–202. 49 Elgart GW: Chromoblastomycosis. Dermatol Clin 1996;14:77–83. 50 Magana M, Magana-García M: Mycetoma. Dermatol Clin 1989;7:203–217. 51 Warren NG: Actinomycosis, nocardiosis, and actinomycetoma. Dermatol Clin 1996;14:85–95. 52 McGinnis MR: Mycetoma. Dermatol Clin 1996;14:97–104. 53 Hay RJ: A thorn in the flesh – a study of the pathogenesis of subcutaneous infections. Clin Exp Dermatol 1989;14:407–415. 54 Elgart ML: Unusual subcutaneous infections. Dermatol Clin 1996;14:105–111. 55 Thappa DM, Venkatesan S, Sirka CS, Jaisankar TJ, Gopalkrishnan, Ratnakar C: Disseminated cutaneous rhinosporidiosis. J Dermatol 1998;25:527–532. 56 Body BA: Cutaneous manifestations of systemic mycoses. Dermatol Clin 1996;14:125–135. 57 Arsura EL, Kilgore WB, Caldwell JW, Freeman JC, Einstein HE, Johnson RH: Association between facial cutaneous coccidioidomycosis and meningitis. West J Med 1998;169:13–16. 58 Conant MA: Fungal infections in immunocompromised individuals. Dermatol Clin 1996;14: 155–162. 59 Hay RJ: Yeast infections. Dermatol Clin 1996;14:113–124. 60 Hernandez AD: Cutaneous cryptococcosis. Dermatol Clin 1989;7:269–274. 61 Kappe R, Levitz S, Harrison TS, Ruhnke M, Ampel NM, Just-Nubling G: Recent advances in cryptococcosis, candidiasis and coccidioidomycosis complicating HIV infection. Med Mycol 1988;36(suppl 1):207–215. 62 Elgart ML: Zygomycosis. Dermatol Clin 1996;14:141–146. 63 Dickinson M, Kalayanamit T, Yang CA, Pomper GJ, Franco-Webb C, Rodman D: Cutaneous zygomycosis (mucormycosis) complicating endotracheal intubation: Diagnosis and successful treatment. Chest 1998;114:340–342. 64 Mitchell SJ, Gray J, Morgan ME, Hocking MD, Durbin GM: Nosocomial infection with Rhizopus microsporus in preterm infants: Association with wooden tongue depressors. Lancet 1996;348: 441–443. 65 Isaac M: Cutaneous aspergillosis. Dermatol Clin 1996;14:137–140.

Cutaneous Mycology 165 66 Myskowski PL, White MH, Ahkami R: Fungal disease in the immunocompromised host. Dermatol Clin 1997;15:295–305. 67 Papouli M, Roilides E, Bibashi E, Andreou A: Primary cutaneous aspergillosis in neonates: Case report and review. Clin Infect Dis 1996;22:1102–1104. 68 Arikan S, Uzun O, Cetinkaya Y, Kocagoz S, Akova M, Unal S: Primary cutaneous aspergillosis in human immunodeficiency virus-infected patients: Two cases and review. Clin Infect Dis 1998; 27:641–643. 69 Ginter G, Petutschnig B, Pierer G, Soyer HP, Reischle S, Kern T, de Hoog S: Atypical cutaneous pseudoallescheriosis refractory to antifungal agents. Mycoses 1999;42:507–511. 70 Rinaldi MG: Phaeohyphomycosis. Dermatol Clin 1996;14:147–153. 71 Weitzman I, Padhye A: Dermatophytes – gross and microscopic. Dermatol Clin 1996;14:9–22. 72 Gupta AK, Sauder DN, Shear NH: Antifungal agents: An overview. Part I. J Am Acad Dermatol 1994;30:677–698. 73 Gupta AK, Sauder DN, Shear NH: Antifungal agents: An overview. Part II. J Am Acad Dermatol 1994;30:911–933. 74 Lesher JL Jr: Recent developments in antifungal therapy. Dermatol Clin 1996;14:163–169.

Dr. Thomas Hawranek, MD, Landeskliniken Salzburg, Department of Dermatology, Müllner Hauptstrasse 48, A–5020 Salzburg (Austria) Tel. ϩ43 662 4482 3023, Fax ϩ43 662 4482 3003 E-Mail [email protected]

1References are mainly restricted to reviews.

Hawranek 166 Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 167–206

Toxins of Filamentous Fungi

Deepak Bhatnagar, Jiujiang Yu, Kenneth C. Ehrlich

US Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, New Orleans, La., USA

Natural toxins in food can be divided into five main categories: mycotoxins, bacterial toxins, phycotoxins, plant toxins and zootoxins. The first three are toxic compounds produced by living organisms, and are formed directly in food or transferred through the food chain, whereas, the latter two are ‘inherent components (of plants or animals) that are harmful to humans and animals’ [1]. Toxins are compounds that are toxic or poisonous to living things, phytotoxins are compounds toxic to plants, and zootoxins are compounds toxic to animals [2]. By the same logic, mycotoxins should be compounds toxic to fungi. However, these are toxic compounds produced by fungi. The term mycotoxin is derived from the Greek words ‘␮␷␬␩␴’ (fungus) and ‘␶␱␰␫␬␱␯’ (arrow-poisons). Most mycotoxins can, therefore, be defined as natural products produced by fungi that evoke a toxic response in higher vertebrates and other animals when fed at low concentrations. Mycotoxins could also be toxic to plants or other microorganisms. However, these compounds are not classified with antibiotics of fungal origin. Biological conversion products of mycotoxins are also called mycotoxins. Mycotoxins have recently been the subject of many reviews [3–5]. Toxic metabolites produced by mushrooms and yeast are not included in this review and have been reviewed by Chu [4]. In this chapter, a comprehensive review has been provided of the most significant mycotoxins, their fungal origin and their toxic effects. The reader will hopefully be able to appreciate the diversity of mycotoxins that have been examined thus far, and will understand the need for research on several of these toxins that exhibit the potential for harmful effects on animals and humans. After cessation of an active growth phase, fungi are known to produce numerous organic compounds called secondary metabolites, which are not required for the growth of the producing fungus. Mycotoxins are low-molecular-weight, nonproteinaceous compounds derived primarily from amino acids, shikimic acid or malonyl CoA, and their effect in animals and humans can be significant. Mycotoxins are generally produced in the mycelia of filamentous fungi, but can accumulate in specialized structures of fungi such as conidia or sclerotia as well as in the environment surrounding the organism. Fungal species that produce mycotoxins are very diverse. However, some mycotoxins are only produced by a single fungal species or even by specific strains of a fungal species or a number of fungal species. The toxic effects of mycotoxins are as diverse as the fungal species that make these toxins. Some mycotoxins have acute toxic effects while others have toxic effects after long-term exposure (chronic effects).

Mycotoxicosis

Mycotoxins can elicit acute toxic, mutagenic, teratogenic and carcinogenic effects, as well as estrogenic effects on animals. The toxic effects of mycotoxins are termed ‘mycotoxicoses’, and the severity depends on the type of mycotoxin, the extent of exposure (duration and dose), age, nutritional status and health of the affected individual, and the synergistic effects of the mycotoxin with other chemical exposure of the individual. Mycotoxicosis is usually mediated by damage to cells of all the major organs, most commonly liver, kidney, lungs and the nervous, endocrine and immune systems. Mycotoxicosis differs from ‘mycosis’, a term that refers to diseases produced by direct pathogenic invasion by fungi. Mycotoxicosis can be labeled acute or chronic depending on the amount of toxin ingested and the length of exposure, with low doses and long-term exposure leading to chronic toxicity, and short-term exposure to extremely high doses leading to acute toxicity. Over 300 mycotoxins have been identified, but only those implicated in mycotoxicoses involving humans have been studied in detail. Table 1 provides a partial list of the best-studied mycotoxins, the fungal source and the reported human mycotoxicosis associated with each toxin. Although most of these mycotoxins have been implicated in human illnesses, a direct connection between the mycotoxin and a corresponding mycotoxicosis has been demonstrated for very few. For example, ochratoxin A, and sterigmatocystin may be carcinogenic to human beings, but there are insufficient or inconclusive data to demonstrate a direct effect [6]. Only in the case of aflatoxins, ergot alkaloids, ochratoxins and to some extent trichothecenes has a relationship between exposure to the toxin and acute and chronic human mycotoxicoses been reasonably well established.

Bhatnagar/Yu/Ehrlich 168 Table 1. Selected mycotoxins and their human and animal mycotoxicoses

Mycotoxin Producing fungi Toxic effect Mycotoxicosis

Alternaria (tenuazonic A. alternata apoptosis onyalai disease acid, alternariol) mutagen Aflatoxin A. flavus apoptosis acute aflatoxicosis, A. parasiticus mutagen hepatocarcinogenesis, hepatotoxin childhood carcinogen cirrhosis, teratogen Rye’s syndrome Cyclopiazonic acid A. flavus nephrotoxin Kodua poisoning P. aurantiogriseum cardiovascular lesion Ergot alkaloids Claviceps sp. neurotrophy St. Anthony’s fire A. fumigatus ergotism P. chermesinum Moniliformin F. verticillioides neurotoxin onyalai disease cardiovascular lesion Ochratoxin A. ochraceus apoptosis Balkan nephropathy, P. niger nephrotoxin renal tumors A. alliaceus teratogen A. terreus P. viridicatum Penicillic acid P. aurantiogriseum neurotoxin – A. ochraceus mutagen Roquefortine P. roquefortii neurotoxin – Rubratoxin P. rubrum apoptosis – P. purpurogenum hepatotoxin teratogen Sterigmatocystin A. versicolor hepatotoxin hepatocarcinogenesis A. nidulans carcinogen A. parasiticus A. flavus Trichothecenes F. sporotrichioides apoptosis alimentary toxic (T-2, deoxynivalenol) Microdochium nivale dermatoxin aleukia, S. atra neurotoxin Akakabi-byo disease, teratogen stachybotryotoxicosis, esophageal cancer, red mold disease, yellow rain Zearalenone F. graminearum genitotoxin cervical cancer, estrogenic premature menarche effects mutagen

Adapted from Bhatnagar et al. [5].

Toxins of Filamentous Fungi 169 Mycotoxicology

Mycotoxicology is the study of the mycotoxins and their corresponding mycotoxicoses. It is a diverse discipline that encompasses basic sciences, such as chemistry, biochemistry and molecular biology, and multidisciplinary subjects, such as plant and animal pathology, toxicology, pharmacology and immunology.

Natural Occurrence of Mycotoxins

Mycotoxins are found worldwide as contaminants of food. Their occurrence depends on geographic and seasonal factors, as well as cultivation, harvesting, storage and transportation practices. Mycotoxin production is also dependent on the factors affecting the susceptibility of the commodity to contamination, e.g., the genetic, environmental and nutritional status of the crop [7]. Physical factors such as time of exposure, temperature during exposure, humidity, and extent of insect or other damage to the commodity prior to exposure determine mycotoxin contamination in the field or during storage. Mycotoxin-producing or other coexisting fungal species, that may be plant pathogens, act as biological factors. These fungi can colonize seeds before and after harvest, thus affecting mycotoxin production based on interactions of existing fungal populations. Chemical factors including the nutritional status of the crops or chemicals (such as fungicides) used in crop management can affect fungal populations, and consequently toxin production. Mycotoxin production is often very variable from the effects of some or all of these factors. The temperature and relative humidity optima for mycotoxin production may differ from that supporting fungal growth. For example, in the field, the temperature range for Aspergillus flavus and A. parasiticus growth varies from 12 to 48 °C (35–37 °C optimum), and water activity requirement could be as low as 0.80 (0.95 optimum). However, aflatoxin production by these fungi requires a narrower range of temperatures (28–33 °C; 31 °C optimum), and water activity (0.85–0.97; optimum 0.90). In general, mycotoxins are produced optimally at 24–28 °C, but some toxins such as T-2 toxin are produced maxi- mally at 15 °C. Contamination during crop storage may be affected by changes in temperature and water activity that allow ecological succession of different fungi. Filamentous fungi are ubiquitous in nature, and are found in soils from temperate and tropical regions. While some of these fungi are pathogenic, others are not. Mycotoxin contamination has been found in cereal plants, oilseed crops and grasses [7–12]. Most commodities have been shown to be

Bhatnagar/Yu/Ehrlich 170 susceptible to mycotoxin contamination. A partial list of these commodities is given in table 2; for an exhaustive list, see Malloy and Mars [6]. Often even when fungal growth cannot be observed, a large amount of mycotoxins can be produced.

History of Mycotoxins

Historical instances of mycotoxicoses can be surmised even though identification of a mycotoxin as a causative agent was unknown at the time of occurrence. For example, at least one of the 10 plagues in ancient Egypt recorded in Exodus could have been associated with mycotoxin contamination of food [13, 14]. The ‘Plague of Athens’ in approximately 430 BC could be attributed to intake of fungal-contaminated food [15]. The festival ‘Robigalia’ in the sev- enth and eighth century BC, established to honor the god Robigus, was cele- brated at the end of April probably to prevent grain and trees from being affected by rust or mildew [16]. Pneumonic plague deaths during the Middle Ages have also been attributed to food poisoning induced by various mycotoxins [17]. For almost 1,000 years, since the ninth century, ergotism from ergot- infected rye has afflicted large populations in Europe [18]. Ergotism was also called ‘ignis sacer’ (sacred fire) or St. Anthony’s fire, because it was believed that a pilgrimage to the shrine of St. Anthony would bring relief from the intense burning sensation caused by the mycotoxin [19, 20]. In the 1850s, ergotism was finally found to be caused by alkaloids from Claviceps purpurea sclerotia. In the USA, the last major outbreak of ergotism was recorded in 1925; but ergot-induced mycotoxicosis was still being reported in France thirty years later [16, 21, 22]. Modern mycotoxicology began in 1960, after 10,000 turkeys died when fed a mycotoxin-contaminated peanut meal (‘Turkey X’ disease). Although major deaths of livestock were reported earlier from consumption of moldy corn in feed of horses in Illinois (1933–1934 where 5,000 horses died) and swine (1,500 lost in Southeastern United States) [23], it was the discovery of aflatoxins, produced by Aspergillus flavus, as the causative agent in the peanut meal causing the ‘Turkey X’ disease (as well as in moldy corn affecting the earlier deaths of livestock) that provided the impetus to associate fungal toxins with illness in animals [24]. In the last 40 years, many new mycotoxins have been identified and characterized, and their biosynthetic origin in various fungi elucidated [25]. It has been estimated that at least 25% of the world’s agricultural product is contaminated with mycotoxins [26]. Modern epidemiology has allowed certain diseases to be linked to ingestion of mycotoxins by documenting

Toxins of Filamentous Fungi 171 Table 2. Natural occurrence of selected mycotoxigenic fungi

Fungal species Commodity contaminated

Aspergillus A. flavus peanuts, corn, most treenuts A. parasiticus cottonseed A. ochraceus figs, cereals, cheese A. versicolor cereals, beans, coffee A. nidulans corn, grains, cheese A. clavatus corn, grains, cheese apple, apple juice, beans, wheat Fusarium F. verticillioides corn, corn products F. crookwellense cereals F. culmorum cereals F. graminearum cereals F. poae cereals F. proliferatum corn, corn products F. sporotrichioides cereals

Penicillium P. citrinum wheat, corn, rice P. aurantiogriseum (P. cyclopium) corn, sorghum, rice P. islandicum corn, hay P. rugulosum corn, hay, peanuts, rice P. viridicatum barley, corn, rice, walnuts P. verruculosum peanut, corn, cheese P. griseofulvum rice, sorghum P. aurantiogriseum (P. puberulum) barley, beans, cereals, coffee P. rubrum apple, apple juice, beans, wheat P. purpurogenum barley, corn P. palitans corn P. roquefortii cheese, hay

Adapted from Scudmore [3], Bhatnagar et al. [5]. occurrence of the mycotoxin contaminations in various parts of the world and correlating outbreaks of illnesses to enteric exposures to mycotoxins. Indoor air pollution by toxigenic fungi may implicate mycotoxins as having a more widespread role in chronic disease than was previously thought possible [27, 28]. Mycotoxins may not be effective tools in biological warfare because acute toxic effects are not very pronounced for most of these toxins [29]. But there

Bhatnagar/Yu/Ehrlich 172 is strong evidence of use of these toxic compounds, such as aflatoxins, in biological terrorism [30, 31].

Classification of Mycotoxins

Mycotoxins are low-molecular-weight organic compounds and have been classified in different ways depending on whether or not the classification was done by chemists, clinicians, mycologists or public health organizations. Chemists have classified these compounds according to their chemical structures. But the number and variety of mycotoxins make this classification challenging even for organic chemists. Mycotoxins have also been classified by toxicologists and clinicians based on the illnesses associated with mycotoxins or organs affected. Mycologists would prefer to classify these toxins based on the fungal genera producing specific mycotoxins. However, since some fungi produce a single mycotoxin and other genera may produce many mycotoxins, and different genera may produce the same mycotoxin, this mode of classification could get confusing. Carcinogenicity has been used by the World Health Organization as an index for classifying mycotoxins [32]. However, the carcinogenic effects of very few mycotoxins have been established or even directly correlated. Therefore, this method of classifying mycotoxins may not be applicable at the present time. But as the carcinogenic risk of more mycotoxins is established based on ongoing research to identify the associations between human or animal consumption of contaminated food and the incidence of associated mycotoxicosis, this classification system may become very relevant. Turner and Aldridge [33] have categorized fungal secondary metabolites not by their properties, but according to their biosynthetic origins. Although diverse in structure and activity, fungal metabolites are derived from a few biosynthetic pathways as shown in table 3.

Economic Impact of Mycotoxins

The most obvious impact of mycotoxins is the inability to sell crops for human or animal food due to contamination with even relatively low levels of certain mycotoxins. In certain developing countries where there is less regulation or monitoring of human and animal exposure to toxins, there are risks of higher human health costs and animal death. Additional losses can be from a number of unseen problems such as reduction in birth rate in certain animals, decline in milk production by dairy cattle and egg production in poultry, loss of

Toxins of Filamentous Fungi 173 Table 3. Representative mycotoxins grouped according to biosynthetic pathways

Biosynthetic origin Mycotoxin Producing fungal genera

Polyketide aflatoxin Aspergillus citrinin Aspergillus, Penicillium citreoviridin Penicillium ochratoxin Aspergillus, Penicillium penicillic acid Aspergillus, Penicillium secalonic acid Claviceps, Penicillium sterigmatocystin Aspergillus, Chaetomium, Bipolaris zearalenone Fusarium Amino acids brevianamide Penicillium cytochalasins Aspergillus, Helminthosporium, Metarhizium, Phoma, Zygosporium ergot alkaloids Claviceps Terpenes penitrem Penicillium trichothecenes Fusarium, Myrothecium, Stachybotris Tricarboxylic acid rubratoxin Penicillium tenuazonic acid Alternaria, Aspergillus

Adapted from Kale and Bennett [222].

quality of animal products, increased susceptibility to infection, losses due to reduced weight gain from toxin-contaminated animal feeds. The perceived significance of mycotoxin contamination of foods for human health has prompted international trade and health organizations and governmental control authorities to regulate toxin levels permitted in commodities for commerce. Since 1977, the Food and Agricultural Organization (FAO) of the United Nations has published over a dozen position papers on mycotoxin regulation and control [34]. Over 50 countries of the world have developed such guidelines. In the United States, only the Aspergillus mycotoxins, aflatoxins, have action levels under the Food, Drug and Cosmetic Act [35]. The US Food and Drug Administration prohibits interstate commerce of feed grain containing more than 20 parts per billion (ppb) aflatoxin B1 and prohibits the sale of milk and eggs containing more than 0.5 ppb aflatoxin M1. Levels varying from zero tolerance to 50 ppb are accepted by at least 50 countries [36] (table 4). And, as stated by Van Egmond and Speijers [37], ‘A recent development in this regard was the establishment in 1994 of the WHO Collaborating Center for Mycotoxins in Food (WHO-CCMF), located in the Albert Ludwigs University School of Medicine in Freiburg, Germany. A particular task of this

Bhatnagar/Yu/Ehrlich 174 Table 4. Limits for aflatoxins in foodstuffs

Countries Maximum allowed aflatoxins, ppb or ␮g/kg

Austria 0.02–1 France 0.1–10 Germany 5 India 30 Japan 10 Malaysia 35 Mexico 20 Netherlands 0.02–5 United Kingdom 10 United States (food and feed) 20 (milk and eggs) 0.5

Adapted from Van Egmond [36].

Center will be to elucidate the role of mycotoxin-contaminated food in human health and disease. In 1997, the National Institute of Public Health and the Environment, Bilthoven, The Netherlands was appointed as a Community Reference Laboratory (CRL) for mycotoxins in animal products.’ The regulatory issues are less well-defined for mycotoxins other than aflatoxins. But with increasing data on specific toxicity of Fusarium mycotoxins such as fumonisins, regulatory guidelines are being considered for these toxins as well. For example, in the USA, advisory limits exist for deoxynivalenol (DON) at 5–10 mg/kg (5–10 parts per million; ppm) in animal feeds [7]. Several countries strictly enforce laws to prevent sale of contaminated commodities, whereas in others regulatory guidelines exist, but are not enforced. Problems related to more precise methods for evaluating mycotoxin contamination of food are still being developed. Errors can result from sampling, subsampling, and analysis [38]. Such errors can lead to accidental exposure to inappropriate levels of mycotoxins or in rejection of sound lots. Additionally, costs of chemical analyses, quality control and regulatory programs, research development, extension services, lawsuits, and the cost of treatment of human illness must be considered in the overall economic burden that mycotoxins has on the economy worldwide. Based on estimates of this burden, one can only justify the need for a major research effort in mycotoxin identification and characterization, prevention and control. The research efforts to date in many

Toxins of Filamentous Fungi 175 areas of the world have made significant progress toward understanding the natural occurrence of several mycotoxins.

Detection and Screening of Mycotoxins

Efforts to minimize mycotoxins in foodstuffs include monitoring, managing and controlling the levels of mycotoxins in agricultural products from farm to fork. Therefore, surveillance programs have been established to reduce the risk of mycotoxin consumption by humans and animals. Analytical testing methods for rapid analyses of a large number of samples of foodstuffs have been developed for several mycotoxins [39]. A single method for analysis of all mycotoxins is not feasible because of the varied chemical nature of mycotoxins, their molecular weights and functional groups. Current analytical techniques for accurate characterization and quantitation of mycotoxins include thin layer chromotography (TLC), high pressure liquid chromatography (HPLC) and gas chromatography (GC). However, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) tests are available, based on sera developed for a majority of the mycotoxins [39–42], for rapid detection of mycotoxins in foods at levels as low as one tenth of a nanogram per milliliter [6]. A dozen commercial test kits are available for field testing of various agricultural commodities for toxins such as aflatoxins.

Selected Mycotoxins

Some of the most prevalent mycotoxins, the producing fungi, and their major toxic effects in humans and animals are discussed below. Although specific mycotoxicoses have been attributed to various mycotoxins, and the mechanisms of toxicity have been established in vitro [43], there are many pitfalls in inter- preting the results of in vitro studies and extrapolating them to in vivo effects, and suggesting a natural disease associated with consumption of a mycotoxin. Riley [44], in describing these difficulties, suggested, ‘… the factors that tend to con- found in vitro mechanisms studied include: (1) failure to differentiate secondary effects from the primary biochemical lesion; (2) failure to relate the effective intracellular concentration in vitro to the tissue concentration of toxin which causes disease in vivo; (3) choice of an in vitro model which is either deficient in the biochemical target or unresponsive due to other inadequacies of the model system; and (4) failure to adequately model the complexity of the in vivo exposure with regard to potential interactions with other toxins, drugs, environmental, and/or nutritional factors’.

Bhatnagar/Yu/Ehrlich 176 Aflatoxins

Aflatoxins are by far the best-characterized class of mycotoxin. They are a group of polyketide-derived bis-furan-containing dihydrofuranofuran and tetrahydrofuran moieties (rings) fused with a substituted coumarin (fig. 1). At least 16 structurally related toxins have been characterized [45]. These toxins are primarily produced by Aspergillus flavus and A. parasiticus on agricultural commodities, and infrequently by A. tamarii and A. nomius [46]. The four major aflatoxins, B1, B2, G1 and G2, were originally isolated from A. flavus hence the name A-fla-toxin. The B toxins fluoresce blue under UV light and the G toxins fluoresce green. Other significant members of the aflatoxin family,

M1 and M2, are metabolites of aflatoxin B1 (AFB1) originally isolated from bovine milk. Attempts to decipher the aflatoxin biosynthetic pathway began with the discovery of the structure of these toxins [45]. However, the major biochemical steps and the corresponding genetics of the AFB1 biosynthesis have been elucidated only in the last decade [47–53]. Starting with the polyketide precursor, acetate, there are at least 23 enzymatic steps in the AFB1 biosynthetic pathway. AFB2, G1, and G2 are synthesized from pathways that diverge from the B1 pathway [54–56]. The genes for almost all the enzymes have been cloned and a regulatory gene (aflR) coding for a DNA-binding, Gal4-type 47-kD protein has been shown to be required for transcriptional activation of all the structural genes [48, 50, 51, 53, 57–65]. It has also been shown by restriction mapping of cosmid and phage DNA libraries of A. flavus and A. parasiticus that all the AFB1 pathway genes are clustered within a 75-kb region of the fungal genome [50, 51, 53, 64]. Since the discovery of the Turkey X disease in 1960, aflatoxins have been established as mutagenic, teratogenic and hepatocarcinogenic in experimental animals. AFB1 is the most toxic of this group of toxins and the order of toxicity is B1ϾG1ϾB2ϾG2. Aflatoxin M1 is 10-fold less toxic than B1, but its presence in milk is of concern in human health [66–68]. AFB1 is also one of the most carcinogenic natural compounds known; therefore, extensive research has been done on its synthesis, toxicity and biological effects [53, 69–72]. Liver is the target organ for aflatoxins, and aflatoxicosis leads to proliferation of the bile duct, centrilobular necrosis and fatty infiltration of the liver, hepatomas and hepatic lesions. The susceptibility of animals to AFB1 varies with species [reviewed in Eaton and Groopman, 72]. In addition to the liver,

AFB1 also affects other organs and tissues, such as the lungs and the entire respiratory system [73–75].

In animal models, activation of AFB1 by microsomal cytochrome P-450 is required for carcinogenicity [72, 76]. One of the cytochrome P-450 monooxy- genases in the liver converts AFB1 to a variety of metabolites of increased

Toxins of Filamentous Fungi 177 a b

R3 O O O

O O O

1 R1 R 4 R

2 O O OCH O H O R H 3

c d R1 R1 H H H H

H H H O H O O O H O H

Aflatoxins Structure R1 R2 R3 R4

B1 a H OCH3 ϭOH M1 a OH OCH3 ϭOH P1 aHOHϭOH Q1 a H OCH3 ϭOOH R0 a H OCH3 OH H R0H1 a H OCH3 OH OH B2 ac H OCH3 ϭOH B2a ad H OCH3 ϭOH M2 ac OH OCH3 ϭOH G1 bH––– G2 bc H – – – G2a bd H – – – GM bc OH – – –

Fig. 1. Chemical structure of aflatoxins. (a) The B-type aflatoxins are characterized by a cyclopentane E-ring. These compounds have a blue fluorescence under long-wavelength ultraviolet light. (b) The G-type aflatoxins have a xanthone ring in place of the cyclopentane.

(c) Aflatoxins of the B2 and G2 type have a saturated bis-furanyl ring. Only the bis-furan is shown. (d) Aflatoxins of the B1a and G1a type have a hydrated bis-furanyl structure.

Bhatnagar/Yu/Ehrlich 178 polarity including AFB1-8, 9-epoxide, the ultimate carcinogen which binds covalently to N7 position of guanine in DNA, resulting in defective repair and DNA damage, mutations and ultimately carcinomas in many animal species [70, 77, 78]. The role of aflatoxins in carcinogenesis in humans is complicated by hepatitis B virus infections in human beings which is also associated with hepatocarcinoma [79–81]. An association of hepatocellular carcinoma and dietary exposure to aflatoxins was established from patients living in high-risk areas of the People’s Republic of China, Kenya, Mozambique, Phillippines, Swaziland, Thailand, and the Transkei of South Africa [72, 78, 82]. The correlation was demonstrated by measuring guanine-aflatoxin adducts in urine samples. The presence of the adducts is considered an indication of the modification of a guanine residue at a mutation hot spot, the transversion of guanine to thymine at the third base of codon 249 in the tumor suppressor gene p53. This gene (p53) is a transcription factor involved in the regulation of the cell cycle which is commonly mutated in human cancers [83]. Since multiple factors may be involved in carcinogenesis [84], aflatoxins in combination with hepatitis B virus may be significant dual etiological factors in hepatocellular carcinoma [85, 86].

Toxic Polyketides Other Than Aflatoxins

Ochratoxins These mycotoxins are a group of dihydroisocoumarins (fig. 2) and are produced by a number of Penicillium and Aspergillus species [87]; the largest amounts of ochratoxins are made by A. ochraceus and P. verruculosum. The two species A. ochraceus and P. verruculosum, occur frequently in nature, and were first reported as ochratoxin producers [88–90]. Other fungi such as Petromyces alliaceus (alternate name Aspergillus alliaceus) [91, 92] and A. niger have also been reported to produce ochratoxins. Ochratoxins are produced primarily in cereal grains (barley, oats, rye, corn, wheat) and mixed feed during storage in temperate climatic conditions. However, this toxin has also been reported to contaminate other commodities such as beans, coffee, nuts, olives, cheese, fish, pork, milk powder, wine, beer and bread [1, 93]. Ochratoxins bind tightly to serum albumin and are carried in animal tissues and body fluids. Consequently, the occurrence of this toxin has been reported in pork sausage made from contaminated organ meats, a product widely consumed in northern Europe and the Balkans. Ochratoxin A, the most toxic member of this group of mycotoxins, has been found to be a potent nephrotoxin causing kidney damage in many animal species as well as liver necrosis and enteritis [4, 89, 94–96]. This toxin also acts as a immunosuppressor [96–98], and demonstrates teratogenic [99],

Toxins of Filamentous Fungi 179 OH O O

R1 C O H

CH3

R2 R3

Ochratoxins R1 R2 R3

AaClH BaHH CbClH A methyl ester c Cl H B methyl ester c H H B ethyl ester b H H 4-hydroxy ochratoxin A a Cl OH ␣ OH Cl H ␤ OH H H

a ϭ C6H5CH2CH(COOH)NH-. b ϭ C6H5CH2CH(COOEt)NH-. c ϭ C6H5CH2CH(COOMe)NH-.

Fig. 2. Structure of the ochratoxins. These metabolites form different classes depending on the nature of the amide group (a–c), and the presence or absence of a chlorine moiety at R2 in the phenyl. mutagenic [100], weak genotoxic [101] effects in test animals. Some studies have shown that ochratoxins may be weakly carcinogenic, affecting both the kidney and the liver [97, 102]. In vitro studies have shown that ochratoxin is an inhibitor of aminoacyl transfer ribonucleic acid (tRNA) synthetase and protein synthesis. Human exposure to ochratoxins can come from ingestion of contaminated sausage meats or cereals, cereal products, coffee, beer and even pulses [4, 103]. Although the role of ochratoxins in human pathogenesis is still speculative, the lesions of nephropathy in humans were reported to be similar to those observed in porcine nephropathy [104]. Outbreaks of kidney disease (Balkan endemic nephropathy) in rural populations in Bulgaria, Romania, Tunisia and the former Yugoslavia were associated with ochratoxin A [104–106]. These correlations were

Bhatnagar/Yu/Ehrlich 180 CH3

H3C N H CH3

H H OH O

NH Fig. 3. Chemical structure of cyclopiazonic acid. based on detection of ochratoxin A in human serum, milk and kidneys in these countries. Among 77 countries which have regulations for different mycotoxins, 8 have specific regulations for ochratoxin A, with limits ranging from 1 to 20 ␮g/kg in different foods [4]. Administration of phenylalanine can reverse some of the toxic effects of ochratoxins. But other efforts to eliminate the presence of this toxin in food have not been successful.

Cyclopiazonic Acid This fungal metabolite is produced by several species of the genus Penicillium, P. aurantiogriseum, P. crustosum, P. griseofulvum, P. camemberti, as well as by A. flavus, A. tamarii, and A. versicolor, but not A. parasiticus [46, 107]. Natural occurrence of cyclopiazonic acid (CPA, fig. 3) has been reported in corn, peanuts and cheese, along with the presence of aflatoxins. The association of CPA with aflatoxins together with a lack of well-developed analytical methods for its determination in foods has prevented an assessment of the possible health effects of CPA. Cyclopiazonic acid apparently causes hyper- esthesia and convulsions as well as liver, spleen, pancreas, kidney, salivary gland and myocardial damage [71, 108]. Kodua poisoning resulting from ingestion in India of Kodo millet seeds contaminated with Aspergillus, has been attributed to CPA.

Patulin Patulin was originally considered desirable for its antibacterial properties. However, its toxicity to mammals precluded its use as an antibiotic. Patulin (fig. 4), a metabolite of many species of Penicillium and Aspergillus, has been detected in damaged apples, apple juice and apple cider made from partially decaying apples and in other fruit juices as well as in wheat. A heat-resistant fungus Byssochlamys nivea, frequently found in foods also produces patulin [109]. Due to highly reactive double bonds that readily react with sulfhydryl

Toxins of Filamentous Fungi 181 O O

H Fig. 4. Structure of patulin showing O the closed-ring tautomeric form.

O HO C O 3 O HO CH2 C C C C C OH CH O 3 O

CH3 C

CH2

Fig. 5. Chemical structure of penicillic acid showing both tautomeric forms. The open-chain form is in equilibrium with the closed form shown on the right in the figure. groups in foods, patulin is not very stable in foods containing these groups [110]. Toxic effects include contact edema and hemorrhage upon ingestion. It appears to be carcinogenic in experimental animals but no instances of human cancers are known [71, 111]. At least 10 countries have regulatory guidelines that limit the presence of patulin to 50 ␮g/kg in various foods and juices.

Penicillic Acid Penicillic acid (fig. 5), also a metabolite of many Penicillium and Aspergillus species, is a contaminant of corn (‘blue eye corn’), beans and even meat. Even though penicillic acid has been shown to be a liver toxin, no instances of animal or human toxicity have been reported. In spite of this, the potential for these toxins to act synergistically with other more potent toxins is a concern since the species that produce these toxins usually produce other toxins as well [71].

Citrinin Frequently associated with the natural occurrence of ochratoxin A, citrinin is produced by Penicillium citrinum and several other Penicillia and Aspergilli [112]

Bhatnagar/Yu/Ehrlich 182 OH H O

Fig. 6. Structure of sterigmatocystin. OMe O O The bis-furanyl structure is similar to that of the aflatoxins except that the E-ring is a H substituted phenol. and Monascus ruber and M. purpureus [113]. As with patulin, citrinin was originally investigated for its antibacterial properties. But, this toxin has been reported to be a nephrotoxin in chickens fed corn contaminated with Penicillium citrinum. This golden-yellow compound is easily detected as a free compound, but upon binding to proteins particularly during the drying of corn, the color is lost, while the toxicity remains. Therefore, significant quantities of citrinin, while part of the diet, could lead to chronic and hard to diagnose kidney disease in susceptible individuals and animals, particularly those that were fed products made from low- quality corn [71].

Sterigmatocystin Sterigmatocystin (ST) is produced by various Aspergilli. But there are reports which indicate that fungi of the genera Bipolaris and Chaetomium, and Talaromyces luteus also produce ST [112]. Structurally related to aflatoxins

(fig. 6), and a precursor of AFB1 [71, 114, 115], ST is a hepatotoxic and carcinogenic mycotoxin. But its carcinogenicity is several order of magnitudes less than that of AFB1 in test animals [116, 117]. ST is a contaminant of cereal grains (barley, rice and corn), coffee beans and cheese [118]. It has been reported as a contaminant in foods from regions of the world with a high incidence of cancer, e.g., esophageal cancer in Linxian province in the People’s Republic of China [119] and liver cancer in Mozambique [117]. Although ST- producing fungi have been isolated from patients with esophageal cancer, the role of ST in human carcinogenesis appears to be indirect and inconclusive [4].

Zearalenone This toxin is produced by several Fusarium spp., in particular by the scabby wheat fungus F. graminearum (roseum) which also makes deoxynivalenol, DON [71]. It has an unusual macrocyclic structure [6(10-hydroxy-6-oxo- trans-1-undecenyl)-beta-resorcyclic acid-␮-lactone] (fig. 7). Zearalenone (also called F-2) is found in the gluten of contaminated wet-milled wheat, and also

Toxins of Filamentous Fungi 183 OH CH3 O H

O Fig. 7. Structure of zearalenone. in corn and corn products. Zearalenone has estrogenic properties and can cause premature onset of puberty in female animals, as well as hyperestrogenic effects and reproductive problems in animals, especially swine. This toxin has also been associated with an increased incidence of cervical cancer. Zearalenone can be toxic to plants; it can inhibit seed germination and embryo growth at low concentrations. Contamination of feed with zearalenone as well as with DON may result in severe economic losses to the swine industry due to both feed refusal and adverse estrogenic effects.

Fumonisins

Fumonisins are a group of toxins produced primarily by Fusarium verticillioides (formerly called F. moniliforme), F. proliferatum and other related species which readily colonize corn all over the world [120–124]. Nine structurally related fumonisins including B1, B2, B3, B4, A1 and A2, have been described (fig. 8). Chemically, fumonisin B1 is a derivative (diester) of propane-1,2,3- tricarboxylic acid of 2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxy- icosane [121–130]. The other fumonisins lack the tricarballylic acid or other ester groups [121, 131, 132]. Fumonisins are chemically similar in structure to toxins (AAL) produced by Alternaria alternata [133]. Production of fumonisins by Alternaria has also been reported [134, 135], and some fumonisin-producing Fusaria have been known to produce AAL toxins [136].

Fumonisins B1, B2 and B3 are the major mycotoxin contaminants in corn and corn-based foods and other grains such as sorghum and rice. The level of fumonisins varies from negligible to greater than 100 ppm, but is generally reported to be between 1 and 2 ppm. Corn frequently contains detectable but low levels of fumonisins, even though there is no visible sign of fungal contamination. In laboratory cultures, highest levels of this toxin are produced on corn, and less on rice; whereas peanuts and soybeans are very poor substrates for fumonisin production [4].

Bhatnagar/Yu/Ehrlich 184 R1 R3 CH3 R CH3

R R2 OH

O O OH O Rϭ ϩN OH HO O

Tricarballylic acid 3-Hydroxypyridinium (3HP)

Fumonisins R1 R2 R3 R4

B1 OH OH NH2 CH3 B2 HOHNH2 CH3 B3 OH H NH2 CH3 B4 HHNH2 CH3 A1 OH OH NHCOCH3 CH3 A2 H OH NHCOCH3 CH3 C1 OH OH H H C4 HHH H P1 OH OH 3HP CH3 P2 H OH 3HP CH3 P3 OH H 3HP CH3

Fig. 8. Chemical structures of different classes of fumonisins. The R residue is

3-hydroxypyridinium in fumonisins P1–P3 while the others are tricarballyl esters.

Like most other mycotoxins, fumonisins are quite stable and are not removed from grains by changes in temperature, pH and salt concentration during food processing. Therefore, significant amounts of fumonisins are usually found in foods and feeds. Stability of fumonisins is of concern because some products, such as gluten from wet milling and distillers’ dried grains from fermentation of contaminated grains, contain higher levels of fumonisins than found in the starting raw material. Acid hydrolysis of fumonisins causes the loss of the tricarballylic acid moiety. Significant amounts of fumonisins are not transmitted to milk, meat or eggs because animals rapidly excrete these toxins [137, 138].

Toxins of Filamentous Fungi 185 Fumonisin biosynthesis has been of significant research interest in the last few years [reviewed in Proctor, 139]. A 20-carbon chain is the backbone of fumonisins which structurally resembles fatty acids and linear polyketides [140]. Studies with 13C- and 14C-labeled acetate have suggested that a polyketide synthase, alanine, methionine and other amino acids are involved in fumonisin synthesis. Four loci (designated fum 1, fum 2, fum 3 and fum 4) involved in fumonisin biosynthesis have been identified by classical genetic analyses utilizing Gibberella fujikuroi, the sexual stage of Fusarium verticillioides [139]. These genes are clustered on chromosome 1 [140, 141].

Fumonisin B1, first isolated in South Africa, is regarded as the most significant of the fumonisins. Fumonisins have been shown to have diverse biological and toxicological effects as discussed below. The mechanism of fumonisin toxicity is not well understood. Studies have shown that fumonisins are inhibitors of ceramide synthase; their effect thus appears to be related to interference with sphingolipid biosynthesis in multiple organs, such as brain, lung, liver and kidney, of the susceptible animals. Fumonisin B1 has also been shown to be a hepatotoxin and a carcinogen in rats resulting in liver cirrhosis and hepatic nodules, adenofibrosis, hepatocellular carcinoma, ductular carcinoma and cholangio-carcinoma [125, 142–145]. Because of its potential role in hepatocarcinoma formation, fumonisin B1 has been classified as a class II carcinogen by the International Agency for Research on Cancer, 1993. In cell culture systems, fumonisin B1 has been demonstrated to be mitogenic and cytotoxic, without genotoxic effects [144, 145]. Kidney cells have also been shown to be targeted by these toxins. Porcine pulmonary edema caused by ingestion of fumonisin-contaminated wheat and corn is one of the recorded toxic effects of this group of mycotoxins. Reports have indicated a possible role of fumonisin

B1 in the etiology of human esophageal cancer in the regions of South Africa, China and northeastern Italy where Fusarium species are common contaminants. Erythro-Leukoencephalomalacia (ELEM) in horses and other Equidae (donkeys and ponies) is the best-characterized toxicological effect of fumonisins, which is a seasonal disease occurring in late fall and early spring. The disease is manifested by neurological changes in animals, including uncoordinated movement and apparent blindness. Clinically, the disease is characterized by edema in the cerebellum. The levels of fumonisins B1 and B2 in feeds associated with ELEM ranges from 1.3 to 27 ppm [4]. Current data suggest that fumonisins may have greater effect on the health of farm animals than on humans, because studies on the impact of levels of fumonisins in human food have not yielded significant correlations [127, 146]. The Mycotoxin Committee of the American Association of Veterinary Laboratory

Diagnostics recommended that permissible levels of fumonisins (B1) in feeds be limited to 5 ppm for horses, 10 ppm for swine and 50 ppm for cattle and poultry.

Bhatnagar/Yu/Ehrlich 186 Trichothecenes

Several species of Fusarium infect corn, wheat, barley, and rice. Under favorable conditions they elaborate a number of different types of tetracyclic sesquiterpenoid mycotoxins that are composed of the epoxytrichothecene skeleton and an olefinic bond with different side chain substitutions (fig. 9). Based on the presence of a macrocyclic ester or ester-ether bridge between C-4 and C-15, trichothecenes are generally classified as macrocyclic (type C) or nonmacrocyclic (types A and B) (table 5). Other fungal genera producing trichothecenes are Myrothecium, Trichoderma, Trichothecium, Acremonium, Verticimonosporium and Stachybotrys. The term trichothecenes is derived from trichothecin, the first compound isolated in this group [115, 147–153]. The biosynthesis of the trichothecenes involves the incorporation of three molecules of mevalonate into the trichothecane nucleus of trichothecin. The precursor farnesyl pyrophosphate can fold in different ways depending on the position and stereochemistry of the three double bonds. The 6, 7-trans olefin is favored to fold to give trichodiene, the first stable intermediate in the biosynthetic pathway, a process catalysed by the enzyme trichodiene synthase. Trichodiene synthases have a high degree of similarity in different trichothecene- producing fungi. Subsequent conversions of the trichothecane nucleus involve a series of specific oxidations of the three rings, mediated by cytochrome P450 oxidases encoded by genes in a gene cluster as has been found for biosynthesis of other fungal secondary metabolites. The most significant oxidation is provided by a cytochrome P450 monooxygenase which introduces the C12, 13 epoxide. At least 12 genes (Tri1 to Tri12) are involved in the biosynthesis of trichothecenes. These genes have been cloned, and are clustered on a 25-kb region of the chromosome. Tri6 is a regulatory gene [139, 154–157]. The trichothecenes constitute a class of over 80 different members that elicit different toxic effects in animals and humans. The trichothecenes causing the most concern for food and feed consumption are nivalenol, deoxynivalenol (DON), T-2 toxin, and the macrocyclic trichothecenes. The most potent trichothecenes are dermal toxins and cause severe blistering and necrosis of the target tissue. Their cytotoxicity parallels their acute toxicity in animals, with T- 2 toxin being more potent than nivalenol. Nivalenol is much more potent as an acute toxin than is deoxynivalenol. DON is of considerable importance to agricultural economies because it is more frequently found in corn and cereal grains, and animals refuse to eat contaminated grain. Economic losses from both rejected feed stocks and underfed animals are significant. Unlike most mycotoxins, T-2 toxin is produced optimally at 15 °C instead of 25–30 °C for other toxins [152, 158]. Contamination of cereal grains such as barley, corn, oats, and wheat with T-2 toxin is less frequent than with DON.

Toxins of Filamentous Fungi 187 H H H

O H3C R1

H R5 CH2 R4 2 CH3 R R3

Trichothecenes R1 R2 R3 R4 R5

Diacetoxyscirpenol OH OAca OAc H H 4-Monoacetoxyscirpenol OH OAc OH H H 15-Monoacetoxyscirpenolb OH OH OAc H H Scirpentriol OH OH OH H H Deoxynivalenol OH H OH OH ϭO Nivalenol OH OH OH OH ϭO Fusarenon-x OH OAc OH OH ϭO T-2 OH OAc OAc H ISVc HT-2 OH OH OAc H ISV T-2 triol OH OH OH H ISV 3Ј-OH-T-2 OH OAc OAc H OH-ISVd T-2 tetraol OH OH OH H OH Neosolaniol OH OAc OAc H OH Roridin A,B,E,H,J H MCa MC H H Roridin K H MC MC H OH Verrucarin A,B,J,K H MC MC H H Verrucarin L H MC MC H OH Satratoxin F,G,H H MC MC H H Verrucarol H OH OH H H

a OAc ϭ OCOCH3. b Macrocyclic. c ISV ϭ OCOCH2CH(CH3)2. d OH-ISV ϭ OCOCH2C(OH)(CH3)2.

Fig. 9. Chemical structures of different trichothecenes. MC ϭ Macrocyclic; ISV ϭ isovalerate; OH-ISV ϭ hydroxyisovalerate.

Bhatnagar/Yu/Ehrlich 188 Table 5. Classification of trichothecenes

Nonmacrocyclic Macrocyclic type A1 type B2 type C3

T-2 toxin deoxynivalenol (DON) roridins HT-2 toxin nivalenol (NIV) verrucarins Neosolaniol (NESO) fusarenon-X stachybotryotoxins Diacetoxyscirpenol (DAS) T-2 tetraol (T-4ol)

1 Contain a hydrogen- or ester-type side chain at the C-8 position. 2 Contain a ketone. 3 Contain a macrocyclic ring.

However, T-2 toxin is at least 20-fold more toxic to animals, and possibly to humans, than DON [4]. The trichothecene mycotoxicoses are difficult to distinguish because they affect many organs, including the gastrointestinal tract, hematopoietic, nervous, immune, hepatobiliary, and cardiovascular systems [reviewed in Chu, 148]. Ingestion of trichothecene-contaminated food and feed causes many types of mycotoxicoses in humans and animals, for example, moldy corn toxicosis, scabby wheat toxicosis (Akakabi-byo disease), vomiting and feed refusal, fusaritoxicosis, and hemorrhagic syndrome. The trichothecenes have now been found to be responsible for the human disease alimentary toxic aleukia in western Siberia prior to World War II, which was then attributed to ingestion of moldy millet and wheat. This contaminated grain contained certain macrocyclic epoxytrichothecenes and the severe blistering agent T-2 toxin. Feed refusal by pigs exposed to moldy corn containing DON (also called vomitoxin) was a severe economic problem in the 1970s in midwest United States. Head scab of wheat caused by F. graminearum was also a serious problem for wheat and barley growers from 1991 to 1997 in the eastern and midwestern US, and in the Canadian provinces of Ontario and Manitoba [159–161]. The major mycotoxin associated with head scab is DON [152, 162]. Red-mold disease caused by contamination of wheat and barley with F. graminearum among other Fusaria was a sporadic problem in Japan and Korea in the 1970s. The combined result of Fusarium mycotoxin contamination is worldwide economic losses in the billions of dollars. Another disease, termed stachybotryotoxicosis, caused the death of thou- sands of horses in the Soviet Union prior to World War II and was attributed to contamination of straw and hay with the fungus Stachybotrys atra.

Toxins of Filamentous Fungi 189 More recently, spores from S. atra growing on moist wood or building materials in houses have been associated with pulmonary hemosiderosis in young infants. Trichothecenes may be involved in this ‘sick building’ syndrome in humans. Symptoms such as headaches, sore throats, hair loss, diarrhea, fatigue and general malaise have been reported in humans living in water-damaged houses [28, 149, 163]. The unusual 12,13-spiroepoxy ring of the trichothecenes has been shown to be necessary for their toxicity [reviewed in Chu, 148]. Their toxicity is mainly due to the inhibition of initiation of polypeptide synthesis or polypeptide chain elongation during protein synthesis. All types of trichothecenes interfere with the action of peptidyl transferase by binding to the 60S subunits of the eukaryotic ribosome. The higher-molecular-weight trichothecenes inhibit initiation by binding to the P site of the ribosome and preventing translocation. The lower-molecular-weight species inhibit both the initiation and elongation steps of protein synthesis.

Alternaria Toxins

Alternaria has been known for centuries to cause various plant diseases such as early blight of potato and various leaf spots and fruit rot. Species of this fungus are widely distributed in soil and on aerial plant parts. Because Alternaria requires high moisture levels (28–34%) for growth, infection of seeds occurs when the seed moisture is high, either in early stages of development or after wetting of crops from rain. More than 20 species of Alternaria are known to produce about 70 secondary metabolites belonging to a diverse chemical group including diben- zopyrones, tetramic acids, lactones, quinones, cyclic peptides. However, only alternariol (fig. 10), tenuazonic acid, altertoxin-I, alternariol methyl ether and altenuene are common natural toxic contaminants of consumable items like fruits (apples), vegetables (tomato), cereals (sorghum, barley, oat) and other plant parts (such as leaves) [118]. The most common species of Alternaria, A. alternata produces all important Alternaria toxins including the five mentioned above and tentoxin, altenuisol, alteniric acid, altenusin, dehydroaltenusin [164, 165]. As mentioned previously, A. alternata f. sp. lycopersici produces a group of host-specific toxins named AAL toxins which are similar to fumonisins. Although most of the compounds produced by Alternaria are generally nontoxic, alternariol monomethyl ether has been shown to be mutagenic in bacteria (Ames tests) [166]. Tenuazonic acid (TA) is a protein synthesis inhibitor and is capable of chelating metal ions and forming nitrosamines. TA is also produced

Bhatnagar/Yu/Ehrlich 190 O OH O

CH 3 OH Fig. 10. Structure of alternariol. by Phoma sorghina and Pyricularia oryzae and may be related to ‘onyalai’, a hematological disorder in humans living south of the Sahara in Africa.

Neurotropic Mycotoxins

Ergot Alkaloids and Related Toxins Ergotism was one of the earliest documented incidences of a disease caused by mycotoxin consumption. Contamination of rye and millet with the ‘ergot’, a name given to the hard, dark purple, sickle-shaped sclerotia from the parasitic fungus Claviceps (C. purpurea and C. paspali) [167], led to accumu- lation of a number of toxic alkaloids that affect the central and peripheral nervous systems and cause vasoconstrictive activity and hallucinations. Two types of ergot epidemics occurred: gangrenous and convulsive. In the gangrenous type, the victim first experienced violent burning pains termed ‘St. Anthony’s fire’ and then numbing and necrosis of the limbs, while the latter type caused severe convulsions and death. Epidemics of convulsive ergotism occurred from the Middle Ages in Europe until as late as 1928 in Russia. In one epidemic in France in the 1700s, it was estimated that 8,000 people died from eating grain containing as much as 25% ergot. Acute human ergotism [168–170] has essentially disappeared as a concern since the modern cleaning and milling processes for grains remove most of the ergot sclerotia. However, ergot poisoning can still be a problem for livestock feeding on wild grasses infected with Claviceps sp. Additional alkaloid-producing fungi are also of concern. Rye grass stag- gers and fescue toxicoses are caused by ingestion of grasses infected with endophytes Neotyphodium sp. and Epichloe sp. These fungi can produce ergot alkaloids, indole diterpenes and indole terpenes. The indoleterpenes can cause tremors in grazing livestock. The ergot alkaloids are derivatives of the four-ring structure of ergoline. These alkaloids can be divided into three groups: derivatives of lysergic acid

Toxins of Filamentous Fungi 191 (e.g. ergotamine and ergocristine); derivatives of isolysergic acid (e.g. ergota- minine); and derivatives dimethylergoline (clavines, e.g. agroclavine) [16]. The most biologically active forms are amide derivatives of D-lysergic acid, an alkaloid whose diethylamide derivative, LSD, is famous in modern times for its use as a recreational hallucinogen. This class of ergot alkaloids also includes the cyclol-type peptide alkaloids in which a cyclic dipeptide forms the amide linkage to lysergic acid. Another class of ergot alkaloids, the clavine alkaloids, lack the carboxyl function at C-17. At least 37 different types of clavine alkaloids are known and are made by Claviceps as well as unrelated fungi, such as Aspergillus fumigatus and Penicillium chermesinum. Ergot alkaloids have even been found in a single family of higher plants, the Convolvulaceae, which includes the common morning glory, whose seeds have been valued for their hallucinogenic properties. The source of the ergot strongly influences the type of alkaloids present, as well as the clinical picture of ergotism [171]. The ergot alkaloids have three types of physiological effects: they cause contraction of smooth muscle, they block the action of serotonin and adrenaline, and they act on the hypothalamic- pituitary system to inhibit the secretion of prolactin. These properties have led to their being used to induce uterine contractions, to relieve migraine headaches, and to treat prolactin-dependent disorders. The biosynthesis of the ergot alkaloids involves condensation of dimethyl- allyl pyrophosphate (derived from mevalonic acid) with tryptophan. Closure of the C- and D-rings of the alkaloid involves specific hydroxylations by cytochrome P450-dependent oxidases and rearrangements. Further modifications involve N-methylation in the presence of S-adenosyl methionine and/or condensation with amino acids and peptides. Coupling of lysergyl CoA with certain peptides forms the peptide alkaloids which are the most bioactive of the ergot alkaloids.

Other Neurotropic Mycotoxins A number of other mycotoxins exhibit neurotoxic activity. In many cases these mycotoxins do not cause noticeable toxicity, but in some cases have tremorgenic activity, as evidenced by uncontrollable shaking and disoriented behavior. In some instances, animals will refuse food, and have lowered resistance to disease. Because the fungi that make some of these toxins, such as several strains of Aspergillus (A. fumigatus, A. flavus) and Penicillium (P. oxalicum) are widespread and contaminate a variety of different grains, their importance in causing mycological disease may be currently underestimated. Like the ergot alkaloids, some of these are derived from tryptophan, for example the tremorgenic toxins fumitremorgin, verruculogen, and roquefortine produced by certain strains of Aspergillus and Penicillium spp. These mycotoxins contain both the

Bhatnagar/Yu/Ehrlich 192 indole ring of tryptophan and a dioxopiperazine ring formed by condensation of two amino acids. The fungus that makes roquefortine, P. roquefortii, is used for the manufacture of roquefort cheese and other blue cheeses. The low levels of toxin in these cheeses are not considered to be a problem for human health. Aspergillus terreus, A. flavus and A. fumigatus have been found to produce tremorgenic toxins territrem A, B, and C, and aflatrem [172]. A. flavus, A. wentii, A. oryzae and Penicillium atrovenetum (as well as Arthrinium sp.) can produce ␤-nitropropionic acid which causes convulsions, congestion in lungs and liver damage [173]. Citreoviridin and xanthomegnin, produced by Penicillium spp. are also frequently found in foods. Toxins causing tremorgenic effects in mice include penitrem (Penicillium crustosum and P. aurantiogriseum) and paspalitrem (also produced by Penicillium spp. and Claviceps paspali) [174–179]. Fusarium verticilliodes also produces mycotoxins such as fusarins [180–182], moniliformin [150, 183], fusaric acid, fusaproliferin and beauvericin. Fusarin C appears to be a potent mutagen. Aspergillus terreus, A. fumigatus and Trichoderma viride also produce gliotoxin that may have immunosuppressive effects in animals [184].

Management of Mycotoxin Contamination

The economic implications of the mycotoxin problem and its potential health threat to humans have clearly created a need to eliminate or at least minimize mycotoxin contamination of food and feed. While an association between mycotoxin contamination and inadequate storage conditions has long been recognized, studies have revealed that seeds are contaminated with mycotoxins prior to harvest [185]. Therefore, management of mycotoxin contamination in commodities must include both pre- and postharvest control measures.

Preharvest Control

Preharvest mycotoxin contamination can be reduced somewhat by appropriate cultural practices such as irrigation, control of insect pests [reviewed in ref. 186–189]. In some cases, changes in cultural practices to minimize toxin contamination are not feasible. For example, inclusion of extra irrigation regimes in desert-grown cotton fields is not feasible because it would add significant cost to the grower even if irrigation were available. However, significant control of toxin contamination is expected to be dependent on a

Toxins of Filamentous Fungi 193 detailed understanding of the physiological and environmental factors that affect the biosynthesis of the toxin, the biology and ecology of the fungus, and the parameters of the host plant-fungal interactions. Efforts are under way to study these parameters primarily for the most agriculturally significant toxins, namely aflatoxins, fumonisins, and trichothecenes [reviewed in ref. 155, 189–191]. While fumonisins may not be essential for virulence, they may play a significant role in fungal infection of corn [190]. And, since Fusarium verticil- loides hyphae are found to be confined to the pedicel of the corn kernel, introduction by genetic engineering or plant breeding and expression of antifungal proteins in the pedicel region of corn kernel could impart resistance in corn against preharvest fumonisin contamination [192]. F. graminearum is the causative organism of head blight (scab) of wheat, and trichothecene produced by this fungus appears to be involved in fungal virulence [157]. This suggests that resistance in wheat against trichothecene production may provide a means of controlling not only head blight but preharvest contamination of crops with this mycotoxin as well [157]. Use of atoxigenic biocompetitive, native Aspergillus flavus strains to out- compete the toxigenic isolates has been effective in significantly reducing aflatoxin contamination [193]. Breakthroughs in the understanding of the molecular biology of aflatoxin biosynthesis have enabled the selective designing of effective biocompetitive agents whenever a native biocontrol strain is not available [189]. Biocompetition may be more feasible in crops such as cotton where resistance against fungal infections is not available due to limited genetic diversity in the cotton germplasm. In the long term, however, significant control of the aflatoxin problem will likely be linked to introduction of resistant germplasm, which is resistant either to fungal invasion or toxin production. Naturally resistant germplasm is available, but the identification of specific biochemical factors linked to resistance against A. flavus are assisting in enhancing the observed resistance levels in the existing germplasm as well as in the identification of novel germplasm that demonstrates the desired characteristics, i.e. the presence of the biochemical factors [189, 194]. However, the aflatoxin contamination process is so complex [187] that a combination of approaches will be required to eliminate or even control the preharvest toxin contamination problem [48].

Postharvest Control

Postharvest mycotoxin contamination is prevalent in most tropical countries due to a hot wet climate coupled with sub-adequate methods of harvesting, handling and storage practices, which often lead to severe fungal growth and

Bhatnagar/Yu/Ehrlich 194 mycotoxin contamination of food and feed [188, 195–197]. Sometimes contaminated food has been diverted to animal feed to prevent economic losses and health concerns. However, this is not a solution to the contamination problem. Irradiation has been suggested as a possible means of controlling insect and microbial populations in stored food, and consequently, reducing the hazard of mycotoxin production under these conditions [reviewed in Sharma, 198]. However, significant emphasis has been placed on detoxification methods to eliminate the toxins from the contaminated lots or at least reduce the toxin hazards by bringing down the mycotoxin levels under the acceptable limits. Detoxification of aflatoxins has been investigated extensively, particularly in corn and peanuts. Comparatively fewer reports are available on detoxification of other mycotoxins, such as zearalenone, tricothecenes, citrinin and ochratoxin. Detoxification of mycotoxins can be achieved by removal or elimination of the contaminated commodities or by the inactivation of toxins present in the commodities through various physical, chemical and biological means. For a successful detoxification procedure, the following basic criteria have been suggested [199]: (1) It must be economical, so that the detoxified commodities may still be sold at competitive prices. (2) The process must be relatively simple, not too time-consuming, and suitable for operation by unskilled labor. (3) It must be capable of removing all traces of active toxin and the chemical or biochemical residues must not constitute a health hazard. (4) The nutritional quality must not be impaired. Various detoxification processes can be categorized as follows: Removal or elimination of mycotoxins. Since most of the mycotoxin burden in contaminated commodities is localized to a relatively small number or seeds or kernels [reviewed in Dickens, 200], removal of these contaminated seeds/kernels is effective in detoxifying the commodity. Methods currently used include (a) physical separation by identification and removal of damaged seeds, mechanical or electronic sorting, flotation and density separation of damaged or contaminated seeds; (b) removal by filtration and adsorption onto filter pads, clays, activated charcoal; (c) removal of the toxin by milling processes; and (d) removal of the mycotoxin by solvent extraction. Inactivation of mycotoxins. When removal or elimination of mycotoxins is not possible, mycotoxins can be inactivated by (a) physical methods such as thermal inactivation, photochemical or gamma irradiation; (b) chemical methods such as treatment of commodities with acids, alkalis, aldehydes, oxidizing agents, and gases like chlorine, sulfur dioxide, ozone and ammonia [201]; and (c) biological methods such as fermentations or enzymatic digestions that cause the breakdown of mycotoxins [202].

Toxins of Filamentous Fungi 195 The commercial application of only some of these detoxifying mechanisms is feasible because, in a number of cases, the methods will be limited by factors such as the toxicity of the detoxifying agent, nutritional or aesthetic losses during treatment, and the cost of the sophistication of the treatment [188, 203]. Moreover, at this time there exist regulatory barriers against interstate commerce of decontaminated commodities, and sale of any mycotoxin- contaminated human food treated by detoxifying agents. Nevertheless, ammo- niation process has been used by some countries and states of the USA for detoxification of aflatoxins in corn and peanut meal for feed purpose.

Dietary Considerations

Modifications to diets, such as use of agents for absorption, distribution and metabolism of mycotoxins, can affect the toxicity of mycotoxins. Dietary additives including anticarcinogenic substances [204–206] and chemoprotective agents [207–211] have been found to inhibit the carcinogenic effects of aflatoxins in test animals by preventing formation of aflatoxin-DNA adducts [212]. Antioxidants such as vitamin C and E reduced the toxic effects of ochratoxin A [213, 214], whereas ascorbic acid provided protection against aflatoxins [215]. The toxic effects of ochratoxin have also been reduced by aspartame because it competitively prevents the binding of ochratoxin to serum albumin [216]. The introduction of hydrated sodium calcium aluminosilicates in diets of animals has reduced the toxicity of various mycotoxins (such as aflatoxins) to animals because these and related compounds (hydrated sodium calcium aluminosilicate or novasil) have a high affinity for mycotoxins [217–221] that prevents the absorption of mycotoxins by animals. Other absorbants that have been tested for mycotoxins such as T-2 toxin included zeolite, bentonite and superactive charcoal.

Summary

Mycotoxins are low-molecular-weight secondary metabolites of fungi. The most significant mycotoxins are contaminants of agricultural commodities, foods and feeds. Fungi that produce these toxins do so both prior to harvest and during storage. Although contamination of commodities by toxigenic fungi occurs frequently in areas with a hot and humid climate (i.e. conditions favorable for fungal growth), they can also be found in temperate conditions. Production of mycotoxins is dependent upon the type of producing fungus and environmental conditions such as the substrate, water activity (moisture and relative humidity), duration of exposure to stress conditions and microbial, insect or other animal interactions. Although outbreaks of mycotoxicoses in humans have been documented,

Bhatnagar/Yu/Ehrlich 196 several of these have not been well characterized, neither has a direct correlation between the mycotoxin and resulting toxic effect been well established in vivo. Even though the specific modes of action of most of the toxins are not well established, acute and chronic effects in prokaryotic and eukaryotic systems, including humans have been reported. The toxicity of the mycotoxins varies considerably with the toxin, the animal species exposed to it, and the extent of exposure, age and nutritional status. Most of the toxic effects of mycotoxins are limited to specific organs, but several mycotoxins affect many organs. Induction of cancer by some mycotoxins is a major concern as a chronic effect of these toxins. It is nearly impossible to eliminate mycotoxins from the foods and feed in spite of the regulatory efforts at the national and international levels to remove the contaminated commodities. This is because mycotoxins are highly stable compounds, the producing fungi are ubiquitous, and food contamination can occur both before and after harvest. Nevertheless, good farm management practices and adequate storage facilities minimize the toxin contamination problems. Current research is designed to develop natural biocontrol competitive fungi and to enhance host resistance against fungal growth or toxin production. These efforts could prevent toxin formation entirely. Rigorous programs for reducing the risk of human and animal exposure to contaminated foods and feed also include economically feasible and safe detoxification processes and dietary modifications. Although risk assessment has been made for some mycotoxins, additional, systematic epidemological data for human exposure is needed for establishing toxicological parameters for mycotoxins and the safe dose for humans. It is unreasonable to expect complete elimination of the mycotoxin problem. But multiple approaches will be needed to minimize the economic impact of the toxins on the entire agriculture industry and their harmfulness to human and animal health.

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Deepak Bhatnagar, USDA/ARS/SRRC, 1100 Robert E. Lee Boulevard, New Orleans, LA 70124 (USA) Tel. ϩ1 504 286 4388, Fax ϩ1 504 286 4419, E-Mail [email protected]

Bhatnagar/Yu/Ehrlich 206 Breitenbach M, Crameri R, Lehrer SB (eds): Fungal Allergy and Pathogenicity. Chem Immunol. Basel, Karger, 2002, vol 81, pp 207–295

Phylogeny and Systematics of the Fungi with Special Reference to the Ascomycota and Basidiomycota

Hansjörg Prillingera, Ksenija Lopandica, Wolfgang Schweigkoflera, Robert Deakb, Henk J. M. Aartsc, Robert Bauerd, Katja Sterflingera, Günther F. Krausa, Anna Marazb

aUniversität für Bodenkultur, Arbeitsgruppe Mykologie und Bodenmikrobiologie, Wien, Austria; bSzent Istvan University, Department of Microbiology and Biotechnology, Budapest, Hungary; cState Institute for Quality Control of Agricultural Products, RIKILT, Wageningen-UR, The Netherlands, and dUniversität Tübingen, Lehrstuhl spezielle Botanik und Mykologie, Tübingen, Germany

In 1965, Zuckerkandl and Pauling [1] argued that sequence comparison of informational macromolecules permits the evaluation of evolutionary relatedness, thereby fomenting a phylogenetic revolution, especially in prokaryotic organisms and protists [2, 3]. Protista were once considered as a distinct third kingdom besides animals and plants by Haeckel [4]. Kimura’s neutral theory of molecular evolution also had an impact on studies of the phylogeny and evolution, especially of microorganisms with an inadequate fossil record [5–7]. Meanwhile, molecular systematics has revolutionized our understanding of the microbial world. Currently, phylogenies of the Eukarya depend principally on small [3, 8, 9–17] or large [18–24] ribosomal RNA (rRNA) subunits, although 5S rRNA [25, 26] and a number of protein sequences [27, 28] also influence phylogenetic interpretations. Based on 18S rDNA sequencing, the Ascomycota and Basidiomycota form monophyletic clades within the kingdom Mycobionta or chitinous Fungi (fig. 1) [14, 29–31]. Defined by a membrane-bounded nucleus, the kingdom Mycobionta is one of several kingdoms within the crown groups of the Eukarya or eukaryotes (fig. 1).

Dedicated to Dr. C.P. Kurtzman on the occasion of his 60th birthday and for his valuable help in establishing the VIAM culture collection. 1 Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 208 The Eukarya constitute one of the three principal domains of life [32]. According to Knoll [33], the Eukarya are an ancient group, as old as the prokaryotic Bacteria and Archea, or nearly so [2, 8]. Paleontological and biogeochemical data suggest that Eukarya were significant organisms of ecosystems at least as early as 1,700–1,900 million years ago [33]. Sequence analyses of protein- encoding genes that duplicated before the divergence of the domains now suggest that the general tree of life should be rooted between the Bacteria and Archea, with the Eukarya bearing a specific phylogenetic relationship to the Archea [34, 35]. Within the Eukarya, the earliest diverging organisms (not shown in fig. 1) are aerotolerant anaerobes, most of which live parasitically or symbiontically within animal hosts (microsporidia, diplomonads, oxymonads, hypermastigids, parabasalia and some others) [3, 8, 36]. Microsporidia contain many promising species for biological control of harmful insects. It is remarkable that microsporidia cluster within the kingdom Fungi or Mycobionta (fig. 1) if gene sequences encoding the largest subunit of the RNA polymerase II or the elongation factors EF-1␣ and EF-2 are used [37]. Doolittle [28] stresses the importance of lateral gene transfers in prokaryotic and eukaryotic evolution. This, however, may complicate phylogenetic interpretations. The diplomonad Giardia infects the human intestine and can cause diarrhea, a disease known as giardiasis, or ‘hiker’s diarrhea’. These organisms have a well-defined nucleus and flagellum apparatus, but no mitochondria or chloroplasts and are included in the kingdom Archezoa [38]. Representatives of the Archezoa have relatively simple cytoskeletons and exhibit a number of ultrastructural (e.g. extranuclear pleuromitosis) [39–41] and biochemical characters more similar to those of prokaryotes than to other eukaryotes [38]. Protists occupying the middle branches of the phylogenetic tree [33] of the Eukarya commonly contain mitochondria, but no chloroplasts. Euglenids are the exception; about one third of them are photosynthetic. Euglenid chloroplasts may be derived from symbiotic green algae [13], implying a relative late acqui- sition of photosynthesis within this group. The amoebaflagellate Naegleria (fig. 1) is considered to be one of the earliest diverging protists with mitochondria. Nuclear rRNA phylogenies support this view. Heterolobosea emerge at

Fig. 1. Phylogenetic tree of eukaryotic organisms based on the primary structure of the 18S rRNA gene. Complete sequences of the 18S rRNA gene were aligned by means of the CLUSTALX program [44]. Software package PHYLIP [45] was used for phylogenetic infer- ences. Distance matrix was constructed in the DNADIST program (Kimura 2 parameter model) and the FITCH program was used for calculating phylogeny. The phylogenetic tree was displayed in TREEVIEW [46]. Branch lengths are proportional to nucleotide differences and the numbers given on branches represent the percentage of frequencies with which a given branch appeared in 100 bootstrap replications. The sequences were retrieved from the nucleotide sequence libraries (EMBL, GenBank and DDBJ).

Systematics of the Ascomycota and Basidiomycota 209 the base of mitochondria-bearing eukaryotes, together with trypanosomids and euglenoids. All three taxa feature extraordinarily long branch lengths [3] (fig. 1). Although predominantly aerobic, organisms in this part of the phylogenetic tree commonly thrive under relatively oxygen-poor conditions [42]. Most eukaryotic diversity is nested within the densely branched crown of the phylogenetic tree (fig. 1) [3, 13, 33, 43]. Major clades that branch near a common point include the kingdoms (fig. 1) Zoobionta or Animalia (Metazoa ϩ unicellular relatives), Chlorobionta (green algae and terrestrial plants), Mycobionta (chitinous or true fungi), Heterokontobionta (Stramenopila or chromophyta: golden brown algae, diatoms, brown algae, oomycetes, slimenets), Rhodobionta (red algae) and Alveolobionta (Alveolates: ciliates, dinoflagellates, apicomplexans; not shown in fig. 1) [3]. Because of rapid diversification, branching order within the crown group of Eukarya remains uncertain. Mitochondria and chloroplast genomes have molecular sequences that ally them to the Bacteria (proteobacteria and cyanobacteria) [47, 48]. The sequence data are congruent with ultrastructural and biochemical evidence supporting the endocytobiotic theory for the origins of these organelles [49, 50]. Molecular data are in agreement with a multiple origin of plastids, with some plastids originating from prokaryotic (simple plastids: chloroplasts, cyanelles, rhodoplasts) and others from eukaryotic (complex plastids: cryptophytes, haptophytes, heterokon- tophytes, euglenophytes, chlorarachniophytes, dinoflagellates) algae [13, 43].

The Kingdom Mycobionta (Eumycota) or True Fungi

Among earlier phylogenetic speculations extensively discussed in Jahrmann and Prillinger [51] and Barr [52], the concept of Cavalier-Smith [53, 54] and Prillinger [41, 51, 55] is noteworthy. Based on a framework of data on cell wall chemistry, biosynthetic pathway of lysine, storage carbohydrates, ultrastructure of mitochondrial cristae, type of motile cells and ploidy of vegetative hyphae, Cavalier-Smith and Prillinger only considered the chytridiomycetes, zygomycetes, ascomycetes and basidiomycetes as true or chitinous fungi and included them in the kingdom fungi or Eumycota. These four fungal groups are characterized by chitinous cell walls [56, 57], the ␣-aminoadipic acid lysine biosynthetic pathway [58, 59], glycogen as storage carbohydrate [54], nondiscoid plate-like mitochondrial cristae [54], the absence of heterocont flagella, and the absence of diploid vegetative hyphal compartments in the higher Ascomycota and Basidiomycota (exceptions: forced heterokaryons: e.g. Aspergillus, Penicillium; solopathogenicity in the smuts: Ustilago; Hymenomycetes: Armillaria [41, 60]; see Oomycota [61]). The primarily heterotrophic origin of this group is extensively discussed by Jahrmann and Prillinger [51]. Within the Eumycota the

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 210 chytridiomycetes are considered basal, the Entomophthorales (zygomycetes) evolved from a chytridiomycete by loss of the flagella [54]. Based on a single pos- terior flagellum (opisthokont), flattened, nondiscoid mitochondrial cristae, a chitinous exoskeleton, storage of glycogen instead of starch, lack of chloroplasts, and the code UGA for tryptophan, not chain termination, in their mitochondria, Cavalier-Smith [54] suggests a common origin of the true fungi with animalia and choanoflagellate protozoa (fig. 1). The oomycetes, hyphochytrids, labyrinthuloids, and thraustochytrids are included in the kingdom Heterokontobionta or pseudofungi [54] based on the presence of cellulose in their cell walls, a tubular mitochondrial crista, heterokont flagella, one decorated with tripartite hairs, and the ␣, ␤-diaminopimelic acid lysine biosynthetic pathway. The slime molds were classified into the kingdom Protozoa [38]. Evidence from complete 18S rDNA sequence divergence (fig. 1) [29, 62] put an end to the discussion on the kingdom Eumycota or true fungi and corroborated the existence of four naturally related phyla or divisions: the Chytridiomycota, the Zygomycota, the Ascomycota and the Basidiomycota within the kingdom Fungi or Mycobionta (fig. 1) [63, 64] or Eumycota [41, 52]. Specific acyclic polyols [65], an exclusively absorptive or lysotroph nutrition [66] and a distinct ultrastructure of the flagellar apparatus of the Chytridiomycota [52] are additional characteristics which support the kingdom Mycobionta. Based on the complete sequence of the 18S rRNA gene and the amino acid sequence of the elongation factor, the Animalia or Zoobionta appeared as a sister group of the Mycobionta or true fungi (fig. 1) [3, 67–72]. Nikoh et al. [73] come to a similar conclusion from a phylogenetic analysis of 23 different proteins. A closer phylogenetic relationship of Zoobionta and Chlorobionta, however, becomes apparent from homologous comparisons of ribosomal proteins [74]. The protozoal Choanoflagellida are phylogenetically closely related to the Zoobionta and Mycobionta (fig. 1) [52]. Prototheca is a ubiquitous achlorophyllous green alga (fig. 1) that lives on decaying organic matter and exhibits a yeast-like growth pattern. Human infection usually involves the skin and underlying tissues. P. wickerhamii (fig. 1) is recovered most often from human specimens, while P. zopfii usually is associated with infections in animals [75]. In the phylogenetic trees of Bruns et al. [29] and Sugiyama [14], the phagotrophic plasmodial slime molds (Myxomycota) and cellular slime molds (Dictyosteliomycota) diverged prior to the terminal radiation of eukaryotes (fig. 1). Presently no data are available on the cellular Acrasiomycota. In contrast, parsimony analysis of amino acid sequences of EF-1␣, a protein involved in the translation of messenger RNA, strongly supports a monophyletic origin of the Dictyosteliomycota and Myxomycota and the amoeboflagellate protostelid

Systematics of the Ascomycota and Basidiomycota 211 Planoprotostelium (kingdom Mycetozoa). Among the multicellular eukaryotes, the Mycetozoa appear closer to Animalia and true fungi than to green plants [27]. The use of EF-1␣ emphasizes the importance of developing multiple sequence data sets. As a conclusion, the phylogeny of the Acrasiomycota, Dictyosteliomycota and Myxomycota remains uncertain at the moment, and additional sequence data are urgently needed. Based on 18S ribosomal DNA sequencing, the plant parasitic slime mold Plasmodiophora brassicae (Plasmodiophoromycota), a severe pathogen of crucifers, may be more closely related to the Alveolobionta than to any of the fungi [76]. The Oomycota (fig. 1, Achlya, Lagenidium, Leptolegnia, Phytophthora, Pythium, Saprolegnia), Hyphochytridiomycota (fig. 1, Hyphochytrium) and net slime molds or Labyrinthulomycota (fig. 1, Labyrinthula, Thraustochytrium, Ulkenia) form a clade with brown algae (Phaeophyceae), diatoms (Bacillario- phyceae), Chrysophyceae, Xanthophyceae, and Chloromonadophyceae. These organisms have heterokont flagella, one decorated with tripartite hairs; autotrophic species contain chlorophylls a and c, and are classified within the kingdom Chromista [38], Heterokontobionta [64] or Stramenopila [63]. The Oomycota lack acylic polyols [65] and differ in sterol biosynthesis from the true fungi [77]. Based on the biosynthesis of sterols, Berg and Patterson [77] suggest a heterotrophic origin of the Oomycota. The labyrinthuloids appear to be basal to other heterokont algae, Oomycota and Hyphochytridiomycota within the Heterokontobionta (fig. 1) [78, 79]. The division Oomycota mainly consists of two orders. The order Saprolegniales comprises aquatic species, some of which are pathogenic to fish. Representatives of the order Peronosporales mostly occur in soil or as parasites of plants [63]. The latter order comprises one species of clinical significance, Pythium insidiosum [80]. Two members of the order Peronosporales, Phytophthora infestans and Plasmopara viticola, have been implicated as allergenic fungi [81].

Morphological Differentiation within the Kingdom Mycobionta

Figure 2 shows a phylogenetic and ontogenetic scheme accounting for the range of morphological organization in the kingdom Mycobionta. Figure 2 is based on a scheme proposed by Pascher [82] for algae, but, unlike the latter, envisages evolution from polykaryotic, via oligokaryotic to mono- and dikaryotic systems [41]. Different types of morphological organization are extensively discussed in Jahrmann and Prillinger [51] and Prillinger [41, 83]. The basal position of the flagellate and rhizopodial types was further corroborated by a compilation of ultrastructural data [41] and sequencing of the ribosomal RNA genes [3, 84]. In figure 2 we used the term flagellate instead of monadal

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 212 Pseudoparenchyma

Plectenchyma

Evolution of vegetative anastomosisHyphal aggregates

Terrestrial Trichal habitat Forcibly discharged secondary spore Hyphal aggregates

Siphonal

Pseudotrichal

Colonies Phylogeny Coccal

Colonies

Rhizopodial Polyphyletic evolution

Colonies

Flagellate

Aquatic habitat

Ontogeny

Fig. 2. Evolutionary scheme for morphological differentiation within the kingdom Mycobionta. Modified from Prillinger [83]. because the first Eucarya was most probably already a chimera of two prokaryotic organisms [49]. Presently, it is not clear whether the Amoebidiales with free-living rhizopodial or amoeboid stages belong to the Zygomycota. The yeast form, denoted by the term ‘coccal’ (i.e. a unicellular organism having a rigid cell wall outside its plasma membrane), occupies a basal position among the Zygomycota, Ascomycota, and Basidiomycota [Oberwinkler, pers. obs.], but seems to be derived in the Chytridiomycota (e.g. Basidiobolus) [83, 85, 86].

Systematics of the Ascomycota and Basidiomycota 213 3 Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 214 A yeast/hypha dimorphism is common in all classes of the Ascomycota (fig. 3: Hemiascomycetes, Protomycetes, and Euascomycetes) and Basidiomycota (fig. 4: Urediniomycetes, Ustilaginomycetes, Hymenomycetes), especially in primitive representatives. It seems to be fundamental for a rapid evolution of the fungi. Similarly, the pseudotrichal pattern is common in many dimorphic Zygomycota, Ascomycota, and Basidiomycota and often reverts to the unicellular condition [51, 83]. The polykaryotic siphonal (coenocytic) type is characteristic for many eukarpic Chytridiomycota and the aflagellate Zygomycota. The forcibly discharged secondary spore (ballistospore in the Basidiomycota) is a specific adaptation of unicellular morphological differentiation to terrestrial life [87]. Its polyphyletic origin in the Entomophthorales and its early exis- tance in the Basidiomycota is discussed by Tucker [88] and Prillinger [83]. Forcibly discharged secondary spores stimulate a faster spreading on new solid habitats and may help to escape or establish parasitic interactions. Septate hyphae, or the trichal type of morphological organization, are well known in advanced groups of the Zygomycota (Dimargaritales, Kickxellales) and filamentous Ascomycota and Basidiomycota, the hyphal compartments may be poly-, oligo- and monokaryotic in the Ascomycota or commonly dikaryotic in the Basidiomycota [41]. The plectenchyma with vegetative anastomoses are characteristic for the fruiting bodies of the higher Ascomycota (except dikaryotic ascogenous hyphae) and Basidiomycota. Pseudoparenchyma, with approximately isodiametric cells and synchronous cell and nuclear divisions, differ from true parenchyma of plants by the absence of a phragmoplast and a meristematic tissue. They are common in the Laboulbeniales and some other meristematic Euascomycetes [89], aecidia of rust fungi, and fruiting structures of diverse Ascomycota and Basidiomycota.

Sexual Differentiation within the Kingdom Mycobionta

Two markedly different concepts of sexuality in Mycobionta have arisen, depending on the primary event(s) or mechanism believed to be involved. According to the view favored here, the primary event is ‘sexual differentiation’ [41, 55, 83]; this being the process which leads to karyogamy and meiosis either within the same strain (homothallism) or after the crossing of two different mating

Fig. 3. Phylogenetic tree of Ascomycota based on the primary structure of the 18S rRNA gene. Alignment, distance matrix and calculation of phylogenetic distances were made by means of different programs as described in the legend of figure 1. Carbohydrate cell wall composition [129] is assigned as follows: ᭹ ϭ Glucose-mannose; ᭡ ϭ glucose-mannose-galactose; ᭿ ϭ glucose, mannose, galactose, rhamnose. Human pathogenic genera are indicated by arrows. T ϭ Teleomorphic species; A ϭ anamorphic species; Y ϭ yeasts or yeast stages.

Systematics of the Ascomycota and Basidiomycota 215 4 Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 216 types (heterothallism). According to the other view, the primary mechanism is the polarity resulting from sexual incompatibility [90], a phenomenon which sup- presses karyogamy and meiosis in monoecious organisms. Fundamental differences between both concepts are extensively discussed in Prillinger [83]. To explain the evolution of sexuality via sexual differentiation in the Mycobionta, two distinct steps seem to be worth discussing. A. A polyphyletic evolution from mitotic to meiotic life cycles within a polykaryotic and coenocytic homothallic organism (primary or primitive homothallism [41, 83] (fig. 5). B. A polyphyletic origin of heterothallism in different groups of fungi [55, 83]. Mycoparasitic interactions which evolve to sexual symbiosis and a lateral gene transfer are considered to be of fundamental importance for the evolution of heterothallism [28, 83]. Burgeff [91, 92] was the first to detect a close relationship between fungal sexuality and parasitism. In contrast to Prillinger [83], however, he considered parasitism as a degenerated form of sexuality. A polyphyletic evolution from heterothallism back to homothallism (secondary or derived homothallism) appears to be very common in fungi [41, 55, 83, 93, 94] (fig. 5). Yun et al. [95] analyzed mating type gene organization, together with a phylogeny from ITS/glyceraldehyde-3-phosphate dehydrogenase gene sequences to show that homothallism in Cochliobolus (C. luttrellii, C. cymbopogonis, C. kusanoi, C. homomorphus; fig. 5) arose independently from heterothallic ancestors. In the Basidiomycota there is in addition some evidence that unifactorial (bipolar) heterothallism as a mating system evolved polyphyletically [90]: (1) primarily via mycoparasitic interactions, as suggested for the Ascomycota (fig. 5) [55, 83]; (2) secondarily from bifactorial (tetrapolar) heterothallism by close linkage of A and B loci, as demonstrated in Ustilago hordei [96]; (3) secondarily from bifactorial mating systems by ‘self-compatible’ mutations in either the A or B factors (or homothallism if both the A and B factors are affected), as has been demonstrated in Coprinus [97]. During recent years, a lot of new information has accumulated in favor of the concept of sexual differentiation: (1) Hijri et al. [98], Hosny et al. [99] and Sanders [100] demonstrated that polykaryotic, coenocytic and highly heterokaryotic homothallic strains without recombination, a prerequiste for the evolution of sexuality according to the

Fig. 4. Phylogenetic tree of Basidiomycota based on the primary structure of the 18S rRNA gene. Alignment, distance matrix and calculation of phylogenetic distances were made by means of different programs as described in the legend of figure 1. Human pathogenic genera are indicated by arrows. T ϭ Teleomorphic species; A ϭ anamorphic species; Y ϭ yeasts or yeast stages.

Systematics of the Ascomycota and Basidiomycota 217 Heterothallism Complex hetero-bifactorial (according to Kües, pers.com.) A : IC, two subunits, multiple highly divergent alleles B : IC? two subunits, multiple highly divergent alleles Schizophyllum commun, Coprinus cinereus Polyphyletic evolution Simple hetero-bifactorial A : EC, two idiomorphs (SP) B : IC, multiple alleles (RP) Ustilago maydis

Unifactorial IC, Two idiomorphs (RP) Polyphyletic evolution Neurospora crassa, Podospora pauciseta Saccharomyces cerevisiae EC, Two idiomorphs?

Secondary homothallism Sexual symbionts Schizosaccharomyces pombe Asterophora ϩ yeasts (Asterotremella) Saccharomyces cerevisiae Nonhaustorial parasites Gelasinospora reticulospora Anixiella sublineata Carcinomyces Neurospora terricola Parasitella, Chaetocladium Neurospora dodgei Neurospora tetraspora Haustorial parasites Podospora pauciseta Tremella, Christiansenia Cochliobolus luttrellii Syncephalis, Piptocephalis Agaricus bisporus Coprinus bilanatus Primary homothallism Haasiella venustissima Benjaminiella multispora Mycena galericulata

Fig. 5. Hypothetical scheme for the evolution of heterothallism and secondary homothallism in Mycobionta. For synchronous nuclear division in Benjaminiella multispora, Cokeromyces and Mucor species with respect to primary homothallism, compare Forst and Prillinger [159]. For molecular details, see Hiscock and Kües [90 and the literature cited therein]. For secondary homothallsim, see Glass et al. [160] and Yun et al. [95]. IC ϭ Intracellular function; EC ϭ extracellular function; SP ϭ structural proteins which are involved in pheromone binding; RP ϭ regulatory proteins which are involved in n-DNA binding and regulation of transcription. concept of sexual differentiation, indeed exist in the Mycobionta within the arbuscular mycorrhiza forming order Glomales of the Zygomycota. The authors detected nuclei in the polykaryotic spores (Scutellospora castanea: approximately 800 nuclei/spore) which were genetically different at the genus level. Since vegetative anastomoses occur in the Glomales [101], it is not clear whether the homothallism of S. castanea is primitive (primary homothallism; fig. 5) or derived (secondary homothallism) [41, 55, 83, 94]. The Glomales are truly ancient, having remained largely morphologically unchanged since plants first colonized the land around 400 million years ago [102]. No sexual stage has been observed in the Glomales life cycle. (2) Four yeast strains were isolated from different species of higher Basidiomycota (Polyporus, Marasmiellus, Ganoderma). In Polyporus, the yeast

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 218 appeared on a cross of isogenized ramarioid inbreeding strains, which had lost their ability to produce fertile hymenia (fig. 6 a, b) [83, 103, 104]. The yeast isolate from Marasmiellus originates from aseptically grown cultures of young fruiting body trama [104]. In Ganoderma two yeast strains were isolated during germination experiments from basidiospores harvested in nature. A spermatia–trichogyne-like recognition reaction (fig. 6 c, d) and a specific homing reaction (fig. 6 e) [104–106] as well as undistinguishable diglobular interphase spindle pole bodies of nuclei (fig. 6 f, g) and closely similar mol% GϩC values suggested conspecificity between the yeasts and the corresponding mycelial fungi. Using genotypic identification methods, we were able to identify all the yeast isolates. All yeast strains belong to Ustilago maydis [106]. Our results were corroborated by breeding experiments with plant pathogenic haploid mating type strains [106]. From our data we conclude that the spermatia- trichogyne fertilization reaction can be traced back to mycoparasitic interactions and has evolved polyphyletically in the Ascomycota and rust fungi (fig. 7 [107]). This is in agreement with a phylogenetic tree of complete sequences of the 18S rRNA gene, where the Uredinales appear as a derived clade which can be traced back to a dimorphic Mixia-like phylogenetic ancestor without morphologically developed sexual organs within the Urediniomycetes (fig. 4). (3) In auxotrophic mutants of Absidia glauca, a specific gene transfer from the parasite Parasitella parasitica to the host A. glauca was detected by Kellner et al. [108] and Wöstemeyer et al. [109]. (4) Prillinger [55] considered at least four different gene types to be involved in sexual differentiation (mating-type genes, homothallism genes, incompatibility genes and sterility genes). A more recent molecular characterization of mating-type genes based on DNA sequence comparisons [90 and literature cited therein] corroborates the interpretation of Prillinger [55] that mating-type genes biochemically differ in function from the incompatibility genes [110–120]. In addition, a similarity of the MAT-1 gene of Cochliobolus heterostrophus with Neurospora crassa mt A-1 protein, the Podospora pauciseta (anserina) mat Ϫ protein and the known DNA-binding region of Saccharomyces cerevisiae MAT ␣1 protein was detected. On the other hand, the MAT-2 gene of C. heterostrophus exhibits similarities to the N. crassa mt a-1 protein, the P. pauciseta mat ϩ protein and the known DNA-binding protein of Schizosaccharomyces pombe mat-Mc [121–123]. In Saccharomyces cerevisiae the cell-type-specific gene regulation in a and ␣-cells clearly corroborates our concept of sexual differentiation [90 and literature cited therein]. There is no evidence for an incompatibility function of the mating type loci in S. cerevisiae. An evolution from extracellular to intracellular functions of mating type genes as was postulated by Prillinger [55] was detected by Bölker et al. [124] and Kämper et al. [125] in Ustilago maydis. Molecular data on mating type genes of

Systematics of the Ascomycota and Basidiomycota 219 a b

c d e

f g

0.1␮m 0.1␮m

Fig. 6. Ustilago maydis isolated from Polyporus ciliatus (a–e) and Ganoderma adspersum (f, g). a Isogenised ramarioid inbreeding strains with nonsporulating fruiting bodies; bottom: haploid parents, top: dikaryotic cross. See Prillinger and Six [103]. b A yeast colony

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 220 Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Neurospora crassa, Ustilago maydis, Coprinus cinereus and Schizophyllum commune [90, 125–128] point to a polyphyletic evolution of heterothallism in different groups of fungi, as suggested by Prillinger [55, 83, 94]. The complexity of mating type genes and their respective number of base pairs nicely correlate with an evolution from the Hemiascomycetes to the Protomycetes and from the Protomycetes on the one hand to the Euascomycetes (fig. 3) and the other hand to the Urediniomycetes, Ustilaginomycetes and Hymenomycetes (fig. 4). (5) Similarly to a polyphyletic evolution of heterothallism, a polyphyletic loss of meiosis becomes obvious in several genera of the Eurotiales, Hypocreales and Onygenales. Using rDNA sequencing, meiotic and strictly mitotic taxa were often recovered clustered together, indicating that multiple independent losses of teleomorphs had occurred: Aspergillus and related teleomorphs [130–132], Penicillium, Geosmithia and their related Talaromyces and Eupenicillium teleomorphs [133, 134], Blastomyces, Histoplasma, Coccidioides, Emmonsia, Trichophyton, Oidiodendron species pathogenic in humans and related Ajellomyces and Myxotrichum teleomorphs [135–137], Fusarium and related Gibberella and Nectria teleomorphs [138–140], Gliocladium, Trichoderma and their Nectria and Hypocrea teleomorphs [141, 142], Acremonium and the phylogenetically different teleomorphs [143], Uredinales [144], and asco- [23] and basidiomycetous yeasts [24]. The molecular phylogenies do not support the existence of the Deuteromycetes or Deuteromycota as a distinct higher taxon within the Myco- bionta. Molecular characters offer the potential for combining the dual classification into one natural classification [145]. Anamorphic genus and species names of the Deuteromycetes, however, may also be necessary in the future for purposes of identification. As shown in figures 3 and 4, mitotic genera could be placed into meiotic orders and families [145].

(arrow) which appeared in the mycelium of the sterile dikaryotic cross. c, d Trichogyne-like approach of a hypha (large arrow) from the Polyporus mycelial culture towards the yeast cells (small arrow) produced on the pseudomycelium by the Ustilago maydis strain. Open star ϭ site of inoculum of Polyporus ciliatus; closed star ϭ site of the Ustilago maydis inoculum; triangle ϭ empty hyphae of the pseudomycelium. e Recognition reaction between pseudohyphae from U. maydis (coming from the left) and true hyphae from P. ciliatus (coming from the right), hyphal annealing (small arrow), possible vegetative anastomosis? (large arrow). For hyphal enveloping and lethal reaction, see Prillinger [104]. f, g Spindle pole body of the nucleus in the hypha of G. adspersum and yeast of U. maydis; the nuclear envelope is arrowed. From Prillinger, H.: Yeasts and anastomoses: Their occurrence and implications for the phylogeny of Eumycota, figure 24.5; in Rayner et al. (eds): Evolutionary Biology of the Fungi. London, Cambridge University Press, 1987. With permission from Cambridge University Press.

Systematics of the Ascomycota and Basidiomycota 221 Zygomycota Ascomycota

Piptocephalis Dispira Parasitella Phycomyces Chaetocladium Penicillium Podospora

ascog Ϫ t ϩ s sp Ϫ sp ϩ t f

ab c d

Sexual symbionts

1 3

2 c

2 b

Non-haustorial parasites

Trichal ap

h E v o l u t i o n Siphonal a

Haustorial parasites

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 222 A problem concerning DNA as the type specimen has been considered by Reynolds and Taylor [146] and Haines and Cooper [147]. Prillinger et al. [148] found that phenotypically identified strains of Sporothrix schenckii may be heterogeneous and stressed the importance of culture collections in modern genotypic identification. (6) Different yeasts were isolated when culturing the pileitrama of young fruit bodies of the two agarics Asterophora lycoperdoides and A. parasitica. Both agarics occur as mycoparasites on Russula nigricans in nature. In A. lycoperdoides, yeasts also appeared endophytically during the germination of chlamydospores [51, 54, 104]. In subcultures of the original yeast isolates from both Asterophora species, agaricoid fruit bodies developed after 4–6 weeks in artificial culture [51, 94, and Prillinger unpubl. obs.]. All attempts to repeat these experiments from single yeast cells or different crossing experiments, however, failed to produce fruit bodies, although some structures which resemble holobasidia were observed [104]. These data suggest that the yeast isolates from pileitrama were contaminated with Asterophora chlamydospores and exclude parasitic interactions. Physiological patterns and the qualitative and quantitative yeast cell wall carbohydrate spectrum of the yeast isolates from the two Asterophora species closely resemble some mycoparasitic Tremella species (e.g. T. encephala) [104, 149]. Using a positive selection vector to clone fungal nuclear DNA in Escherichia coli together with random fragment hybridization analysis, the conspecificity of the Asterophora yeasts and hyphae was excluded [150]. Based on mt-DNA RLFP [83] and nDNA/nDNA hybridization as well as ribosomal DNA restriction fragment analysis [455] the yeast isolates from A. lycoperdoides and A. parasitica can be considered as distinct species. Based on complete sequences of the 18S rDNA (fig. 4) and partial sequences of the 26S rDNA [24], the genus Cryptococcus appeared heterogeneous with respect to the type species C. neoformans (fig. 4). We therefore have included C. humicola and the two genotypically distinct yeast isolates from the agarics A. lycoperdoides and

Fig. 7. Evolution of heterothallism in the kingdom Mycobionta. a Haustorial parasites (after Jeffries and Young [153]); ap ϭ appressorium; h ϭ haustorium; arrow shows cell wall of fungal host. b, c Nonhaustorial parasites (after Burgeff [91, 92]); hatched ϭ nuclei and cytoplasm of the host; stippled ϭ nuclei and cytoplasm of the parasite; hatched and stippled ϭ heterokaryosis; the arrow indicates septum formation in the parasite. d Early successive stages in the gametangiogamy of Phycomyces nitens (Orban [154], Burgeff [92]). e Successive stages of gametangiogamy (arrows) in Talaromyces (Penicillium) stipitatus (after Emmons [155]); note the budding asci (a). f Spermatia-trichogyne fertilization in Podospora fimbriata (after Zickler [156, 157]: P. fimbriata (Bombardia lunata) after Mirza and Cain [158]); s ϭ spermatia; insert shows a spermatogonium (sp) fusing with a trichogyne (t).

Systematics of the Ascomycota and Basidiomycota 223 A. parasitica in the new genus Asterotremella as As. humicola, As. lycoperdoides and As. parasitica [Prillinger et al., in preparation]. We interpret As. lycoperdoides and As. parasitica together with the agarics from which they were isolated as sexual symbionts and missing links in the polyphyletic evolution from mycoparasitism to heterothallism (fig. 5, 7). Haustorial mycoparasites are common in the closely related genus Tremella. As a conclusion, the trinity system Russula, Asterophora, and Asterotremella is remarkable, and may be fundamental in the evolution of sexuality, especially primary homothallism and subsequently heterothallism in fungi. (7) Based on 18S ribosomal DNA sequencing, we have traced back the uni- nucleate ascomycetous yeasts (Kluyveromyces, Saccharomyces) to a polykaryotic (Eremothecium) ancestor [152]. Our arguments in favor of a sexual differentiation corroborate the idea that genetic engineering was not discovered by molecular biologists of the 20th century: it is common in nature and fundamental in the evolution of heterothallism in the Mycobionta.

Phylogenetic Relationships among the Chytridiomycota and Zygomycota

In the view of many mycologists, it is believed that the Chytridiomycota are the most primitive fungi within the Mycobionta, because they are zoosporic (fig. 2), and sexual reproduction has been reported to be accomplished by a variety of different methods (isogamy, anisogamy, oogamy, gametangiogamy, and somatogamy [63]. This view has been corroborated recently by the detection of the new order Neocallimasticales [161] which consists of species of obligately anaerobic chytrids that inhabit the rumen of herbivorous animals. While some species have typical uniflagellate zoospores, others are exceptional within the chytrids because they are polyflagellate with more than 10 flagella observed. In contrast to the other orders of the Chytridiomycota, the zoospores of the Neocallimasticales lack the microbody-lipid globule complex and mitochondria [63, 162]. The order appears to be monophyletic based on preliminary results from cladistic analysis of structural and molecular characters [161, 163, 164] and is, furthermore, ecologically distinct. Our phylogenetic analysis of the Chytridiomycota and Zygomy cota (fig. 1) corroborates the view of Nagahama et al. [86], Sugiyama [14], Tanabe et al. [165] that both phyla are not monophyletic and instead suggest that losses of flagella occurred in several lineages during the course of fungal evolution. The Blastocladiales (Allomyces, Blastocladiella) form a clade distinct from the Monoblepharidales (Monoblepharis, Monoblepharella),

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 224 Spizellomycetales (Spizellomyces), Neocallimasticales (Neocallimastix) and Chytridiales (Chytridium) [165]. Basidiobolus ranarum an aflagellate entomophthoralean fungus, which has a yeast stage [85] must be included in the Chytridiomycota (fig. 1) [86]. As McKerracher and Heath [166] already mentioned based on ultrastructural data, the morphology of the nucleus-associated organelle of Basidiobolus is unusual since no other nonflagellated organism contains microtubules as structural components of their nucleus-associated organelle. Within the Zygomycota three different clades appear. Mucor racemosus (Mucorales: M. mucedo, Mycotypha microspora, Rhizopus oligosporus, Syncephalstrum racemosum) [165] forms a distinct clade with three representatives of the Entomophthorales (Entomophthora muscae, Conidiobolus coronatus and Zoophthora radicans; fig. 1) [86]. The insect-pathogenic Entomophthorales (Conidiobolus, Entomophaga, Entomophthora, Eryniopsis, Pandora, Strongwellsea, Zoophthora) form a monophyletic group except Basidiobolus [165]. Based on Micromucor and Mortierella, the Mucorales appear polyphyletic [165]. The arbuscular mycorrhizal fungi (Acaulospora, Gigaspora, Glomus: Glomales) form a clade together with the ectomycorrhizal fungus Endogone (Endogonales) and Geosiphon pyriforme, a fungus forming endocytobiosis with Nostoc (Cyanobacteria; fig. 1) [165, 167]. As already indicated by similarities in septal pore ultrastructure, cell wall structure, asexual reproductive apparatus, and serological affinity [168], the Harpellales (Smittium, Furculomyces), an order of the Trichomycetes, have a close relationship to the Kickxellales (Coemansia, Martensiomyces, Linderina, Kickxella; fig. 1) [169]. Spiromyces can be excluded from the Kickxellales; it forms a distinct clade related to the Harpellales and Kickxellales (fig. 1) [169]. The monophyly of the mycoparasitic Dimargaritales received strong bootstrap support [165]. Also the mycoparasitic and zooparasitic Zoopagales in which Syncephalis, Thamnocephalis, and Rhopalomyces form a sister group to Piptocephalis and Kuzuhaea appear monophyletic [165]. Basidiobolus ranarum (fig. 1) as a representative of the Chytridiomycota rarely causes subcutaneous infections in humans [170]. B. haptosporus and B. meristosporus are additional species of clinical importance [75]. Conidio- bolus coronatus (fig. 1; Entomophthorales, Zygomycota), also known as Delacroixia coronata, is a pathogen causing nasal granuloma in man [80, 171, 172] and other higher mammals [173]. C. incongruus is an extremely rare agent of systemic mycosis with a pulmonary portal of entry [80]. No molecular information is presently available for some other medically important genera of the Zygomycota (e.g. Apophysomyces, Absidia, Cunninghamella, Rhizomucor and Saksenaea; also see De Hoog and Guarro [80]. Allergen characterization has been reported only for Rhizopus nigricans [81].

Systematics of the Ascomycota and Basidiomycota 225 Although Tanabe et al. [165] present a good overview of the phylogeny of Chytridio- and Zygomycota, further studies of molecular systematics, chemotaxonomy and ultrastructure will be necessary to establish a phylogenetic system of the Chytridiomycota and Zygomycota.

Phylogenetic Relationships among the Ascomycota and Their Anamorphs

About 70,500 species of true fungi or Mycobionta have been described; however, some estimates of total numbers suggest that 1.5 million species may exist [174, 175]. Most of them so far belong to the Ascomycota (32,300) and the mitosporic fungi (14,100) which can generally be included in the Ascomycota using sequencing of ribosomal DNA. There are numerous hypotheses on the phylogeny and evolution of higher fungi [for references, see 51, 107, 176–178]. Among these, Savile’s [179] phylogenetic considerations of higher fungi [107, 179] have attracted many mycologists. Savile suggested that Taphrina was the closest survivor of a common ancestor of the Euascomycetes and the Basidiomycota. He suggested that two major lineages evolved from ‘Prototaphrina’, a common ancestor. One major lineage led to the present- day Taphrina and the higher Ascomycota – today’s Euascomycetes – whereas another major route led to the Basidiomycota (the Uredinales line and the parasitic Auriculariaceae line) through a ‘Protobasidiomycete’. Phylogenetic trees inferred from 18S rDNA sequence divergence indicate the existence of two distinct phyla or divisions among the higher fungi (fig. 1), the Ascomycota and the Basidiomycota, e.g. [12, 14, 16, 29, 30, 180–182]. Using Mucor racemosus as an outgroup, our FITCH tree inferred from approximately 1,600 alignable sites of the 18S rRNA gene sequence from about 200 selected species (fig. 3, 4) supports that both phyla appear to be monophyletic. As already mentioned above, the use of the Deuteromycota or Deuteromycotina as a formal taxon decreases [14, 16, 145, 175, 183, 184]. Molecular sequence data clearly demonstrate that ascoma characters traditionally used to delimit ascomycete orders or classes converge. Eriksson [185] focused attention on the orders of ascomycetes and discouraged the use of supraordinal taxa. Plectomycetes, Pyrenomycetes, Loculoascomycetes, Discomycetes and other traditional class level categories are no longer used formally in the fungal classification system [14, 175]. Similarly, the classical dipartite systems of the Ascomycota (Hemiascomycetes, Euascomycetes) [177] and Basidiomycota (Heterobasidiomycetes, Homobasidiomycetes) [186] are not corroborated by molecular and biochemical data, instead tripartite systems appear in molecular phylogenies. Based on complete or nearly complete 18S rDNA sequencing,

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 226 the qualitative and quantitative monosaccharide pattern of purified cell walls (fig. 3) [129], the ultrastructure of septal pores and urease activity [Prillinger unpubl. obs., and [187], three classes seem promising among the Ascomycota (fig. 3): the Hemiascomycetes, the Protomycetes and the Euascomycetes [16, 188–190]. We cannot confirm the concept of the basal Ascomycetes or Archiascomycetes suggested by Berbee and Taylor [30], Nishida and Sugiyama [181], and Sugiyama [14]. Based on our polyphasic biochemical and molecular approach, the Euascomycetes and Protomycetes appear as a sister group (fig. 3), whereas the Hemiascomycetes occupy a basal position. The following data of the literature are in favor of our interpretation [also see Cai et al., 191]: (1) the nuclear genome size of the Hemiascomycetes appears to be about one-third the size of Aspergillus, Schizosaccharomyces and the basidiomycetous yeasts [192, 193]. More recent data from the whole genome sequencing project, however, suggest that the genome of Saccharomyces cerevisiae and Schizosaccharomyces pombe are similar in size (S. cerevisiae 13.4 Mb [194]; Sch. pombe about 14 Mb [195]). The 14 Mb are organized in 3 compact chromosomes in Sch. pombe, which resemble higher eukaryotes. In S. cerevisiae 16 rather primitive chromosomes (see 2 and 5) were found. (2) S. cerevisiae shows a rather unique cell cycle with the doubling of the spindle pole body and the formation of a short mitotic spindle already in the

S-phase.The G2 phase is missing. On the other hand, the cell cycle of Sch. pombe resembles the higher eukaryotes with a characteristic G1, S, G2 and mitose phase [196]. (3) S. cerevisiae has a very compact nuclear genome with very few introns (223 introns) [197, 198]. The higher frequency of introns in the genes of Sch. pombe resembles the situation in the higher eukaryotes. (4) S. cerevisiae is one of the few eukaryotes that can live without functional mitochondria. On the other hand, Sch. pombe requires functional mitochondria for survival as do the higher eukaryotes. (5) The centromers of S. cerevisiae are smaller and lack the repeated sequences which are typical for higher eukaryotes. Sch. pombe has centromers which resemble those in ‘higher’ ascomycetes and have a similar function [199]. (6) The length of the mating type loci: S. cerevisiae has the shortest known idiomorphs for the mating type loci (a: 640 bp, ␣: 750 bp). The respective lengths for Sch. pombe are P: 1.1 kb, M: 1.1 kb; for Podospora pauciseta matϩ: 3.8 kb, mat–: 4.7 kb, for Neurospora crassa A: 5.3 kb, a: 3.2 kb; for Ustilago maydis a1: 4.5 kb, a2: 8 kb; and for the hymenomycetous yeast Cryptococcus neoformans mat ␣ 35–45 kb (the mat-a locus of C. neoformans is not yet determined) [200]. (7) The lack of fruit bodies among the Hemiascomycetes could be interpreted as a primitive rather than as a reduced character. Among the

Systematics of the Ascomycota and Basidiomycota 227 Protomyces/Schizosaccharomyces group the Neolectales produce club-shaped fruit bodies, which are up to 7 cm tall and differ from other ascomycetous fruit bodies mainly by the absence of sterile hyphae (paraphyses) between the asci, the lack of ascogenous hooks (crosiers) prior to ascus development, and the unusual combination of inoperculate asci having amyloid ascus walls [201]. (8) The coenzyme Q of the Hemiascomycetes contain a variable number of isoprene units ranging from Q-5 to Q-9 (Q-10 was found in Lipomyces lipofer). On the other hand, most strains of the Protomyces/Schizosaccharomyces clade analyzed so far contain coenzyme Q-10 (with the exception of Schizo- saccharomyces octosporus, which has Q-9), resembling the Euascomycetes, which, in most cases contain coenzyme Q-10 and Q-10 (H2). Coenzyme Q-9 was found only rarely in some euascomycetous strains (e.g. Capronia parasitica, Symbiotaphrina spp.). No strain with less isoprene units was found within the Euascomycetes so far. Basidiomycetous yeasts possess coenzyme Q systems with Q-7, Q-8, Q-9, Q-10 and Q-10 (H2) [202 and references therein]. (9) The Hemiascomycetes include morphologically primitive fungi (within the genus Eremothecium) with coenocytic ‘siphonal’ [41, 83, 152] ontogenetic stages which resemble the Zygomycota and Chytridiomycota. (10) The haplo-diplontic life cycle of S. cerevisiae also shows some similarities with the Chytridiomycota, where in contrast to the Euascomycetes, this type of life cycle and diploid stages, are common [63].

Hemiascomycetes

Within the Hemiascomycetes, Kurtzman and Fell [202] presently accept a single order, Saccharomycetales (Endomycetales), only. Based on the qualitative and quantitative monosaccharide pattern of purified yeast cell walls and complete 18S rDNA sequences (fig. 3) [203], we accept four different orders: Saccharomycetales, Dipodascales, Lipomycetales and Stephanoascales. Whereas the Saccharomycetales and Lipomycetales can be delimited by the cell wall monosaccharide pattern and the presence or absence of extracellular amyloid compounds (Saccharomycetales: glucose, mannose, extracellular amyloid compound Ϫ; Lipomycetales: glucose, mannose, galactose, extracellular amyloid compound ϩ) it is not possible to separate the Dipodascales and Stephanoascales based on the cell wall monosaccharide pattern. Within both orders, glucose, mannose and galactose dominate; however, species with the glucose mannose pattern appear intermingled [203]. Sequences of the complete 18S rRNA gene are important to decide, whether a species belongs to the Dipodascales or the Stephanoascales. Extracellular amyloid compounds (starch formation) are absent in the Dipodascales and Stephanoascales. Kurtzman and

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 228 Fell [202] extensively discuss the problem of using Saccharomycetales instead of the older order Endomycetales. The order Dipodascales was already introduced by Batra [204]. However, based on molecular characteristics, many genera suggested by Batra cannot be included into this order. Additional complete 18S rDNA sequences are necessary to corroborate the orders Dipodascales, Lipomycetales and Stephanoascales [203]. Within the Dipodascales, four yeast species are of medical importance: Dipodascus capitatus and its anamorph Geotrichum capitatum, Yarrowia lipolytica (anamorph: Candida lipolytica), G. candidum and G. clavatum. Komagatella (Pichia) pastoris is a biotechnologically interesting yeast which exhibits the glucose mannose cell wall monosaccharide pattern and comes close to the Dipodascales [203] and [Prillinger unpubl. obs.). K. pastoris became an important host for the expression of recombinant DNA in recent years [202]. Its gene expression system has been developed to produce large amounts of medically and industrially important proteins [205]. D. capitatus is associated with human lung disorders. It is increasingly being found in blood of immunocompromised hosts, particularly in cases of leukemia [80, 202]. C. lipolytica has been isolated from patients with fungemia [75]. The main human disorders caused by G. candidum are bronchial or pulmonary infections, in humans as well as in nonhuman mammals [206]. Smith et al. [207] and Prillinger et al. [208] have shown that Galactomyces geotrichum and its anamorph G. candidum are genotypically heterogeneous. G. candidum should be conserved for a bene- ficial species common in cheese and other dairy products. G. clavatum is involved in human mycoses, particularly in connection with pulmonary disorders. Within the Saccharomycetales, the following species are of clinical importance: Candida albicans, C. parapsilosis, C. tropicalis, C. viswanathii, Debaryomyces fabryii (anamorph: C. famata var. flareri), Pichia guilliermondii (anamorph: C. guilliermondii) are phylogenetically closely related and may be included within the family Debaryomycetaceae (fig. 3) [23]. Pichia norvegensis (anamorph: C. norvegensis) and Issatchenkia orientalis (anamorph: C. krusei), Kluyveromyces marxianus (C. kefyr), S. cerevisiae, and C. glabrata, Clavispora lusitaniae (C. lusitaniae) and C. haemulonii are additional groups of phylogenetically related species which can be included in the Saccharomycetales (fig. 3) [23]. Last but not least Pichia anomala and its anamorph C. pelliculosa are representatives of the Saccharomycetales. As can be seen from figure 3, Kurtzman and Robnett [23] and Suzuki et al. [203], within the Hemiascomycetes, the genera Candida and Pichia are still heterogeneous. It was not possible to separate the genus Issatchenkia genotypically from the genus Pichia represented by its type species P. membranifaciens (fig. 3) [23]. Genotypically, the genera Kluyveromyces, Saccharomyces, Torulaspora and Zygosaccharomyces appear intermingled (fig. 3) [23, 191].

Systematics of the Ascomycota and Basidiomycota 229 K. delphensis is the teleomorphic species closest to Candida glabrata (fig. 3) [23, 191]. C. dubliniensis was recently described as a new species [209] isolated from 60 HIV-infected and 3 HIV-negative persons. Although C. dubliniensis closely resembles C. albicans phenotypically, it could be distinguished genotypically [23, 209]. Rapid identification of C. dubliniensis with commercial yeast identification systems was described recently [210]. However, Sullivan et al. [209] did not compare their isolates with strains representing the many synonyms of C. albicans, so it is possible that the species may be synonymous with an earlier described species. C. albicans commonly occurs in the digestive tract. Candidiasis is by far the most important mycosis. Vaginal candidiasis is extremely frequent. Mucocutaneous candidiasis occasionally leads to osteomyelitis [211]. Further information on the pathogenicity of C. albicans can be found in De Hoog and Guarro [80] and Murray et al. [75]. C. albicans and Saccharomyces cerevisiae were also considered as allergenic yeasts [81]. S. cerevisiae has been isolated from deep infections in debilitated patients and in patients with impaired immunity, both natural and acquired [212, 213]. De Hoog [213] presents an excellent overview of risk assessment of fungi reported from humans and animals. An interesting process of ‘budding meiosis’ was reported by van der Walt and Johannsen [214] in Candida albicans and C. tropicalis. Diploidization of the sexually active haplophase appeared to involve somatogamous autogamy or autodiploidization. The site of reduction divisions was identified when it was shown that the diplophase formed multinucleate cells on which the haplophase was delimited externally as buds. ‘Budding meiosis’ in ascomycetous yeast may be a phylogenetic precursor of the concept of ‘yeast basidia’ which was introduced by Prillinger et al. [215–217] and seemed to be fundamental in the evolution of basidia in the Basidiomycota. Although the presence of sexuality in C. albicans was corroborated recently by the presence of a mating-type-like locus [128], the existence of ‘budding meiosis’ in hemiascomycetous yeasts needs further confirmation by cytological and ultrastructural data. C. glabrata is often involved in urogenital infections [218]. It can also be involved in deep infections (heart [219]; lungs, occasionally with sepsis [220]; osteomyelitis [221]. There is evidence that C. glabrata may emerge after anti- C. albicans therapy [222]. Debaryomyces hansenii var. fabryii and its anamorph C. famata var. flareri were reported to be pathogenic by Vazquez et al. [223] and Nicand et al. [224]. Using random amplified polymorphic DNA analysis (RAPD-PCR), Prillinger et al. [208] considered D. hansenii var. fabryii as a distinct species, i.e. D. fabryii. A compilation of the pathogenicity of C. haemulonii, C. parapsilosis, C. tropicalis, C. viswanathii, Clavispora lusitaniae, Issatchenkia orientalis,

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 230 Kluyveromyces marxianus, Pichia anomala, P. guilliermondii, and P. norvegensis can be found in De Hoog and Guarro [80] and Murray et al. [75]. Blaschke- Hellmessen [225] gives an overview of the habitats of pathogenic Candida species. Based on ribosmal DNA sequencing, Messner et al. [21] and Prillinger et al. [152] included the two filamentous and plant parasitic species Eremothecium ashbyi and E. gossypii as well as the two dimorphic plant parasites E. coryli and E. sinecaudum in the family Saccharomycetaceae (fig. 3). These data clearly indicate that yeasts cannot be separated taxonomically from filamentous fungi. Within the Stephanoascales Stephanoascus ciferrii and its anamorph C. ciferrii are the only pathogenic species so far. The species is often associated with animals and occasionally isolated from clinical specimens [226] as an agent of human onychomycosis [227]. Some strains are strongly hyphal and produce conidia from characteristically inflated, denticulate heads. This anamorph has been described as Sporothrix catenata. No species of the order Lipomycetales is known to be pathogenic in humans so far. The order comprises typical soil yeasts (Babjevia, Lipomyces) and mycelial species (Dipodascopsis).

Protomycetes

Figure 3 shows there is good bootstrap support for the new class of the Protomycetes in our FITCH tree. A similar tree topology was obtained when the programs neighbor-joining and maximum likelihood of the PHYLIP packages were used for tree construction [16, 151]. Presently, four orders are accepted within the Protomycetes. These are the Neolectales, the Pneumocystidales, the Schizosaccharomycetales and the Taphrinales [228]. A fifth order, the Protomycetales, suggested by Eriksson and Winka [228] and Kurtzman and Fell [202], is not supported by biochemical and molecular data [182, 229, 230]. Prillinger et al. [229, 230] consider the Protomycetaceae as a family of the Taphrinales. Whereas the order Taphrinales was already introduced in 1928 by Gäumann and Dodge [see Eriksson and Winka, [228], the Schizosaccharomycetales, Neolectales, and Pneumocystidales were suggested only recently based on molecular characters. The order Schizosaccharomycetales was introduced by Prillinger et al. [229]. It was accepted by Kurtzman [193], Eriksson et al. [231], and Kurtzman and Fell [202]. The orders Neolectales and Pneumocystidales (fig. 3) were suggested by Landvik et al. [232] and Eriksson [233]. Based on the qualitative and quantitative monosaccharide pattern of purified cell walls, Prillinger et al. [215, 216, 229, 234] consider the Protomycetes (Protomyces-Typ, Schizosaccharomycetales) ancestral to the Euascomycetes and Basidiomycota,

Systematics of the Ascomycota and Basidiomycota 231 especially the Urediniomycetes sensu Swann and Taylor [10]. Morphological and ultrastructural data of Mixia osmundae [235 and Bauer unpubl. obs.] and 5S rRNA sequences from Protomyces inundatus [25] and Taphrina deformans [236] are additional characteristics which give support to the concept that the Protomycetes are ancestors of the Basidiomycota, as was originally suggested by Savile [107]. Based on the presence of fucose in cell walls of T. vestergrenii [217, 229], we consider T. vestegrenii a missing link on the route from the Protomycetes to the Urediniomycetes. Based on complete sequences of the 18S rDNA T. vestergrenii occupies an intermediate position between the genera Protomyces and Taphrina. We have suggested the new genus Fucotaphrina for this species. Based on the qualitative and quantitative monosaccharide pattern of purified cell walls (glucose: 70, mannose: 23, galactose: 7) and rDNA sequencing (fig. 3) [237], the anamorphic pigmented yeast Saitoella complicata unequivocally belongs to the Protomycetes and can be excluded from the Uredin- iomycetes where it was included originally. A two-layered cell wall, a negative diazonium blue B test and positive urease activity are additional characteristics of yeasts and yeast stages which belong to the Protomycetes [238]. Enteroblastic budding of S. complicata [14], however, suggests affinities to the Basidiomycota. The Neolectales are so far the only group of the Protomycetes where morphologically distinct fruting bodies, apothecia similar to clavarioid basidiocarps, are produced. Landvik et al. [232] and Landvik [239] excluded the apothecial ascomycetes Neolecta vitellina and N. irregularis from the Euascomycetes (fig. 3). Asci of N. vitellina are occasionally filled with numerous conidia and the ascospores become conidiogenous by producing a single apical collarette from which the phialoconidia emerge [201]. These features are similar to budding of ascospores within the ascus of Taphrina, and therefore do not conflict with the proposed molecular phylogeny. The Pneumocystidales is the only order of the Protomycetes which harbors species pathogenic in humans. Pneumocystis carinii is a unicellular eukaryotic organism with a tropism for growth on respiratory surfaces of mammals [75]. Epidemic pneumonia may occur in institutional housing, such as orphanages, under conditions of overcrowding and malnutrition. P. carinii has emerged as one of the most common pulmonary infections in AIDS patients in recent years; the presence of Pneumocystis has become one of the first indications for the disease [80]. The molecular phylogeny and systematics of P. carinii have been controversial for a long time (fig. 3 [75, 240, 241]). P. carinii has a number of features that are atypical for fungi (e.g. cholesterol instead of ergosterol) [75]. There is emerging molecular evidence that there are different varieties, and possibly different species of Pneumocystis.

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 232 The Schizosaccharomycetales comprise three distinct fermentative species which reproduce by fission; none of them are of medical importance [202]. Schizosaccharomyces pombe is known from tropical millet beer. The Taphrinales (fig. 3) comprise the Protomycetaceae and the Taphrinaceae [230]. All species are dimorphic fungi with the mycelial phase parasitic on ferns and especially woody dicotyledons, and the yeast phase saprophytic [176]. The mycelia and meiosporangia of the Protomycetaceae are polykaryotic, within the Taphrinaceae mycelia are commonly dikaryotic and the young meiosporangia contain one nucleus only. Prillinger et al. [229] regard the ascus of Taphrina as a ‘siphonal’ germination state of a chlamydospore. This siphonal germ tube acts as a meiosporangium, where an evolution from an unde- termined number of meiotic nuclei in the case of Protomyces to a single meiotic nucleus represented by the Taphrina species becomes obvious.

Euascomycetes

The Euascomycetes with comparatively well-developed fruiting bodies or ascomata comprise the plectomycetes, pyrenomycetes, loculoascomycetes, laboulbeniomycetes, and discomycetes based on traditional morphological classifications. In the monophyletic euascomycete lineage (100% bootstrap support in our FITCH tree fig. 3) [14], two major lineages, the Plectomycetidae with closed ascomata (cleistothecia) and the Pyrenomycetidae with flask- shaped ascomata (perithecia), appeared monophyletic, each receiving 100% bootstrap support (fig. 3). The tree topology in figure 3 supports the monophyly of the Plectomycetidae and Pyrenomycetidae as already detected by Berbee and Taylor [242] and Nishida and Sugiyama [181]. Our phylogenetic tree (fig. 3) does not support the concept of Gargas and Taylor [243] that the apothecial Pezizales (Ascobolus, Peziza, Gyromitra, Inermis, Morchella, Plectania) are ancestral to the cleistothecial and perithecial forms. Based on the apothecial Neolectales, however, Nannfeldt’s [244] phylogenetic hypothesis of primitive apothecial ascomata with subsequent evolution of cleistothecial and perithecial forms cannot be excluded. The cleistothecial Erysiphales which come close to the Leotiales, however, appear as an exception (fig. 3) [63]. Within the Hypocreales there are in addition some cleistothecial taxa, such as Heleococcum, Mycoarachis and Roumegueriella, which have to be excluded from the Plectomycetidae [142]. Analyses of 18S rDNA support that neither the loculoascomycetes (fissitunicate ascomycetes) nor the discomycetes (apothecial ascomycetes) are monophyletic (fig. 3) [16, 89, 243, 245–248]. Within the fissitunicate ascomycetes, the loculoascomycete order Pleosporales (fig. 3) appears as a monophyletic group including the families Pleosporaceae and

Systematics of the Ascomycota and Basidiomycota 233 Lophiostomataceae; similarly, the loculoascomycete order Dothideales (fig. 3) may also constitute a monophyletic group, however, with weaker statistical support [89, 247]. On the other hand, the fissitunicate Chaetothyriales appear as a sister group of the Plectomycetidae or the lichen-forming Lecanorales and Peltigerales (fig. 3) [16, 89, 246–249]. Within the Laboulbeniales, the Pyxidiophora-Rickia lineage (obligate parasites of arthropods) lies outside the other perithecial ascomycetes among loculoascomycetes and discomycetes where taxon sampling is still incomplete [250]. At the moment, therefore, previous hypotheses including Pyxidiophora in the Hypocreales, the Ophiostomatales or the Hemiascomycetes are not supported [250]. For allergenic and human-pathogenic Euascomycetes, the following three orders are of special importance: Chaetothyriales, Eurotiales and Onygenales (fig. 3). Additional species can be found within the Dothideales, Hypocreales, Leotiales, Microascales, Ophiostomatales, Phyllachorales, Pleosporales and Sordariales. Extensive explanations of terms and types of mycosis used in clinical pathology can be found in De Hoog and Guarro [80].

Chaetothyriales There is molecular evidence that at least the families Chaetothyriaceae and Herpotrichiellaceae belong to the Chaetothyriales [249]. Unexpectedly, the Chaetothyriales were found to be remote from the remaining Loculoas- comycetes such as Dothideales and Pleosporales and relatively close to the Onygenales and Eurotiales (fig. 3) [246] or the lichen-forming Lecanorales and Peltigerales [249]. The diversity of anamorphs in Chaetothyriales is remarkable and it is often difficult to distinguish them from anamorphs of the Dothideales [246]. Many species are dimorphic and are able to grow in yeast form (black yeasts). Similarly to the fissitunicate ascomycetes, the black yeasts are polyphyletic as well, and occur within the Chaetothyriales, Dothideales and Pleosporales. Calcium regulates in vitro dimorphism in chromoblastomycotic fungi [251]. Basic morphological differences are associated with differences in thallus structure and maturation [252], which explains why anamorphs of Chaetothyriales are smaller and more homogeneously pigmented than those of Dothideales [246]. Whereas Capronia species, a teleomorphic genus of the Herpotrichiellaceae, were described from plants such as Ericaceae [253], most anamorphs, however, have been associated with a wide spectrum of human diseases. Chromoblastomycosis is a disease which is not found outside the Herpotrichiellaceae [246]. Untereiner and Malloch [187] discussed the patterns of substrate utilization within the Herpotrichiellaceae. Cladophialophora (C. arxii, C. bantiana, C. boppii, C. carrionii, C. devriesii), Exophiala (E. berg- eri, E. castellani, E. dermatitidis, E. jeanselmei, E. lecanii-corni, E. moniliae, E. pisciphila, E. salmonis, E. spinifera), Fonsecaea (F. compacta, F. pedrosoi),

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 234 Phaeoannellomyces (P. elegans), Phialophora (P. bubaki, P. macrospora, P. repens, P. richardsiae, P. verrucosa), Ramichloridium (R. mackenziei, R. schulzeri), Rhinocladiella (R. aquaspersa, R. atrovirens), and Sarcinomyces (S. phaeomuriformis) are important human pathogenic genera and species within the Herpotrichiellaceae [75, 80, 246]. It was not possible to differentiate S. phaeomuriformis from E. dermatitidis using complete sequences of the 18S rDNA. Within the Herpotrichiellaceae, however, none of these genera appeared phylogenetically homogeneous based on complete 18S rDNA sequences [246]. The genus Sarcinomyces is polyphyletic, S. crustaceus and S. petricola have to be excluded from the Herpotrichellaceae [89]. The genus Wangiella was not accepted by Haase et al. [246]. It is considered as a synonym of Exophiala. Cladophialophora modesta takes a somewhat external position with respect to the Herpotrichiellaceae and comes close to the Chaetothyriaceae. This is remarkable, since the species was isolated from mycosis in the brain of a human patient [254].

Eurotiales Benny and Kimbrough [255] redefined the classical morphological class of the Plectomycetes with emphasis on centrum development and mode of discharge of the asci, and recognized six orders (Ascosphaerales, Elaphomycetales, Eurotiales, Microascales, Onygenales and Ophiostomatales). Molecular data, however, suggest that only the Ascosphaerales, Elaphomycetales, Eurotiales and Onygenales can be accepted within the subclass Plectomycetidae (fig. 3). Presently, it is not clear whether the Ascosphaerales which lack ascocarps and the hypogeous Elaphomycetales can be accepted as distinct orders or families within the Eurotiales (fig. 3) [63, 228, 256]. Eriksson and Winka [228] suggest five families within the Eurotiales (Ascosphaeraceae, Elaphomycetaceae, Eremascaceae, Monascaceae and Trichocomaceae) based on molecular characterization. The plectomycete family Trichocomaceae within the Eurotiales includes cleistothecial teleomorphic genera which are associated with economically and medically important anamorphs, such as Penicillium, Geosmithia, Merimblia, Aspergillus, Peacilomyces and related genera [257, 258]. The teleomorphic genera associated with a Penicillium anamorph are Talaromyces, Hamigera, Eupenicillium, Trichocoma, Penicilliopsis and Chromocleista [14, and literature cited therein]. Berbee et al. [259] and Sugiyama [14] gave a good overview of molecular phylogenetic studies in the Trichocomaceae. According to these studies, the genus Penicillium is not monophyletic; one group diverged first within the Trichoco- maceae cluster and contains different Talaromyces species with the Penicillium- producing Talaromyces flavus and the Geosmithia-producing T. bacillisporus. The second group consists of the Penicillium-producing Eupenicillium javanicum, the Aspergillus-producing Eurotium rubrum and Neosartorya fischeri as well as the Basipetospora-producing Monascus purpureus.

Systematics of the Ascomycota and Basidiomycota 235 Emericella (Aspergillus) nidulans is a remarkable fungus which lacks synap- tic meiosis. Prillinger [41, 83] considers this fungus besides S. pombe important for a polyphyletic evolution of meiosis within the Eumycota or Mycobionta [260, 261]. Within the genus Aspergillus especially three species are of clinical importance A. flavus, A. fumigatus and A. terreus (fig. 3 ) [262]. A. flavus is one of the main agents of human allergic bronchial aspergillosis. The species also occurs in the external ear and may be involved in otitis [263]. It is a common agent of mycotic sinusitis [80]. Systemic infections occur in leukemic patients [264]. Together with A. parasiticus, A. flavus is well known for the production of the mycotoxin aflatoxin [63]. A. fumigatus is the main agent of aspergillosis in immunocompromised patients. It causes a typical inhalation mycosis, whereby colonization and invasion are commonly accompanied by allergic reactions. De Hoog and Guarro [80] gave a good overview of the clinical importance of A. fumigatus. This species has a natural habitat in rotten plant material at higher temperatures. It is especially common in air during biological waste treatment and compost formation [265]. A. terreus causes allergic or invasive bronchopulmonary aspergillosis [80]. De Hoog and Guarro [80] discuss the clinical importance of 29 additional Aspergillus species (A. alliaceus, A. caesiellus, A. candidus, A. carneus, A. clavato-nanicus, A. clavatus, A. conicus, A. deflectus, A. janus, A. japonicus, A. niger, A. ochraceus, A. oryzae, A. restrictus, A. sclerotiorum, A. sydowii, A. tamarii, A. ustus, A. versicolor). Some of them have teleomorphs in Eurotium (E. amstelodami, E. chevalieri, E. herbariorum, E. repens), Emericella (E. nidulans, E. quadrilineata, E. unguis), and Fennellia (F. flavipes, F. nivea). Neosartorya spinosa is a rare opportunistic human pathogen with an Aspergillus anamorph. Cases of pulmonary infection and endocarditis have been reported [80]. Within the genus Penicillium only P. marneffei, a member of the subgenus Biverticillium, is a true and common pathogenic fungus. P. marneffei occurs naturally in bamboo rats in Southeast Asia. It is the third most common cause of disseminated opportunistic infection in patients with AIDS in parts of Southeast Asia. The species is unique among the genus Penicillium in being dimorphic and forming a unicellular yeast stage that reproduces by planate division (fission) in tissues [75, 80]. The mycosis is acquired by inhalation and is mostly fatal when untreated. Ten additional Penicillium (P. chrysogenum, P.citrinum, P. commune, P. decumbens, P. expansum, P. griseofulvum, P. purpurogenum, P. rugulosum, P. spinulosum, P. verruculosum) species are known as rare opportunistic pathogenic fungi [80]. Within the genus Paecilomyces seven species (P. crustaceus, P. fumosoroseus, P. javanicus, P. lilacinus, P. marquandii, P. variotii, P. viridis) are rare opportunistic fungi pathogenic in humans [80]. The genus Paecilomyces has teleomorphs in Byssochlamys, Talaromyces, and Thermoascus. Paecilomyces variotii (fig. 3) causes pneumonia [266], sphenoid sinusitis [267], soft tissue infection of the

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 236 heel [268] and cutaneous hyalohyphomycosis [269] in humans. Further human infections are discussed by De Hoog and Guarro [80]. P. tenuipes, a parasitic fungus of moth larvae and pupae can be excluded from the genus Paecilomyces based on 18S rDNA sequencing [270]. The data suggest P. tenuipes may be the anamorph of an entomogenous fungus of the genus Cordyceps (Hypocreales). Eremomyces langeronii with its anamorph Arthrographis kalrae is a cleistothecial species whose inclusion in the Eurotiales has to be confirmed by ribosomal DNA sequence information. The species causes an onychomycosis and is mainly isolated from human skin and nails [80, 271].

Onygenales Eriksson and Winka [228] suggest three families within the Onygenales (fig. 8; Arthrodermataceae, Gymnoascaceae and Onygenaceae). The Arthroder- mataceae and Onygenaceae harbor two important groups of human pathogenic fungi: the dermatophytes and the dimorphic systemic fungi. Dermatophytes which were traditionally classified within the Hyphomycetes produce thallic, one-celled microconidia in addition to multicelled macroconidia. Teleomorphs of dermatophytes belong to the genus Arthroderma (e.g. Arthroderma simii is the teleomorph of Trichophyton simii; Arthrodermataceae) [75, 80]. They have spherical evanescent asci containing 8 ascospores; the ascoma wall is often a loose network of hyphae with complicated branching and ornamentation [80, 272]. Dermatophytes are keratinophilic fungi which are capable of invading the keratinous tissues of living mammals. They are grouped into three categories on the basis of host preference and natural habitat. Anthropophilic species almost exclusively infect humans, rarely animals. Zoophilic species are essentially pathogens of nonhuman mammals or birds, although animal to human transmission is not uncommon. Geophilic species are soil-associated organisms, and soil per se or soil-borne keratinous debris is a source of infection for humans as well as other animals. Epidermophyton floccosum, Microsporum audouinii, M. ferrugineum, Trichophyton concentricum, T. gourrilii, T. kanei, T. megninii, T. metagrophytes, T. raubitschekii, T. rubrum, T. schoenleinii, T. soudanense, T. tonsurans, T. violaceum and T. yaoundei are the most important anthropophilic species. M. canis, M. equinum, M. gallinae, T. equinum, T. simii and T. verrucosum are zoophilic species and M. nanum, M. persicolor, M. praecox, M. vanbreuseghemii and T. terrestre are geophilic species. The pathogenicity of these species is extensively discussed in De Hoog and Guarro [80]. The dimorphic systemic fungi are phylogenetically closely related to the dermatophytes and can be included in the family Onygenaceae (fig. 3, 8) [135, 273, 274]. Five clearly different genera can be distinguished: Blastomyces, Coccidioides, Emmonsia, Histoplasma and Paracoccidioides. Each comprises only a very few species and all are pathogenic. Species of Blastomyces

Systematics of the Ascomycota and Basidiomycota 237 8 Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 238 and Histoplasma have a teleomorph in Ajellomyces; for the other genera no teleomorph is known so far. The natural habitat of all species are warm-blooded animals. Humans are infected by inhalation of dry propagules or by trauma. In healthy persons, symptoms are mild and mostly heal spontaneously [80]. Blastomyces dermatitidis is the agent of a chronic, granulomatous blastomycosis of the skin, mostly originating as a pulmonary infection or possibly also from trauma. Coccidioides immitis causes a coccidioidomycosis. Emmonsia parva is the agent of a adiaspiromycosis. Adiaspores are liberated conidia, which, after inhalation, enlarge in the alveoli of the host [80, 275]. Histoplasma capsulatum causes histoplasmosis. The species is the agent of an intracellular mycosis of the monocyte-macrophage system. Budding yeast cells are produced within phagocytosing cells. Paracoccidioides brasiliensis causes a paracoccidioidomycosis. The species is responsible for a systemic, chronic disease. It may cause painful, erosive stomatitis with loss of teeth, frequently associated with swollen lymph nodes. The fungus is abundantly present with yeast cells in pus and tissues. All species show a temperature-dependent yeast-hypha dimorphism. In the environment, they all produce a filamentous mycelial form at room temperature; at 37 °C they reproduce as yeasts in the tissues. Chrysosporium zonatum was detected recently as the agent of a disseminated infection in a patient with chronic granulomatous disease. C. zonatum is the anamorph of the heterothallic ascomycete Uncinocarpus orissi [276]. Although a dimorphism is absent, this species is phylogenetically closely related to Coccidioides immitis [277]. It produces abundant arthroconidia and degrades cellulose as well as keratin [277]. Aphanoascus fulvescens (Onygenaceae), Arachnomyces nodososetosus (anamorph: Onychocola canadensis; Gymnoascaceae), Gymnoascus dankaliensis, Gymnascella hyalinospora (Gymnoascaceae), Myxotrichum deflexum, and Neoarachnotheca keratinophilum (anamorph: Myriodontium keratinophilum; Onygenaceae) are five rare opportunistic clinical fungi which belong to the Onygenales [80, 278].

Hypocreales The Hypocreales are pyrenomycetous ascomycetes with unitunicate asci produced within fleshy, lightly or brightly colored, typically ostiolate perithecial ascocarps [142]. However, as already mentioned above, they include

Fig. 8. Phylogenetic tree of pathogenic yeasts and fungi based on the partial sequences of the 18S rRNA gene. Alignment, distance matrix and calculation of phylogenetic distances were made by means of different programs as described in the legend of figure 1. Approximately 400 bp long DNA fragments were compared corresponding to the position 582–1,006 bp in Saccharomyces cerevisiae.

Systematics of the Ascomycota and Basidiomycota 239 some cleistothecial species within the Bionectriaceae too [142]. Eriksson and Winka [228] and Rossman et al. [279] accept five families within the Hypocreales (Bionectriaceae, Clavicipitaceae, Hypocreaceae, Nectriaceae, Niessliaceae). Fusarium solani and its teleomorph Nectria haematococca is a common clinical species causing keratitis, endophthalmitis and disseminated and cutaneous infections (fig. 3) [80]. It is also known as an allergenic fungus [81]. Besides Cladosporium cladosporioides, C. sphaerospermum and Alternaria alternata, species of Fusarium are the most common allergenic fungi in Canada [280]. F. aquaeductuum, F. chlamydosporum, F. dimerum, F. incarnatum, F. oxysporum, F. proliferatum, F. sacchari, F. tabacinum, and F. verticillioides are rare opportunistic Fusarium species [80]. Based on ribosomal DNA sequencing, F. dimerum makes the genus Fusarium polyphyletic [139]. Genotypic identification methods are necessary to identify Fusarium species unequivocally [281]. Cylindrocarpon is an additional anamorph of Nectria species which is phylogenetically closely related to Fusarium [139]. C. destruc- tans (teleomorph: Nectria radicicola) and C. lichenicola are two rare opportunistic clinical fungi within the Hypocreales. No sequence data are available for C. cyanescens recovered from a human mycetoma [80]. Species of the genus Trichoderma (e.g. T. viride) are potentially pathogenic, toxigenic, and implicated in allergy or hypersensitivity pneumonitis [80, 282]. Teleomorphs are known in different Hypocrea species [80, 283]. Trichoderma viride, T. longibrachiatum, T. pseudokoningii, T. koningii and T. harzianum were reported in recent years to occur in humans [262]. As in the case of Fusarium genotypic identification methods are necessary to identify these species unequivocally [284, 285, Kubicek, pers. commun.]. Based on ribosomal DNA sequencing, Acremonium was shown to be a highly polyphyletic genus affiliated to at least three ascomycetous orders [143]. Teleomorphs of Acremonium are found in several genera of the Euascomycetes (Emericellopsis, Hapsidospora, Nectria, Nectriella, Neocosmospora, Pronectria and Thielavia). A larger number of species including the type species A. alter- natum and the human pathogenic A. kiliense exhibit affinities to the Hypocreales. A. kiliense has been described to cause ulcerative, nodulous hyalohyphomycosis, mycetomes and keratitis [80]. In A. strictum, a rare opportunistic human pathogen, phylogenetically closely related to A. kiliense, we have recently detected a yeast stage. A. alabamense is the known anamorph of Thielavia ter- restris. It is a rare opportunistic fungus pathogenic in humans and phylogenetically related to the Sordariales [80, 143]. No molecular data are available for some additional more or less rare opportunistic Acremonium species (A. blochii, A. curvulum, A. falciforme, A. hyalinulum, A. potronii, A. recifei, A. roseogri- seum) pathogenic in humans [80].

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 240 Conidia germinates on host cuticle Hypha penetrates host

New infection

Red colouring due to oosporein production

Saprophytic growth New sporulation on Blastospores proliferation in the soil? mummified larva white grub dies

Fig. 9. Life cycle of the entomopathogenic fungus Beauveria brongniartii.

Stachybotrys chartarum is an anamorphic soil and indoor air toxigenic fungus which especially degrades cellulose. It has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio (USA), area [286]. A teleomorph, Melanomma pomiformis, is known only in S. albipes. It is included in the Niessliaceae of the Hypocreales based on morphology. Beauveria bassiana and Metarhizium anisopliae are anamorphic soil fungi well known as insect pathogens. Both genera have attracted a great deal of attention because of their biological control potential [63]. Beauveria and Metarhizium both produce mycotoxins, and the destruxins, a group of secondary metabolites produced by M. anisopliae, are considered an important new generation of insecticides [287]. Although the proteinaceous insect cuticle is an effective barrier to many fungi, insect pathogens, including Beauveria and Metarhizium, have a series of extracellular proteolytic enzymes that degrade native insect cuticle [288]. Whereas B. bassiana and M. anisopliae are polyphage and attack a wide host range, B. brongniartii acts specifically against the cockchafer (Melolontha melolontha) and is used as a biological control agent (fig. 9). After the fungus has penetrated the cuticle and reached the hemocoel, yeast-like blastospores are produced, most probably to overcome the host

Systematics of the Ascomycota and Basidiomycota 241 defense system. After the death of the host the filamentous form will be expressed again. This dimorphism is similar to that of some human pathogens (e.g. Histoplasma capsulatum, Sporothrix schenckii). In addition, B. bassiana and M. anisopliae cause rare infections in humans and have been identified as agents of keratitis. Evidence that B. bassiana is an invasive human pathogen is doubtful because in the only reported case, the isolated mould was described as having greenish colonies and had microscopic features inconsistent with those of B. bassiana [75]. rDNA analyses place these anamorphic fungi among the Hypocreales [142, 289]. Verticillium is a further heterogeneous anamorph genus of many species which are pathogenic in insects and plants. Although Messner et al. [290] suggested a relationship between the common plant pathogen V. dahliae and the Hypocreales, partial sequences of the 28S rDNA [142] and a more comprehensive phylogenetic tree of complete 18S rDNA sequences (fig. 3) exclude V. dahliae from the Hypocreales. In contrast, the entomopathogenous V. lecanii clusters within the Hypocreales [142].

Ophiostomatales This order is usually characterized by perithecial ascocarps, but with cleistothecia in one genus (Europhium) [291, 292] and evanescent asci. A yeast stage is known in many species [148, 293]. The qualitative and quantitative monosaccharide patterns of purified yeast cell walls resemble those of Protomyces and Taphrina species containing rhamnose [148]. Based on cell wall sugars, sensitivity to cycloheximide, rDNA sequencing (fig. 3) and some additional characteristics [63], species of Ophiostoma are phylogenetically distinct from morphologically similar Ceratocystis (Microascales; fig. 3) species [294, 295]. Genotypic methods are important to distinguish species unequivocally [148, 293]. Many species are associated with scolytid and platypodid bark beetles in woody tissues where they occur as saprophytic blue stain fungi. O. novo-ulmi is a highly virulent plant pathogenic fungus causing Dutch elm disease [63]. Ophiostoma stenoceras causes onychomycoses in humans [296]. Sporothrix schenckii (fig. 3) is an anamorphic species which is the agent of human sporotrichosis; in addition it is known as an allergenic fungus [297]. Characteristic lesions at regional lymph nodes or localized cutaneous infection are common [80]. Species with a Sporothrix anamorph are also known within the Hemiascomycetes (Stephanoascales: Stephanoascus ciferrii) and Basidiomycota (Cerinostereus cyanescens).

Phyllachorales The characteristics of the perithecial order Phyllachorales are not clear-cut and await further molecular characterization and investigation of additional

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 242 species. The order is accepted based on Alexopoulos et al. [63] and Eriksson and Winka [228] for Glomerella, Phyllachora and Polystigma species. Glomerella cingulata, more often encountered as the anamorph Colletotrichum gloeosporioides, has been reported as a parasite of over 100 angiosperms. Sutton [298] provided descriptions for almost 40 species of Colletotrichum; however, morphological criteria are of little use when information on a plant pathogenic isolate is needed. Colletotrichum coccodes, C. dematium and C. gloeosporioides are known as rare opportunistic fungi causing keratitis in humans [80].

Sordariales The perithecial order Sordariales is less important from the economic point of view; however, it harbors genera well known from experimental mycology (Neurospora, Podospora, Sordaria; fig. 3). Neurospora sitophila (Sordariaceae), the red bread mould, is known to infest bakeries and cause considerable contamination. In culture, the fungus literally lifts the lid of petri dishes, and contaminates by rapid growth and the production of enormous numbers of pinkish air-dispersed conidia. N. sitophila is also known as an allergenic fungus [297]. Species of the Chaetomiaceae differ from Sordariaceae by their usually globose or ovoid asci that lack an apical ring and deliquesce within the perithecium or cleistothecium. In addition, the best-known species have conspicu- ous hyphal appendages on the ascocarp surface. Chaetomium atrobrunneum, C. funicola and C. globosum are known as rare opportunistic human pathogens [80]. Members of the Chaetomiaceae are cellulolytic and occur naturally on paper, tapestries, and cotton fabrics, sometimes causing considerable damage. C. globosum is also known as an allergenic fungus [297]. Corynascus heterothallicus, with its anamorph Myceliophthora ther- mophila, is another species of the Chaetomiaceae. This fungus was recovered from a disseminated infection in a leukemic patient [80]. The genus Phaeoacremonium was introduced by Crous et al. [299] to distinguish Acremonium species with pigmented vegetative hyphae and conidiophores. P. parasiticum, originally described as Phialophora parasitica, is known as the agent of phaeohyphomycoses or mycetomes [80]. Partial sequences of the 26S rDNA of P. parasiticum corroborate the exclusion of the genus Phialophora [300]. De Hoog [pers. commun.] suggests a relationship with Sordariales. Lecythophthora hoffmannii, L. mutabilis, Phialemonium curvatum and P. obovatum are rare opportunistic clinical fungi for which morphological and molecular data suggest an affinity to Sordariales (Coniochaetaceae) [80, Prillinger and Lopandic unpubl. obs.].

Systematics of the Ascomycota and Basidiomycota 243 Microascales Members of this order are characterized by a lack of stromata, perithecia in most species, but some possess cleistothecia. Previously, Microascaceae were placed among plectomycetes by some mycologists because of the mature condition of evanescent, scattered asci [255]. More recently, Barr [301] placed the family among the pyrenomycetes. The work of Berbee and Taylor [242, 302] and Spatafora and Blackwell [295] using DNA analysis has clearly placed the group within the perithecial ascomycetes (Pyrenomycetidae, fig. 3). The conidia of Microascaceae are blastic, and conidiogenous cell proliferation is percurrent or sympodial. Several anamorphs include Scopulariopsis, Scedosporium and Wardomyces. Pseudallescheria boydii is a cleistothecial species which is frequently encountered as a saprophyte in soil, manure and polluted water. The species is reported worldwide as the agent of white grain mycetomes. In addition, the fungus causes systemic infections in immunocompromised hosts or occurs in the respiratory tract where it triggers allergic reactions, sinusitis, pneumonia or systemic pseudallescheriasis [80]. Species causing onychomycosis include Scopulariopsis brevicaulis, by far the most important as both a pathogen and a regular contaminant, S. candida, Microascus cirrosus (fig. 3) and M. cinereus [75, 80]. S. brumptii is increasingly found as a pulmonary invader in patients with impaired cellular immunity [80]. S. acremonium, S. asperula, S. flava, S. fusca and S. koningii are known as rare opportunistic pathogenic fungi in humans [80]. Microascus manginii has been reported in several cases of onychomycosis [80]. Scedosporium prolificans has been frequently isolated from subcutaneous lesions; in addition, it was recovered from a fatal case of endocarditis [80]. Ceratocystis fimbriata (fig. 3) is an aggressive primary pathogen with a worldwide distribution that causes diseases in a wide range of plants (sweet potato, rubber, coffee, quaking aspen, prune, apricot) [303]. Its long-necked perithecia are morphologically closely similar to those of Ophiostoma species [63]. In contrast to Ophiostoma, no yeast stage is known for Ceratocystis species.

Dothideales Nannfeldt [244] first segregated the classical Loculoascomycetes (which he called ascoloculares) from the other filamentous ascomycetes. While the other filamentous ascomycetes usually have thin-walled asci with a single functional wall layer, the Loculoascomycetes have thick-walled asci with two sep- arable wall layers (fissitunicate) [63]. Luttrell [304] established the subclass Loculoascomycetes to grant formal taxonomic status to Nannfeldt’s group. He placed all other filamentous ascomycetes among the Euascomycetes. Barr [253] accepted the Loculoascomycetes as a class and presented a highly structured, hierachical view of its taxonomic subdivisions. Based on rDNA

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 244 sequencing, Berbee [247] and Sterflinger et al. [89] showed that the Loculoas- comycetes are not monophyletic and can be separated into three distinct orders: the Chaetothyriales, the Dothideales and the Pleosporales. Although the jack-in-the-box-type ascus is a good marker for large, monophyletic loculoascomycete orders (fig. 3), it must have evolved at least twice or been lost at least once [247]. Whereas species of the genus Cladosporium pathogenic in humans show a phylogenetic relationship to the Chaetothyriales (Herpotrichellaceae) and were therefore assigned to the genus Cladophialophora by Masclaux et al. [300], endophytic, mycoparasitic, plant pathogenic, and saprophytic species of Cladosporium can be included in the Dothideales (fig. 8). Cladosporium herbarum was proven to represent the anamorph of Mycosphaerella tassiana [300]. C. cladosporioides and C. sphaerospermum belong to the most common allergenic fungi in Canada [282]. Mycosphaerella tassiana, C. herbarum, C. macrocarpum and C. cladosporioides had the same partial 26S rRNA sequence. C. sphaerospermum was found to have 14 base differences [300]. C. cladosporoides and C. herbarum were commonly found as endophytes of grapevine [305]. Hortaea werneckii is a dimorphic black yeast which exclusively causes tinea nigra palmaris on one or both hands or on the sole. It is restricted to tropical, subtropical and mediterranean areas [80]. H. werneckii is halotolerant having its natural habitat in salty environments [306]. Complete sequences of the 18S rDNA (fig. 3) [89] as well as partial sequences of the 26S rDNA suggest a relationship of H. werneckii with the Dothideales. Aureobasidium pullulans, a common dimorphic endophyte of grapevine [305] and saprophyte on plant leaves, occurs in addition as an allergenic fungus [297, 307] and as a rare opportunistic pathogen in humans, where it caused keratitis, pulmonary infection, systemic infections, cutaneous infection, peritonitis, and invasive mycosis in an AIDS patient [80]. Complete 18S as well as partial 26S rDNA sequences corroborate a relationship of Aureobasidium species with Dothideales (fig. 3) [16, 89, 300]. Hormonema dematioides is a very similar dimorphic fungus, but can be differentiated from A. pullulans by the absence of synchronous conidiation, by different physiological profiles and genotypic approaches like RAPD-PCR [151]. It is also occasionally pathogenic in humans [262]. Madurella grisea and M. mycetomi are the main agents of human black grain eumycetoma [80]. Presently no molecular data are available which corroborate an affinity to Dothideales. Nattrassia mangiferae (synanamorph: Scytalidium dimidiatum) is known as a plant pathogen but is also commonly reported from human superficial infections in subtropical and tropical countries. In humans it causes extensive hyper- keratosis with scaling of the skin of the extremities, as well as onychomycoses

Systematics of the Ascomycota and Basidiomycota 245 [308]. Scytalidium hyalinum probably comprises a hyaline mutant of S. dimidiatum causing similar clinical symptoms [80, 308]. Lasiodiplodia theobromae is a rare opportunistic human pathogen causing keratitis, onycho- and phaeohyphomycosis [80]. Botryosphaeria rhodina is known as a teleomorph of L. theobromae. B. rhodina clusters with B. ribis (fig. 3.) in the Dothideales [247]. Neotestudina rosatii and Piedraia hortai are two human pathogenic species where morphological data suggest a classification among the Dothide- ales. N. rosatii occurs in soil of tropical countries and causes mycetoma in humans [80]. P. hortai is the agent of black piedra on scalp hair [80]. Cenococcum geophilum is a cosmopolitan fungus and is the most widespread ectomycorrhizal fungus. It has an extremely broad host range and habitat. Parsimony and distance analyses positioned C. geophilum as a basal, intermediate lineage between the two Loculoascomycete orders, the Pleosporales and the Dothideales [309]. At least four independent lineages of mycorrhizal fungi were identified among the Ascomycota examined (compare Elaphomyces, Tuber; fig. 3).

Pleosporales The Pleosporales form a monophyletic group with high bootstrap support (fig. 3) [247]. In phylogenetic trees based on complete 18S rDNA sequences, they commonly appear as a sister group of the Dothideales (fig. 3). This, however, may change if partial sequences are used (fig. 8). Alternaria alternata is a saprophyte on dead plant material and a common endophyte [305], but it may also cause skin lesions in humans after a trauma. Rare cases of systemic infection, onychomycosis and endophthalmitis following eye surgery were reported [80]. Besides Cladosporium and Fusarium species A. alternata is the most common allergenic fungus in Canada [282]. Breitenbach et al. [310] reported nucleotide sequences of three cDNA clones coding for 53-, 22-, and 11-kD allergens. All of these allergens are homologous to Cladosporium herbarum allergens and are abundant, cytosolic housekeeping proteins. Teleomorphs of Alternaria species are known in Pleospora and Lewia [311]; they can be included in the Pleosporales (fig. 3; Pleosporaceae) [311] based on complete sequences of 18S rDNA (fig. 3). Eriksson and Winka [228] accept four families within the Pleosporales (Leptosphaeriaceae, Lophiostomataceae, Melanommataceae and Pleosporaceae). A. chlamydospora, A. dianthicola, A. infectoria, and A. tenuissima are additional Alternaria species of clinical importance [80]. Ulocladium morphologically closely resembles Alternaria. Ulocladium chartarum was recovered from extensive infection of subcutaneous human tissues and is considered to be allergenic [80]. Further molecular data are necessary to clarify the phylogenetic relationship between Alternaria and Ulocladium.

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 246 Species of Phoma are known to have teleomorphs in Leptosphaeria (e.g. Phoma lingam) or chlamydospores with muriform septation, resembling the conidia of Alternaria (e.g. Phoma glomerata). Partial sequences of the 18S rDNA of Phoma species exhibit a relationship to the teleomorph Cucurbitaria which can be included in the Pleosporales (Leptosphaeriaceae; fig. 3) [16]. Further sequence data are necessary to clarify whether the genus Phoma is heterogeneous. Phoma cava, P. cruris-hominis, P. eupyrena, P. glomerata, P. herbarum, P. minutella, P. minutispora, P. oculo-hominis and P. sorghina are known as rare opportunistic pathogens in humans [80]. P. betae is considered to be an allergenic fungus. Epicoccum nigrum is an allergenic fungus; molecular data suggest that it is a synanamorph of Phoma epicoccina. It was isolated from Vienna monument surfaces [Sterflinger and Prillinger, unpubl. obs.]. Lehrer et al. [322] reported that E. nigrum is one of the most important sources of spores isolated outdoors. It is a frequent sensitizing agent in the Scandinavian population [307]. Leptosphaeria senegalensis and L. thompkinsii cause mycetoma in Africa [80]. Coniothyrium fuckelii is the anamorph of L. coniothyrium. It is known as a plant pathogen especially on Rosaceae and from human infections [80]. The genus Cochliobolus harbors many fungi pathogenic in plants and humans [80, 311]. Based on ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences, Berbee et al. [311] support a suggestion by Tsuda and Ueyama [312] to separate the genus into two closely related genera: Cochliobolus and Pseudocochliobolus. Additional molecular sequences, especially, of species pathogenic in humans, however, are necessary to corroborate this concept. Species of Cochliobolus exhibit a Bipolaris anamorph. For species of Pseudocochliobolus, two anamorphs (Bipolaris, Curvularia) are known [311]. Although De Hoog and Guarro [80] used the anamorph genus Drechslera for different Cochliobolus species (D. hawaiensis, D. spicifera and D. australiensis), Berbee et al. [311] restricted Drechslera to Pyrenophora species, which again cluster within the Pleosporales (fig. 3). There is some molecular evidence [311] that highly virulent species pathogenic in plants are common within Cochliobolus (C. carbonum and C. victoriae) and species pathogenic in humans cluster within Pseudocochliobolus (P. australiensis, P. geniculatus, P. hawaiiensis, P. lunatus and P. verruculosus). Presently, no molecular data are available for C. spiciferus (anamorph: Bipolaris spicifera) an agent of human and animal sinusitis and cutaneous phaeohyphomycoses [80]. Bipolaris hawaiiensis is a common saprophyte on plant material. Sinusitis and pulmonary and cerebral mycosis have been reported [80]. B. australiensis and B. papendorfii are rare opportunistic fungi pathogenic in humans [80]. Curvularia geniculata was found after traumatic implantation in the eye and as the agent of allergic sinusitis [80]. Curvularia lunata is a ubiquitous saprophyte on plant material. It is known from allergic bronchopulmonary disease

Systematics of the Ascomycota and Basidiomycota 247 [313], sinusitis, keratitis, phaeohyphomycosis, onychomycosis or mycetomas [80]. C. brachyspora, C. clavata, C. pallescens, C. senegalensis and C. verrucu- losa are considered as rare opportunistic human pathogenic fungi [80]. Exserohilum is an anamorphic genus with teleomorphs in Setosphaeria [311]. The genus comprises plant pathogenic species, mainly occurring on grasses. Human mycoses mostly concern cases of sinusitis, partially with cerebral involvement. E. mcginnisii, E. longirostratum and E. rostratum are known from human infections [80]. Helminthosporium halodes is the anamorph of Setosphaeria rostrata. It causes allergic bronchopulmonary mycosis [314]. Stemphylium is the anamorph which belongs to the teleomorphic Pleospora species (fig. 3) [311]. S. macrosporoideum is recovered from a mixed infection in antromycosis [315]. Together with Alternaria species, Stemphylium botryosum is considered as one of the most important mold allergens in the United States [307]. There is some molecular evidence that the genus Pleospora (P. herbarum, P. rudis) [89] is heterogeneous. Botryomyces caespitosus is a rare opportunistic fungus which causes a chromoblastomycosis-like subcutaneous infection after trauma in humans [316].

Leotiales The Leotiales are the largest of the orders of inoperculate discomycetes. They are characterized by either cup- or disk-shaped apothecia and asci that have more or less thickened apices. Although the apothecial discomycetes are not a monophyletic group based on molecular characters [243], there is support that the Leotiales, Lecanorales and Pezizales are monophyletic orders within the apothecial Euascomycetes. Many members of the Leotiales live saprobically on the soil, some are parasitic on plants and belong to the worst fungal pathogens. Among these are Monilinia fructicola, the cause of brown rot of stone fruits and Sclerotinia sclerotiorum, the cause of lettuce drop and other vegetable diseases (fig. 3). Moserella radicicola produces hypogeous apothecia [317]. Botrytis cinerea and its teleomorph Botryotinia fuckeliana is known as gray mould of lettuce and strawberries. The fungus is also known from dessert wines, grapes are left in vineyards purposely to become infected with B. cinerea, the ‘noble rot’, which enhances the sweetness of the grapes. In addition, B. cinerea is a common allergenic fungus [297]. Based on partial sequences of the 18S rDNA, B. cinerea can be assigned to the Sclerotiniaceae [318]. Ochroconis gallopava is a common agent of encephalitis in poultry. In addition, it is known to cause subcutaneous phaeohyphomycosis and endocarditis in humans [80]. O. constricta, O. humicola and O. tshawytschae are known as rare opportunistic pathogenic fungi [80]. Presently, no molecular data are available which corroborate the inclusion of the genus Ochroconis in the Leotiales.

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 248 Interestingly, the cleistothecial powdery mildew Blumeria graminis (fig. 3; Erysiphales) clusters with Sclerotinia sclerotiorum [319]. Additional complete 18S rDNA sequences of powdery mildew fungi are necessary to clarify the phylogenetic relationship between Erisyphales and Leotiales. It is, however, remarkable that many representatives of the Leotiales have asci with thick- walled apices [Oberwinkler, pers. commun.].

Pezizales Pezizales are a large monophyletic order that contains the species commonly called operculate discomycetes as well as derived hypogeous forms that have evanescent asci with ascospores spread by mycophagy. O’Donnell et al. [320] have recently investigated phylogenetic relationships among ascomycetous truffels and the true and false morels. The results indicate that the hypogeous ascomycetous truffle and truffel-like taxa studied represent at least five independent lineages within the Pezizales. The data also suggest that several epigeous and most hypogeous taxa have been misplaced taxonomically. There is strong support for a Tuberaceae-Helvellaceae clade which is a monophyletic sister group of a Morchellaceae-Discinaceae clade (fig. 3). Members of the Morchellaceae, the common morel (Morchella esculenta) and Tuberaceae (Tuber melanosporum) are well known as food. There are only few poisonous species (e.g. Gyromitra esculenta) of clinical importance. Recently, a very rare and interesting fungus, Calyptrozyma arxii, was isolated from a case of esophagitis. This fungus is typically dimorphic, initially developing as a yeast and then producing hyphae. Both sexual and vegetative reproductive structures are produced in the same thallus. Sexual reproduction is represented by naked asci, and both blastic and thallic conidia are produced [321]. Recent 5.8S rRNA sequence analysis suggests a close relationship with Pezizales [262].

Phylogenetic Relationships of the Basidiomycota and Their Anamorphs

Within the Basidiomycota neither the dipartite classical system of Patouillard [323], which was recently improved by Oberwinkler [186, 324, 325], nor the tripatite systems, which are based on the morphology of the basidium according to Lowy [326], Talbot [327] and Donk [328, 329], could be corroborated by biochemical and molecular data. In contrast to Lowy, Talbot and Donk, Patouillard and Oberwinkler considered the mode of basidiospore germination important for the definition of different classes of the Basidiomycota.

Systematics of the Ascomycota and Basidiomycota 249 a b

Fig. 10. A yeast stage in the agarics Collybia cirrata and C. tuberosa. a C. tuberosa on decaying agaric; arrow indicates purple sclerotium. b C. cirrata: yeasts develop from basidiospores on an acidic (pH 4.5) malt extract medium. From Prillinger et al.: Expel Mycol 1993;17:26. With permission from Academic Press.

Dörfler [330] and Prillinger et al. [215–217, 234] detected three phylogenetically distinct cell wall sugar patterns which correlate perfectly with the complete 18S rDNA sequences of Swann and Taylor [10, 331], Schweigkofler and Prillinger [16] or the partial 26S rDNA sequences of Begerow et al. [22], leading to three distinct classes: Urediniomycetes, Ustilaginomycetes and Hymenomycetes. Yeasts or yeast stages are known in all three classes of the Basidiomycota (fig. 4). In addition, a yeast stage could be genotypically demonstrated in three species of the agaric Collybia (fig. 10) [150]. Meanwhile yeast stages are also known from symbiontic agarics (Agaricales) of leaf-cutting ants (e.g. different Cyphomyrmex species) [332]. A yeast/hypha dimorphism is considered of major importance in the evolution of Zygomycota, Ascomycota and Basidiomycota. With respect to basidia, Prillinger et al. [216] introduced two different types of holobasidia. Whereas simple holobasidia are known in all three classes of the Basidiomycota, complex holobasidia are reported from the Hymeno- mycetes only [215, 216, 234]. Complex holobasidia can be traced back by partially septate basidia to tremelloid basidia (e.g. Syzygospora) [216, 333]. Yeast cells were considered to be the most primitive basidia within the

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 250 Basidiomycota [215–217]. Similarly, the forcibly discharged basidiospore was already established in yeast cells of the ‘Sporobolomyces’ type. Ballistospores stimulate a faster spreading of yeast colonies on solid habitats and may help to escape or establish parasitic interactions. In Melanotaenium endogenum, all stages of transition from a mitotic ballistospore to the meiosporangium of smuts can be observed [334]. Similarly, in the Zygomycota the structures of the mitosporangium were used to disperse meiospores [63]. The new concept of the Urediniomycetes, Ustilaginomycetes and Hymenomycetes can also be corroborated by ultrastructural data on septa and spindle pole body morphology [325, 335, 336 and references cited therein; also see Hibbett and Thorn, 337]. In tables 1–3 we have compiled some recent data of our cell wall sugar analyses [129]. The Urediniomycetes are characterized by dominant amounts of mannose and commonly the presence of fucose (table 1). Rhamnose, which is characteristic of the Protomyces type [239], may occur sporadically together with fucose (table 1). The absence of fucose in the Nahoidea/Sakaguchia clade (table 1, fig. 4) needs further corroboration. Rhodotorula yarrowii is so far the only species among the 64 investigated strains where xylose was detected as well (table 1) [338]. In Mixia osmundae we cannot corroborate the data of Sjamsuridzal et al. [182], who detected rhamnose instead of fucose. Based on dominant amounts of mannose and the presence of fucose (table 1), M. osmundae unequivocally belongs to the Microbotryum-type [217, 234], but its position is uncertain. Morphological as well as ultrastructural data [235] suggest that M. osmundae is a rather primitive representative of the Uredinio- mycetes. Sadebeck [339] already observed a Mixia-like exogenization of spore formation in Taphrina carpini. The qualitative and quantitative monosaccharide patterns of purified yeast cell walls of Sterigmatomyces halophilus closely resemble those of some Saccharomyces species (table 1). They can be distinguished from those of the Saccharomyces type by the absence of glucose fermentation and a positive diazonium blue B and urease test (Microbotryum type) [217, 234]. The glucose mannose cell wall sugar pattern also appears in extremely derived Hymenomycetes (table 3), but is different from that of the Urediniomycetes, as it exhibits very high amounts of glucose (e.g. symbiotic yeast isolates from the leaf-cutting ants Cyphomyrmex). It seems that the glucose mannose pattern is the alpha and omega in the evolution of the Basidiomycota. Glucose is low at the beginning and high at the end of evolution. Among different representatives of the Ustilagionomycetes, the qualitative and quantitative cell wall sugar patterns are commonly very homogeneous (table 2) [215, 217, 340]. Glucose dominates over mannose, and galactose is commonly present. Among the Hymenomycetes we have included 10 filamentous species representing

Systematics of the Ascomycota and Basidiomycota 251 Table 1. Cell wall sugars of yeasts or dimorphic Basidiomycota which belong to the Urediniomycetes

Species Strain Cell wall sugars

GLC MAN GAL XYL FUC RHA

Urediniomycetes Nahoidea/Sakaguchia clade Erythrobasidium hasegawianum Y HB 62T 28 70 2 – – – Occultifur externus Y HB 262T 23 74 3 – – – Rhodotorula minuta Y HB 477T 25 71 4 – – – Agaricostilbales Bensingtonia yuccicola Y HB 419T 27 70 1 – 1 – Kurtzmanomyces nectairei Y HB 106T 12 87 0.4 – – – K. tardus Y HB 268T 17 81 2 – – – Sporobolomyces ruber Y HB 317T 14 75 6 – 5 – S. xanthus Y HB 316T 12 83 2 – 3 – Sterigmatomyces elviae Y HB 104T 19 80 1 – – – St. halophilus Y HB 100T 39 61 – – – – Microbotryomycetidae Bensingtonia intermedia Y HB 417T 15 80 3 – 2 – Microbotryum salviae Y HB 315 10 59 7 – 24 – M. succisae Y HB 313 10 45 12 – 33 – Rhodotorula auriculariae Y HB 413T 16 79 1 – 1 3 R. glutinis var. glutinis Y HB 476T 14 86 – – tr – R. glutinis var. glutinis Y HB 462 20 78 1 – 1 – Uncertain position Rhodotorula yarrowii Y HB 705T 20 58 2 5 2 13 Kriegeria eriophori Y HB 263 22 49 16 – 9 4 Mixia osmundae Y HB 748 33 55 10 – 2 – M. osmundae Y HB 749 35 55 7 – 2 – Platygloea disciformis Y HB 267 23 73 1 – 1 –

Y=Yeast stage.

the Agaricales (Asterophora, Clitocybe, Mycena, Pholiota), Hymenochaetales (Phellinus), Polyporales (Fomes, Laetiporus, Phanerochaete, Polyporus) and Schizophyllales (Schizophyllum; fig. 4, table 3). Additional species can be found in O’Brien and Ralph [341], Prillinger et al. [216, 217] and Messner et al. [340]. Dominant amounts of glucose and the presence of xylose are well- established characters of the Hymenomycetes. Presently, only Coniophora puteana (Boletales) [341] deviates from this pattern, showing glucose, mannose and galactose as cell wall monosaccharides.

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 252 Table 2. Cell wall sugars of dimorphic Basidiomycota which belong to the Ustilaginomycetes

Species Strain Cell wall sugars

GLC MAN GAL XYL FUC RHA

Ustilaginomycetes Doassansiales Nannfeldtiomyces sparganii Y HB 304 87 2 11 – – – Rhamphospora nymphaeae Y HB 405 85 0.7 14 – – – R. nymphaeae Y HB 406 86 0.4 14 – – – Exobasidiales Kordyana cubensis Y HB 16 69 19 12 – – – Ustilaginales Schizonella sp. nov. Y HB 3 88 5 7 – – – S. cocconii Y HB 112 93 2 5 – – – S. melanogramma Y HB 195 87 4 9 – – – Sporisorium ophiuri Y HB 19 96 2 2 – – – Sp. reilianum Y HB 303 96 1 3 – – – Ustilago avenae Y HB 302 71 27 2 – – – U. bullata Y HB 296 72 25 3 – – – U. hordei Y HB 297 84 13 3 – – –

Y ϭ Yeast stage.

Urediniomycetes

The Urediniomycetes presently comprise four distinct clades: the Agaricostilbales, the Microbotryales, the Uredinales and the Cystobasidiales. The Septobasidiales appear as a distinct order within the Uredinales clade. All four clades are well supported by bootstrap factors close to 100% (fig. 4). Swann et al. [11] recently introduced the subclass of the Microbotryomycetidae which includes Heterogastridium pycnidiodeum (fig. 4) and Kriegeria eriophori (table 1). In contrast to Swann and Taylor [10] and in agreement with Sjamsuridzal et al. [342], the Cystobasidiales (Nahoidea/Sakaguchia clade) occupy a basal position in our phylogenetic tree (fig. 4). Other characters that generally corroborate the Urediniomycetes are the 5S rRNA secondary structure of type A [236], plate-like spindle pole bodies, the cell wall monosaccharide pattern (table 1) and simple septa tapering towards the pore or poreless septa [22, 217, 234, 335, 336, 340, 343–348]. The members of this class are predominantly dimorphic except the Uredinales

Systematics of the Ascomycota and Basidiomycota 253 Table 3. Cell wall sugars of dimorphic and filamentous Basidiomycota which belong to the Hymenomycetes

Species Strain Cell wall sugars

GLC MAN GAL XYL FUC RHA

Hymenomycetes Hymenomycetidae Polyporus ciliatus M MB 15 83 12 – 5 1 – Phanerochaete chrysosporium M MB 57 73 13 4 8 2 – Fomes fomentarius M MB 79 77 13 2 8 – – Laetiporus sulfureus M MB 80 89 6 – 3 0.7 0.5 Phellinus torulosus M MB 125 87 8 1 3 0.5 0.5 Schizophyllum commune M MB 148 91 9 – – – – Asterophora parasitica M MB 29 95 4 – 1 – Clitocybe phyllophila M MB 95 88 6 2 2 1 1 Collybia tuberosa Y Pr 1986/93 97 2 – 1 – – C. cookei Y Pr 1987/146 94 3 – 2 1 – Mycena gallopus M MB 140 91 4 – 3 2 – Pholliota squarrosa M MB 111 88 6 2 4 – – Lepiotaceae Y.i. Cyphomyrmex minutus HB 667 98 2 – – – – Y.i. C. salvini HB 666 97 3 – – – – Tremellomycetidae Asterotremella lycoperdoides Y HB 81T 83 10 – 6 – – A. parasitica Y HB 82T 85 10 1 4 – – A. humicola Y CBS 571T 84 10 – 3 – – Atractogloea stillata Y HB 260 91 7 – 3 – – Captotrema sp. Y HB 259 57 13 4 25 – 1 Christiansenia pallida Y HB 91 62 22 – 12 4 – Filobasidiella neoformans Y HB 420T 82 14 0.5 3 – –

Y.i. ϭ Yeast isolate; M ϭ mycelium; Y ϭ yeast.

[5,000–8,000 species; Oberwinkler, pers. commun.] and produce rhodotorulic acid as siderochrome [349]. The formation of secondary spores (fig. 2) is common in rust fungi [324]. Teliospores, thick-walled resting spores of the rust and smut fungi in which karyogamy occurs, are present or absent. Based on M. osmundae, the Urediniomycetes can be traced back to the Upper Carboniferous by coevolution and a fossil records of the Osmundaceae (Discopteris, Todeopteris and Kidstonia) [229].

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 254 Cystobasidiales Oberwinkler [pers. commun.] suggested to include basidiomycetes which cluster within the Nahoidea/Sakaguchia or Erythrobasidium clade in the order Cystobasidiales (fig. 4) [Bauer et al., in preparation, table 1]. Sampaio et al. [350] call the teleomorphic nature of Erythrobasidium hasegawianum [351] into question and consider Erythrobasidium as an anamorphic genus. They interpret the proposed holobasidium as a conidiogenic structure. E. hasegawianum and Sporobolomyces elongatus are phylogenetically closely related and so far the only basidiomycetous yeasts that possess a hydrogenated ubiquinone

Q-10 (H2) [24, 351]. This ubiquinone system is common to many filamentous Euascomycetes [352]. Sexual cycles within the Cystobasidiales differ. Sakaguchia (Rhodosporidium) dacryoideum produces teliospores that germinate to a 2- to 4-celled phragmobasidium with repetitively budding basidiospores. Occultifur externus is a non-teliospore-forming fungus with a yeast stage that produces auricularioid basidia with ballistospores and physiologically resembles the anamorphic yeast Rhodotrula minuta [350]. O. internus is an interesting mycoparasite within fruiting bodies of the Dacrymycetales [353]. R. minuta is a species of clinical importance within the Cystobasidiales. R. minuta was isolated from bronchoscopy specimens and from postoperative endophthalmitis [80]. The genus Rhodotorula, however, is heterogeneous and has representative species at least in three different clades of the Urediniomycetes (Microbotryum, Sporidiobolus and Erythrobasidium clades) [24]. Genotypic methods are necessary to identify a Rhodotorula species unequivocally [208].

Microbotryales The order Microbotryales was proposed by Bauer et al. [335] as ‘phyto- parasitic members of the Basidiomycota having transversely septate basidia with multiple production of sessile basidiospores and only intercellular hyphae’. The order especially comprises phragmobasidial smut fungi from dicotyledonous host plants (Liroa, Microbotryum, Sphacelotheca and Zundeliomyces) and phragmobasidial species from monocotyledonous host plants (Aurantiosporium, Bauerago, Fulvisporium and Ustilentyloma) [24, 217, 234, 335]. In contrast to the phragmobasidiate members of the Ustilaginomycetes, they do not produce intracellular hyphae or haustoria [335]. Parasitic species occurring on dicots have poreless septa, whereas parasitic species on monocots have simple septal pores [335]. Yeast stages from phyto- parasitic smut fungi of the Microbotryales commonly have a narrower oxydative degradation spectrum of carbon and nitrogen compounds than yeasts of smuts from the Ustilaginales [149, 234]. Celerin et al. [354] noted a specific glycosylation pattern that is unique to fimbriae from the Microbotryales. Based on partial sequences of the 26S rDNA, Colacogloea peniophorae and Kriegeria

Systematics of the Ascomycota and Basidiomycota 255 eriophori as well as Heterogastridium pycnidioideum, which were traditionally placed among the Platygloeales and Heterogastridiales [355], respectively, and the basidiomycetous yeasts Leucosporidium, Mastigobasidium, Rhodotorula, Bensingtonia, Sporobolomyces and Reniforma are closely related to the Micro- botryales [24]. Presently, it is not clear whether the red pigmented teliosporic yeasts Rhodosporidium and Sporidiobolus and their related anamorphs in the genera Rhodotorula and Sporobolomyces form a distinct clade as suggested by partial sequences of the 26S rDNA (Sporidiobolus clade) [24] or whether they have to be included in the Microbotryales, which is supported by complete sequences of the 18S rRNA gene (fig. 4). Reniforma strues is unique among the basidiomycetous yeasts due to the presence of kidney-shaped vegetative cells and the presence of ubiquinone Q-7 [24]. Rhodotorula glutinis is a common saprophyte on various substrates; disseminated cases in patients with compromised innate immunity do occur. Sepsis due to the use of indwelling catheters has repeatedly been reported. The species is also implicated in cases of keratitis and dacryoadenitis [80]. R. mucilaginosa is an additional species of clinical importance [262]. Genotypic methods are necessary to identify these species unequivocally [208]. Sporidiobolus johnsonii and its anamorph Sporobolomyces holsaticus are implicated in dermatitis [356]. The species is homothallic and forms simple holobasidia with diploid basidiospores. Reduction division occurs with the formation of dikaryotic hyphae with clamp connections [202].

Uredinales Fungi belonging to the order Uredinales (fig. 4) commonly are referred to as rust fungi. Approximately 5,000 species belonging to about 140–150 different genera which occur on spikemosses (lycophytes), ferns, gymnosperms and angiosperms are known. They are especially important from the economic point of view. All are parasitic on plants, often causing great losses to many cultivated crops [63]. Based on traditional morphological systematics, the Uredinales were considered for a long time as primitive Basidiomycota [357 and also see ref. 51, 83 and the literature cited therein]. New molecular data, however, suggest that the Uredinales include many modern and advanced taxa without yeast/hypha dimorphism, probably arising from simple-septate primitive auricularioid parasites on mosses and ferns (fig. 4) [342] within the Urediniomycetes. Deml et al. [358, 359] and Prillinger et al. [216] isolated many different tremelloid yeasts specifically from spermogonia of different rust fungi. To the best of our knowledge, there are no fungi of clinical importance within the Uredinales.

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 256 Ustilaginomycetes

Although this clade comprises the least species of the three major lineages of the Basidiomycota based on complete sequences of the 18S rRNA gene (fig. 4), sufficient data from 5S rRNA [236], cell wall sugars [215, 217, 340] (table 2), ultrastructure [335, 336], 18S rRNA gene (fig. 4) and partial sequences of the large subunit rDNA (D1/D2 domain; [22, 24, 360] corroborate the class of the Ustilaginomycetes. The species are usually dimorphic. Besides some species pathogenic in humans (Malasseziales), almost all members of the Ustilaginomycetes are known as phytoparasites. They share type B of 5S rRNA secondary structure with the Hymenomycetes [236]. In addition, there are some similarities in spindle pole body morphology with the Hymenomycetes (fig. 6e, f) [336]. Figure 6e shows a characteristic hemispherical spindle pole body of U. maydis. The spindle pole bodies of the Ustilaginomycetes roughly resemble in their form those of the Hymenomycetes, but they have in common with those of the Urediniomycetes an internal layering. Based on ultrastructural data and partial sequences of the 26S rDNA there are three major lineages which can be considered as subclasses: Entorrhizomycetidae, Exobasidio- mycetidae and Ustilaginomycetidae [22, 335]. Teliospores are present or absent. Presently, the Entorrhizomycetidae comprise one order: Entorrhizales, the Ustilaginomycetidae two orders: Urocystales and Ustilaginales, and the Exobasidiomycetidae seven orders: Malasseziales, Georgefischerales, Tilletiales, Entylomatales, Microstromatales, Doassansiales, and Exobasidiales [22, 335, 361]. Complete 18S rDNA sequences are urgently needed to corroborate these orders further. Except for five species, the host range of the Ustilaginomycetes is restricted to angiosperms. The lycophytes with species of Selaginella represent the most primitive host group of the Ustilaginomycetes. A new genus Melaniella with two species M. oreophila and M. selaginellae was recently described [362]. The origin of lycophytes can be dated back to the Lower Devonian, about 400 million years ago [363]. According to Bauer et al. [362], there are two possibilities to explain the occurrence of Ustilaginomycetes on lycophytes: either it is the result of a jump, or the Ustilaginomycetes arose as parasites of at least early vascular plants and the parasitic smut fungi of Selaginella species represent extant representatives of this ancestral ustilaginomycetous group. Because the systematically different hosts of the Doassansiales are all paludal or aquatic plants, Bauer et al. [362] believe the Doassansiales represent a good example for evolution bound to an ecosystem, but not to a specific host relationship and favor the jump hypothesis. Exoteliospora (Ustilago) osmundae is another representative of the Ustilaginomycetes which occur on primitive leptosporangiate ferns [364]. As already mentioned for Mixia osmundae (Urediniomycetes), the

Systematics of the Ascomycota and Basidiomycota 257 Osmundaceae can be traced back to the Upper Carboniferous. Earlier known reports of leptosporangiate ferns are in the Lower Carboniferous. Subsequent major filicalean radiations during the early Mesozoic resulted in several families with extant representatives, but it was obviously not until the Upper Cretaceous that much of the extant diversity has appeared [365]. Based on coevolution, the Urediniomycetes and Ustilagionmycetes appeared as distinct lineages at least since the Carboniferous. Species allergenic and pathogenic in humans are known among four orders within the Ustilaginomycetes; representatives of the Malasseziales are of special importance.

Malasseziales The order was introduced by Moore [366]. A separate position of the different Malassezia species is in agreement with morphological, physiological, ultrastructural, and molecular characteristics [24, 361]. The cell wall of the Malassezia yeasts is thick, multilamellate and reveals a unique substructure with a helicoidal band that corresponds to a helicoidal evagination of the plasma membrane [367–369]. The lipophilic, dimorphic yeast genus Malassezia is presently divided into seven different species (M. furfur, M. pachydermatis, M. sympodialis, M. globosa, M. obtusa, M. restricta, M. slooffiae) [369–371]. Of these, six are strictly lipid dependent with a requirement for long-chain fatty acid supplementation in the medium to ensure their growth, and one (M. pachydermatis) for which the lipids present in rich media such as Sabouraud glucose agar are sufficient. Whereas M. pachydermatis is isolated only rarely from humans, the six lipid-dependent species are commonly found on human skin. The yeasts are members of the normal human cutaneous flora and can be cultured from almost all body areas. Under the influence of predisposing factors they become pathogenic and are associated with several diseases such as pityriasis versicolor, Malassezia folliculits, seborrheic dermatitis, some forms of atopic dermatitis, some forms of confluent and reticulate papillomatosis, and even systemic infection [80, 371]. M. pachydermatis has occasionally been implicated in cases of systemic infection. A rapid and inexpensive identification method has been established to separate the seven species routinely on the basis of morphological and physiological differences [371]. The new taxonomy must be applied to epidemiological surveys before one can conclude whether all seven Malassezia species have clinical importance.

Georgefischerales Species of Tilletiopsis are frequently found as epiphytes on leaves, especially those infected with powdery mildew or rust fungi [372, 373].

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 258 T. pallescens is a potential biological control agent of Spaerotheca fulginea, a powdery mildew which is common on cucumber leaves [373]. Among the Ustilaginomycetes only the Melanotaeniaceae of the Ustilaginomycetidae, the Georgfischeriaceae and Tilletiariaceae of the Georgefischerales and the Entylomatales form Tilletiopsis-like pseudohyphal anamorphs that produce ballistoconidia. This indicates that the genus Tilletiopsis is highly polyphyletic [24, 361, 374]. T. albescens and T. pallescens are members of the Exobasidio- mycetidae, but they cannot be assigned to any known order. T. minor is a member of the Georgefischerales which caused subcutaneous infection in an immunosuppressed patient [361, 375]. Based on the qualitative and quantitative monosaccharide pattern of purified cell walls, T. minor fits well in the Ustilaginomycetes [215].

Microstromatales The genus Cerinosterus was erected by Moore [376] for basidiomycetous hyphomycetes previously classified in Sporothrix. This transfer was necessary because the type species of Sporothrix, S. schenckii, had been shown to be a member of the euascomycetes order Ophiostomatales [377]. Two species were accepted in Cerinostereus, with C. luteoalba as the type of the genus [376]. This species is the anamorph of Ditiola pezizaeformis (Femsjonia luteoalba), which belongs to the Dacrymycetales. The second species, C. cyanescens was excluded from the Dacrymycetales; 25S rRNA sequencing suggests an affinity to Microstromatales [378]. Cells which produce conidia sympodially on denticles, singly or in short chains, therefore can be found at least in four unrelated orders of the Ascomycota and Basidiomycota: Stephanoascales (Hemiascomycetes), Ophiostomatales (Euascomycetes), Microstromatales (Ustilaginomycetes) and Dacrymycetales (Hymenomycetes). C. cyanescens has occasionally been isolated from human skin and blood and was involved in nosocomial infections in patients with pneumonia [80]. Experimental inoculation showed low virulence [379]. Based on partial ribosomal DNA sequencing, Rhodotorula bacarum, R. hinnulea and R. phylloplana can be excluded from the genus Rhodotorula. Together with Sympodiomycopsis paphiopedili (fig. 4), they can be included in the Microstromatales [24]. Presently there are no morphological and ultrastructural data to circumscribe this order [Oberwinkler, pers. commun.].

Ustilaginales The genus Ustilago is representative of this order. Ustilago species are commonly dimorphic phragmobasidial smut fungi parasitic on seeds and flowers of many cereals and grasses [380]. Smut spores may be inhaled and therefore may be isolated from sputum specimens. Based on partial sequences

Systematics of the Ascomycota and Basidiomycota 259 of the LSU rDNA, the genus Ustilago is heterogeneous. U. maydis appears to be distinct from U. hordei, the type species of the genus [22, 24]. Rhodotorula acheniorum is a candidate for reclassification. It clusters with the Ustilaginales based on partial sequences of the LSU rDNA [24]. The species of Pseudozyma are anamorphs of the Ustilaginales [360, 361]. Ustilago tritici is an allergenic species of the Ustilaginales [297].

Hymenomycetes

Representatives of the Hymenomycetes have dolipore septa with various types of pore caps (without, cupulate, continuous or perforate) [325, 336], their spindle pole bodies have a true globular morphology lacking obvious internal differentiation [336]. In addition, they have a type B secondary structure of the 5S rRNA (cluster 5) [236]. A cell wall sugar analysis commonly exhibits dominant amounts of glucose and the presence of xylose (table 3) [216, 217, 340]. Based on sequence analysis of the small subunit rDNA, Swann and Taylor [10] recommended two subclasses among the Hymenomycetes: (1) the Tremel- lomycetidae, which are commonly dimorphic and often yeast-like, and (2) the Hymenomycetidae, containing the non-yeast-like macrofungi including the mushrooms and puffballs. Nuclear (nuc) rDNA studies all support or are consistent with the view that the classical Homobasidiomycetes plus Auriculariales s. str., Tualsnellales, and Ceratobasidiales form a monophyletic group (fig. 4) [337], and that the Tremellomycetidae and Dacrymycetales are at the base of the hymenomycete lineage. Based on complete sequences of the 18S rDNA [381], the Tremellomycetidae comprise two distinct orders: the Tremellales and the Cystofilobasidiales (fig. 4). This is in contrast to partial sequences of the 26S rDNA where Fell et al. [24] accept the Tremellales, the Trichosporonales, the Filobasidiales and the Cystofilobasidiales (fig. 4). Presently, however, no ultrastructural data give support to the Filobasidiales and Trichosporonales [Bauer, unpubl. results]. Takashima and Nakase [381] subdivide the Tremellales into six different lineages: the Filobasidium lineage, the Trichosporon lineage, the Cryptococcus luteolus lineage, the Fillobasidiella lineage, the Bulleromyces lineage and the Sterigmatosporidium lineage. Additional complete 18S rDNA sequences especially of Tremella species are necessary to clarify systematic relationships within the Tremellales unequivocally. The Hymenomycetidae include the Auriculariales as well as all groups of the classical Homobasidiomycetes. Cantharellus tubaeformis, however, causes some problems (fig. 4). In a phylogram which is based on combined nuc-ssu and mt-ssu rDNA sequences, C. tubaeformis clusters together with Hydnum repandum and Clavulina cristata (Cantharelloid clade) inside the

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 260 Hymenomycetidae [337, 382, 383]. In a phylogenetic tree based on nuc-ssu rDNA, only C. tubaeformis remains distinct (fig. 4). As discussed by Pine et al. [383], the rate of sequence evolution of nuc-ssu rDNA appears to be greater in the cantharelloid clade than in most other Homobasidiomycetes, which makes it very likely that long branch attraction is responsible for the placement of C. tubaeformis near the base of the tree. We have observed a similar phenomenon with complete 18S rDNA sequences from Tulasnella pruinosa and T. violea. We therefore have excluded these sequences from our phylogram presented in figure 4 Pine et al. [383] present cytological (stichic nuclear division in basidia) [384] and molecular (combined data for mit-ssu rDNA and nuc-ssu rDNA) evidence that the Cantharellales (Cantharellus, Craterellus, Clavulina, Hydnum, Stichoclavaria) are a well-established clade within the Hymenomycetidae. Presently, it is not clear whether the Dacrymycetales should be included in the Hymenomycetidae as suggested by Swann and Taylor [10] or not (fig. 4). The classical Homobasidiomycetes were tentatively separated into eight major clades by Hibbett et al. [382] and Hibbett and Thorn [337] based on molecular data. In accordance with Oberwinkler [186], these clades commonly comprise resupinate, bracket-like, club-shaped or coralloid, pileate and gasteroid (secotioid gasteroid and hypogeous gasteroid) fruiting bodies with various types of hymenia (e.g. corticoid, toothed, poroid, agaricoid, gleba chambers). Similarly to Oberwinkler [186], we used distinct orders to circumscribe these clades: Agaricales, Boletales, Cantharellales, Gomphales, Hymenochaetales, Polyporales, Russulales, Thelephorales. Some of these orders have already been accepted by Gäumann [385] (Agaricales, Cantharellales, Dacrymycetales, Polyporales) and Kreisel [386] within his subclass Hymenomycetidae (Agaricales, Boletales, Cantharellales, Dacrymycetales, Polyporales, Russulales) but the genera included generally differ remarkably if molecular characters are used in addition. In contrast to Hibbett et al. [382], the Schizophyllales appear as an additional distinct group in our phylogram (fig. 4). The oldest unambiguous homobasidiomycete fossils are from the mid- Cretaceous, but indirect evidence, including molecular clock dating, suggests that the higher Hymenomycetes may have existed by the late Triassic (ca. 200 million years) [30, 387].

Tremellales Cryptococcus neoformans is a zoopathogenic basidiomycetous yeast which has a teleomorph in Filobasidiella (F. neoformans) but is usually encountered in the imperfect state [388, 389]. Fell et al. [390] proposed to conserve Cryptococcus with C. neoformans (Sanfelice) Vuillemin as the neotype species. Cryptococcosis is an inhalation mycosis, occurring nearly exclusively in

Systematics of the Ascomycota and Basidiomycota 261 immunocompromised patients. Pleural effusion is a first indicator of AIDS. Dissemination leads to chronic meningitis which is usually fatal when untreated [80]. Based on genetic recombination in the F1 generation C. neoformans consists of the following two varieties according to the current classification: C. neoformans var. neoformans, with serotypes A, D and C. neoformans var. gattii, with serotypes B, C. According to Boekhout et al. [389], the two varieties differ in karyotype, RAPD-PCR patterns, in a number of physiological characteristics, morphology of basidiospores, and in sensitivity to killer toxins of Cryptococcus laurentii. The two varieties also differ in geographic distribution and habitat. C. neoformans var. neoformans occurs worldwide and is frequently isolated from bird droppings. C. neoformans var. gattii is restricted to the tropics and the southern hemisphere; it is usually associated with Eucalyptus species [389]. Differentiation between the two varieties is usually performed on L-canavanine-glycine-bromthymol blue medium [391, 392] or by testing D-proline assimilation [393]. Boekhout et al. [389] considered the two varieties as distinct species, Filobasidiella neoformans and F. bacillispora. All species of Cryptococcus are nonfermentative aerobes exhibiting a positive urease test, a positive diazonium blue B test, and the presence of extracellular amyloid compounds. Cryptococcus differs from Trichosporon by the presence of capsules and the absence of arthroconidia. Genotypic methods are necessary to identify the species unequivocally [208]. C. laurentii is an additional species of clinical importance (pulmonary abscess) which belongs to the Tremellales [24, 80, 262, 381]. Based on partial 26S rDNA and complete 18S rDNA sequences, the genus Cryptococcus is polyphyletic and occurs in at least five different clades of the Tremellales, and within the Cystofilobasidiales [24, 381] (fig. 4). Filobasidium (Cryptococcus) uniguttulatum, Cryptococcus albidus, and C. ater are three species of clinical importance which belong to the Filobasidium lineage of the Tremellales [24, 262, 381] (fig. 4). The Filobasidium lineage may be considered as a distinct family (Filobasidiaceae) within the Tremellales. Although F. uniguttulatum has been isolated from human diseased nails or other clinical specimens; it has not been documented to cause invasive disease [394]. Cases of meningitis and pulmonary infections have been reported to be caused by C. albidus [80]. The type strain of C. ater was isolated from multiple ulcers on the leg of a young man [202]. Species of Trichosporon are characterized by the presence of arthroconidia, positive urease and diazonium blue B tests and the absence of extracellular amyloid compounds. The septa have dolipores, which may or may not have tubular/vesicular parenthesomes. Except for T. pullulans, which is a member of the Cystofilobasidiales (fig. 4), all species form a coherent clade within the Tremellales, which suggests a distinct family (Trichosporonaceae) [15, 24, 381,

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 262 395, 396]. Genotypic methods are essential to identify a Trichosporon species unequivocally [208]. There are six Trichosporon species of clinical importance (T. asahii, T. asteroides, T. cutaneum, T. inkin, T. mucoides, T. ovoides). T. beigelii is a synonym of T. ovoides [202]. Two strains of Fissuricella filamenta isolated from human skin showed DNA homology values around 85% with T. asteroides strains [395]. The groups of strains are thus genetically close, despite the fact that F. filamenta strains were entirely composed of meristematic cells, while such cells were absent from the strains of T. asteroides [202]. T. asahii, T. inkin and T. mucoides are regularly isolated from clinical specimens (white piedra), whereas the remaining species cause only occasional infections [80, 262]. Cryptococcus humicola was isolated from human skin [262]. It is related to the genus Trichosporon [24, 381, 397]. Based on 26S rDNA sequencing of the D1/D2 region, C. humicola differs only by 2 bp from two yeast isolates of the agarics Asterophora lycoperdoides and A. parasitica [151]. Prillinger et al. [190] considered these yeast isolates, together with the agarics from which they were isolated, as sexual symbionts and missing links in the evolution from mycoparasitism to sexuality (primary homothallism and heterothallism; fig. 5, 7). Based on nucleotide divergence of the complete 18S rDNA from C. neoformans, the type species of the genus Cryptococcus, we have placed the two yeast isolates from the agarics and C. humicola in the new genus Asterotremella (fig. 4) [Prillinger et al., in preparation].

Cantharellales The Cantharellales (fig. 4) include cantharelloid to agaricoid (Cantharellus, Craterellus), hydnoid (Hydnum), clavarioid to coralloid (Clavulina), clavarioid (Multiclavula), and corticioid (Botryobasidium) fungi [337]. A distinctive feature of the Cantharellales is the possession of ‘stichic’ basidia [383, 384]. Analyses of mt-rDNA sequences suggest that the classical heterobasidiomycete order Tulasnellales belongs in the cantharelloid clade [398]. Based on morphological and ultrastructural data, Oberwinkler [pers. commun.] has some doubts on this suggestion. Our alignments of complete 18S rDNA sequences suggests a higher evolution rate in two Tulasnella species (see above). Except for Botryobasidium, the species of this group have dolipores with perforate parenthosomes [325, 399, 400]. These data suggest that Botryobasidium occupies a systematic position at the base of this group. Most members of the Cantharel- lales are known or presumed to be mycorrhizal, but Multiclavula is a basidiolichen. Cantharellus cibarius and Craterellus cornucopioides are delicious edible mushrooms. Based on molecular data [383], there is no evidence for the ‘Clavaria theory’ of Corner [401]. Corner [401] treats the cantharelloid and clavarioid

Systematics of the Ascomycota and Basidiomycota 263 fungi as a basal paraphyletic group from which all other Homobasidiomycetes derived. Presently, the Cantharellales comprise about 170 described species of Hymenomycetes.

Gomphales This order originally was introduced by Jülich [402] based on comparative morphology and a dark green reaction of the fruiting body plectenchyma when treated with an aqueous solution of ferric sulfate; however, Jülich does not include gasteroid genera and families as suggested by Pine et al. [383]. The Gomphales (fig. 4) [337, 383, 398] include club-shaped fungi (Clavariadelphus), cantharelloid forms (Gomphus), coralloid fungi (Lentaria, Ramaria), hydnoid resupinate fungi (Kavinia), gilled mushrooms (Gloeocantharellus), and some Gasteromycetes (false truffels: Gauteria; earthstars: Geastrum; cannon-ball fungus: Sphaerobolus; stinkhorns: Clathraceae, Phallaceae). The corticioid fungus Ramaricium probably also belongs in this group [337]. The gomphoid-phalloid clade includes presently about 350 described species.

Thelephorales The thelephoroid clade (fig. 4) [337, 398] is a morphologically diverse group that includes corticioid fungi (Tomentella), clavarioid forms (Thelephora), and pileate fruiting bodies with poroid (Boletopsis), toothed (Hydnellum, Sarcodon), smooth to wrinkled or tuberculate (Thelephora), or lamellate hymenophores (Lenzitopsis). Oberwinkler [186] suggested that the agaricoid fungus Verrucospora is related to the Thelephorales based on spore morphology. Characters used by Donk [403] to support the Thelephoraceae include dark, ornamented spores with an angular outline, pigmentation of the fruiting body, and the presence of thelephoric acid [404]. Thelephoric acid, which is a terphenylquinone product of the shikimate-chorismate pathway is found in Bankera, Boletopsis, Hydnellum, Phellodon, Polyozellus, Pseudotomentella, Sarcodon and Thelephora. This is consistent with molecular characters, which strongly support monophyly of the Thelephorales. Nevertheless, thelephoric acid also occurs in Suillus and Rhizopogon (Boletales), Omphalotus and Lampteromyces (Agaricales) and Trametes (Polyporales) [337]. All members of this group are thought to be ectomycorrhizal or orchid symbionts [398]. The phylogenetic relationship to the Bankeraceae (Phellodon) awaits further molecular studies. So far only dolipores with perforate parenthesomes are known in the Thelephorales [399, 400]. The Thelephorales presently include about 240 described species of Hymenomycetes.

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 264 Polyporales The polyporoid clade (fig. 4) [337, 382] is primarily composed of corticioid fungi and polypores, but also includes the gilled mushrooms Lentinus, Panus and Faerberia as well as the ‘cauliflower fungus’ Sparassis. The brown rot root parasite Sparassis spathulata appears to be phylogenetically related to the brown rot polypores Laetiporus sulphureus, Phaeolus schweinitzii and Antrodia car- bonica [337]. The phylogenetic relationship of the brown rot genera Gloeophyllum and Neolentinus (Lentinus lepideus) to this clade (fig. 4) [337], is presently not clear, however. Although the polyporoid clade as a whole is weakly supported, there are four groups within the polyporoid clade that are strongly supported and have corroborating anatomical and physiological characters. They may be candidates for a new family delimitation (Fomitopsis-Daedalea Piptoporus: brown rot, unifactorial mating system; Fomes Polyporus-Lentinus- Ganoderma: white rot, bifactorial mating system; Bjerkandera-Phanerochaete- Ceriporia-Phlebia: white rot; Antrodia-Phaeolus-Laetiporus-Sparassis: brown rot) [405]. Dolipores with perforate parenthosomes are common in the Polyporales [406, 407]; Phanerochaete so far is the only genus having imperforate parenthesomes [400]. A perforate parenthesome, however, was found in Phanerochaete cremea [Bauer, unpubl. obs.]. Dimitic or trimitic hyphal systems and hyphal pegs, fascicles of sterile hyphae that emerge from the hymenium are common anatomical characters in the Polyporales [186]. Haploid apomixis was considered to be an extreme type of homothallic breeding systems [41, 55, 83, 94], and its occurrence in the Basidiomycota in nature was reviewed by Prillinger [93]. Using Polyporus ciliatus, we were able, after eight inbreeding generations, to obtain isogenic strains which develop fertile apomictic pileate fruiting bodies which could be distinguished from the dikaryotic fruiting bodies only by microscopical investigations [103]. After 2 years of apomictic propagation, a Phlebia-like resupinate mutant (corticioid morphology) appeared on a petri dish inoculated with one of these haploid- apomictic pileate strains (fig. 11a). The Phlebia-like mutant exhibited a fertile and predominantly bisporic apomictic hymenium, but it had still not lost its mating capacity. It was therefore easy to obtain the three additional mating types of the resupinate mutant by crossing with a compatible pileate strain (fig. 11a). P. ciliatus is a complex hetero-bifactorial white rot fungus (fig. 5). Compatible crosses of two resupinate strains yielded a morphologically similar Phlebia-like dikaryotic strain, which, in contrast to the haploid-apomictic strains, microscopically exhibited a clamped mycelium and predominantly four-spored basidia (fig. 11b) [94]. This result clearly confirmed the polygenic control of fruit body formation suggested by Prillinger and Six [103] and excluded the existence of a ‘fruiting initiation gene’ which was postulated by Esser et al. [408]. A genetic analysis of 200 haploid single-spore cultures of a

Systematics of the Ascomycota and Basidiomycota 265 a b

Fig. 11. A Phlebia-like resupinate monogenic mutant of Polyporus ciliatus. The resupinate mutant appeared in a culture of a haploid pileate fruiter strain after 2 years of apomictic propagation. a Different mating types of the resupinate mutant were obtained from a cross between the resupinate haploid mutant and a pileate haploid fruiter strain (bottom); from the dikaryon (top) it is obvious that the resupinate mutant behaves as a recessive gene. b A dikaryotic resupinate strain was obtained (top) in a cross of two compatible haploid resupinate strains. For additional information, see Prillinger [94] and text.

Table 4. A monogenic fertile resupinate mutant of Polyporus ciliatus

Dikaryotic cross Spores Haploid progeny

Resupinate ϫ haploid number germinated resupinate haploid fruiting bodies fruiting bodies

A1B1 ϫ A2B2 50 49 21 28 A1B2 ϫ A2B1 50 45 22 21 A2B1 ϫ A1B2 50 48 28 20 A2B2 ϫ A1B1 50 47 24 23 Total 200 189 95 92

cross of resupinate with pileate strains yielded a clear-cut 1:1 (95:92) segrega- tion pattern (table 4), indicating a typically monogenic character. In view of this monogenic difference between resupinate and pileate strains with a corticoid and poroid hymenium, respectively, these resupinate fruiting bodies can be interpreted as atavistic only [94]. Our data are in favor of a concept of Oberwinkler [186] which suggests that the different fruiting bodies of the classical Homobasidiomycetes have evolved repeatedly from morphologically simple resupinate ancestors. Different types of haploid fruiting bodies (resupinate,

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 266 clavarioid, coralloid, pileate) detected in single-spore cultures of P. ciliatus are a valuable tool to trace the phylogeny of the Polyporales (fig. 6a) [103]. Within Lentinus tigrinus (fig. 4) Rosinski and Robinson [409] detected complete intercompatibility between monokaryotic isolates of the pileate L. tigrinus and the gasteromycete Lentodium squamulosum. They accept Le. squamulosum as a recessive mutant of L. tigrinus and recognized it as a variety. The polyporoid clade contains roughly 1,350 described species of the Hymenomycetes, including about 90% of the morphologically described Polyporaceae and 25% of the Corticiaceae, as well as all Ganodermataceae and Sparassidaceae [337]. Ganoderma applanatum, G. lucidum and G. meredithae are known to cause allergy in humans [81]. Anamorphs of polypores and corticoid fungi are encountered with increasing frequency in clinical mycology. Identification to species has been problematic, especially in the case of haploid monokaryons [410]. Molecular techniques like ribosomal DNA sequencing may faciliate the identification of filamentous haploid Hymenomycetes in the future. Bjerkandera adusta is one of the most common Hymenomycetes isolated in North America from sputum, skin, urine and air [410]. Other Polyporales (Phlebia rufa, Phanerochaete chrysosporium, anamorph: Sporotrichum pruinosum) are rarely isolated from human sputum [410].

Hymenochaetales The order Hymenochaetales (fig. 4) was introduced by Oberwinkler [186] and corroborated by Hibbett and Thorn [337] by molecular data. The Hymenochaetales comprise the classical Hymenochaetaceae and some additional genera morphologically placed in the Corticiaceae (Basidioradulum, Hyphodontia) and Polyporaceae (Trichaptum, Oxyporus, Schizopora). Thus, the Hymenochaetales include resupinate and bracket-like poroid, toothed, and corticoid forms. In addition, the clavarioid Clavariachaete belongs to the Hymenochaetales, based on anatomical features [186]. Based on the following characters the Hymenochaetaceae have been considered monophyletic: clampless generative hyphae, fruiting bodies darkening in KOH, production of white rot, presence of thick-walled tapering cystidia (setae). In addition, most members of the Hymenochaetales have been shown to have imperforate parenthosomes (Basidioradulum, Hyphodontia, Inonotus, Phellinus, Schizopora, Trichaptum) [325, 411–413]. Coltricia is a remarkable exception having perforate parenthesomes and being the only ectomycorrhizal member of the Hymenochaetales [406]. In our phylogram (fig. 4), Schizopora paradoxa clusters outside the Hymenochaetales. In a phylogram of combined data for mit-ssu rDNA and nuc-ssu rDNA, S. paradoxa is within the Hymenochaetales

Systematics of the Ascomycota and Basidiomycota 267 [337, 382]. Presently, there are no morphological-anatomical data to include Basidioradulum, Hyphodontia, Oxyporus, Schizopora and Trichaptum in the Hymenochaetales [Oberwinkler, pers. commun.]. The Hymenochaetales presently include about 630 described species of Hymenomycetes.

Russulales Based on comparative anatomy and morphology, Oberwinkler [186] redefined the Russulales and discussed older concepts of this order. The russuloid clade has a remarkable diversity of fruiting body morphologies (fig. 4) [337, 382]. There are resupinate (Stereum), coralloid (Clavicorona), and pileate (Russula) forms with smooth (Stereum), toothed (Hericium), lamellate (Lentinellus, Russula), or poroid hymenophores (Bondarzewia). Gasteroid forms are also common (e.g. Martellia, Macowanites, Zelleromyces). The russuloid clade is also ecologically variable, having ectomycorrhizal (Lactarius, Russula), parasitic (Heterobasidion), saprophytic (Auriscalpium, Stereum) and possibly lichenized (Pleurogala igapoensis) [414] species. Heterobasidion annosum (fig. 4) is one of the most important root pathogens in European spruce forests [415]. Species of Hericeum occur as parasites (H. erinaceus) and saprophytes (H. clathroides) and cause a white rot. As discussed by Donk [403] and Oberwinkler [186], many members of the Russulales have spores with amyloid ornamentations and gloeoplerous cystidia or hyphae, but these characters are variable within the group. Hibbett and Thorn [337] estimate that the russuloid clade contains approximately 1,000 described species of Hymenomycetes, including roughly 20% of the morphologically described Corticiaceae. Lactarius deliciosus and Russula cyanoxantha are well known delicious, edible mushrooms.

Boletales The history and concept of the Boletales is extensively discussed in Bresinsky [64]. The delimitation of the order is based on morphological (spindle-shaped basidiospores), chemical (pigments: shikimate-chorismate pathway derivatives), wood decay (brown rot), specific mycoparasites (Sepedonium) and more recently and better reliable on molecular data [382, 398, 416–421]. Based on a sequence database of the mitochondrial large subunit rRNA gene, Bruns et al. [398] divided the Boletales into six groups. Complete 18S nuclear ribosomal DNA sequences and additional species may be useful to corroborate this groups at the family level: group 1 (Boletaceae): Boletus, Chamonixia, Gastroboletus, Leccinum, Strobilomyces, Tylopilus, Xerocomus; within group 1 the genera Boletus and Xerocomus appeared to be heterogeneous; group 2 (Paxillaceae): Chalciporus, Paxillus s. str., Paragyrodon; group 3 (Tapinellaceae): Hygrophoropsis, Serpula,

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 268 Tapinella; group 4 (Coniophoraceae): Coniophora; group 5 (Gyrodonaceae): Gyrodon, Gyroporus, Phaeogyroporus, Pisolithus; group 6 (Suillaceae): Alpova, Brauniellula, Chroogomphus, Gomphidius, Hymenogaster, Melanogaster, Rhizopogon. The bolete clade contains resupinate fungi (Coniophora, Serpula), cantharelloid forms (Hygrophoropsis), gilled mushrooms (Gomphidius, Paxillus, Tapinella), false truffels (Alpova, Hymenogaster, Melanogaster, Rhizopogon), secotioid fungi (Brauniellula, Chamonixia, Gastroboletus, Gastrosuillus), and puffballs (Pisolithus, Scleroderma), including the unusual stalked, gelatinous puffball Calostoma [337]. The shikimate-chorismate pathway produces a number of compounds that are characteristic of the Boletales (atrotomentin, pulvinic acid derivatives, cyclopentanoids, polyprenylquinones). These compounds have been found in diverse corticoid, resupinate, cantharelloid, lamellate, boletoid, and gasteroid taxa including Boletus, Chamonixia, Chroogomphus, Coniophora, Gomphidius, Gyrodon, Hygrophoropsis, Leucogyrophana, Paxillus, Phylloporus, Pisolithus, Rhizopogon, Scleroderma, Serpula, Suillus, and some others [422]. Atrotomentin, pulvinic acid derivatives, and cyclopentanoids have also been found in the ligni- colous white rot agarics Omphalotus and Lampteromyces [423]. However, analyses of rDNA sequences suggest that Omphalotus and Lampteromyces belong to a distinct family Omphalotaceae within the Agaricales [421]. In addition, atrotomentin and cyclopentanoids are found outside the Boletales in Albatrellus, a heterogeneous genus within the Polyporales and Russulales [337, 398] and Hydnellum (Thelephorales). These observations imply that the production of atrotomentin, pulvinic acid derivatives, and cyclopentanoids has evolved repeatedly. The Boletales presently include about 840 described species of Hymenomycetes. Many species of the Boletales are known as tasty (Boletus edulis, Xerocomus badius) and a few as poisonous (B. satanas, Paxillus involutus). P. involutus caused immunohemolytic anemia after repeated consumption of its carpophores. Bresinsky and Besl [424] present an extensive documentation of poisonous mushrooms and fungal intoxications. Jarosch and Bresinsky [425] investigated the problem of speciation and cryptic species in P. involutus. Boletus species and Serpula lacrymans are known as allergenic fungi [297, 426, this volume]. The brown-rot fungus S. lacrymans is primarily important in Europe, where the dreaded dry rot causes tremendous damage to wooden structural elements and floors in houses and other buildings.

Schizophyllales Different from the combined analysis of nuc-ssu and mt-ssu rDNA of Hibbett et al. [382] and Hibbett and Thorn [337] Fistulina hepatica and Schizophyllum commune form a distinct clade with high bootstrap support,

Systematics of the Ascomycota and Basidiomycota 269 closely related to the Agaricales in our phylograms of complete 18S nuc-DNA and partial sequences of the 26S rDNA (fig. 4) [Schweigkofler unpubl.]. We tentatively use the order Schizophyllales originally suggested by Nuss [427] based on comparative morphology for this clade. Additional sequences, especially from cyphelloid fungi may be useful to corroborate or reject this order. S. commune is probably the best-known basidiomycetous agent of infection. Reports involving the lung include fungus ball of the lung [428], a case of allergic bronchopulmonary mycosis in an otherwise healthy female [429] and repeated isolation of S. commune from the sputum of a patient with chronic lung disease [430]. Other reports of S. commune infections include cases of meningitis [431], sinusitis [432–434], ulcerative lesions of the hard palate [435], and possible onychomycosis [436] in both immunocompetent and immunosuppressed hosts. The presence of clamp connections on dikaryotic hyphae and the development of fruiting bodies in culture are primary characters which allow the identification of S. commune in human infections. The diagnostic problems presented by a monokaryotic nonclamped, nonfruiting isolate from a dense mass in the right upper lobe of the lung in a female with a past history of pulmonary tuberculosis and diabetes are described and discussed by Sigler et al. [428]. Genotypic identification methods like partial sequencing of ribosomal DNAs [16] are a promising tool to solve these problems in the future.

Agaricales The Agaricales sensu Fries have long been recognized as an artificial taxon, but the number of independent evolutionary lines of gilled mushrooms, and their order relationships, have remained controversial [186, 402, 437, 438]. In a molecular analysis Hibbett et al. [382] suggest that agaricoid forms evolved at least six times independently within the Hymenomycetes. Our present understanding of the phylogeny of the Hymenomycetes is only partially consistent with Corner’s [401] ‘Clavaria theory’; agarics are polyphyletic, but there is no indication that they have all derived from paraphyletic grades of club and coral fungi. The molecular data of Hibbett et al. [382] and Hibbett and Thorn [337] agree well with a phylogenetic concept of Oberwinkler [186] and genetic experiments (table 4, fig. 11) of Prillinger [94] which suggest that the agarics have evolved polyphyletically from morphologically simple corticioid or resupinate ancestors. The Agaricales contain the majority of the gilled mushrooms in the Agaricales sensu Singer [438]. The largest groups of gilled mushrooms outside the Agaricales are Lentinus (Polyporales), Lactarius, Russula (Russulales), Gomphidius, Paxillus, Tapinella (Boletales). Although the pigments of Lampteromyces, Omphalotus and Ripartites point to a Boletales affinity, these species were included in the Agaricales by Binder et al. [421] based on ribosomal

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 270 DNA sequencing. Pegler [439] restricted Lentinus to dimitic species, and therefore, transferred the monomitic shiitake fungus, traditionally known as Lentinus edodes, into Lentinula which is close to Collybia within the Agaricales [440, 441]. In addition, ribosomal DNA sequence information suggests that species of the clavarioid genera Clavaria, Clavulinopsis and Typhula belong to the Agaricales too [382, 383]. Whereas Hibbett and Thorn [337] present evidence to include at least some representatives of the Ceratobasidiales in the Agaricales, data from Lee and Jung [442] and our investigations (Rhizoctonia solani; fig. 4), however, are not in agreement with this suggestion and warrant further investigations. Classical Gasteromycetes that have been sampled in the Agaricales include bird’s nest fungi, puffballs and many secotioid fungi. Lycoperdon, Calvatia, and Tulostoma form a weakly supported monophyletic group within the Agaricales which is phylogenetically closely related to Lepiota procera [382]. The exact placement of the bird’s nest fungi (Nidulariaceae) is not resolved with confidence [382]. Further gasteroid species or genera which have been studied include Hydnangium (H. carneum, H. sublamellatum, H. microsporium), Podohydnangium sp. which is closely related to Laccaria (L. bicolor, L. oblon- gospora, L. laccata, L. trullisata, L. gomezii, L. vinaceobrunnea, L. glabripes, L. proximella), Montagnea arenaria and Podaxis pistillaris, which are closely related to Coprinus comatus (Coprinus appeared to be heterogeneous) and Leratia and Weraroa, which show affinities to Stropharia [422, 443, 444]. According to Hibbett and Thorn [337], the Agaricales contain approximately 7,400 species of gilled fungi and 1,025 species of the classical Aphyl- lophorales and Gasteromycetes. These species represent more than half of all known classical Homobasidiomycetes, including approximately 87% of all known gilled mushrooms [175]. Although symbionts of plants (mycorrhizae: Amanita, Cortinarius, Hebeloma, Inocybe) dominate, symbionts of animals (Amylostereum, Termitomyces), saprotrophs (Agaricus bisporus, Pleurotus ostreatus, Lentinula edodes), pathogens (Armillaria mellea, Crinipellis perniciosa, Mycena citricolor, Oudemansiella mucida, Pholiota aurivella), and mycoparasites (Asterophora lycoperdoides) can be found as well [337]. Agaricus bisporus, Calvatia cyathiformis, Coprinus comatus, C. quadrifidus, Lentinula edodes, Pleurotus ostreatus, Psilocybe cubensis are known allergenic fungi [81, 426, this volume]. Psilocybe mexicana and other related species have been referred to collectively as the ‘sacred or divine mushrooms’ or ‘teonanácatl’ used for centuries in certain religious rites of endemic peoples of Mexico. The halucinogenic compounds present in these fungi were first isolated and identified by Hofmann et al. [445] and named psilocin and psilocybin. The hallucinogenic properties of both compounds are similar to d-lysergic acid

Systematics of the Ascomycota and Basidiomycota 271 diethylamide (LSD). They are extensively discussed in Bresinsky and Besl [425] and Alexopoulos et al. [63]. Phylogenetic analyses of partial sequences from nuclear 26S rDNA indicate monophyletic Pleurotaceae, consisting of the monophyletic genera Pleurotus and Hohenbuehelia. The attack and consumption of nematodes (nematophagy) support the monophyly of this family [441]. Agaricus campestris, Amanita caesarea, Coprinus comatus, Macrolepiota procera, Marasmius scorodonius, Pholiota mutabilis and Rozites caperata are well-known edible mushrooms. Bresinsky and Besl [424] present an overview of many poisonous species (e.g. Amanita muscaria, A. phalloides, Cortinarius orellanus, Inocybe patouillardi, Lampteromyces japonicus, Omphalotus olearius) and different fungal intoxications.

Genotypic Identification

A number of fingerprinting methods based on the analysis of genomic DNA polymorphism have been developed in the last decades for genotypic species identification and delimitation. In our laboratory, we use two techniques successfully. RAPD-PCR is a simple and highly specific method. It was introduced independently by Welsh and McClelland [446] and Williams et al. [447]. We used RAPD-PCR to separate different species of yeasts like Mrakia and Sterigmatomyces [340], Kluyveromyces [448], Metschnikowia [449], Saccharomyces [450, 451], to identify known and new species of yeasts from nature [148, 452], and to identify or describe new species of filamentous microfungi (Fusarium [281]; Ophiostoma [293]; Verticillium [290]; endophytic fungi from grapevine [16, 305]). Rapid DNA extraction without digestion or removal of RNA yields the template for RAPD analysis, which is routinely used to determine the concentration of chromosomal DNA and the DNA to RNA ratio on the basis of the intensities of ethidium-bromide-stained bands. Since the amplification reaction for RAPD analysis cannot be completely standardized, the absolute numbers and sizes of the fragments formed are by themselves not significant markers for strain differentiation. Therefore, all of the strains that are to be compared must be processed with one stock solution of premixed reagents in one run of the thermocycler used and loaded onto one gel for maximum information output [340]. Amplified fragment length polymorphism (AFLP), developed by Vos et al. [453], is also a new technique for DNA fingerprinting. This technique is based on the selective amplification of restriction fragments (SARE) by PCR from a digest of total genomic DNA.

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 272 AFLP involves four steps: (a) restriction enzymes digestion; (b) ligation of adapters to the restriction fragments; (c) PCR amplification of the restriction/ ligation fragments, and (d) detection and analysis of the amplified fragments. The restriction fragments are generated by two restriction enzymes, one of them is a rarely cutting one and the other is a frequently cutting one. Specific adapters are ligated to the restriction fragments at the temperature at which restriction enzymes are still active to avoid concatamer formation of the restriction fragments. Two different adapters are used, one specific for the rare cutting enzyme and one specific for the frequent cutting enzyme. After ligation of the adapters, the fragments have appropriate attachment sites for the selective PCR primers. One of the two primers used is labeled (by isotopes or fluorescently). Amplified fragments are detected according to the labeling technique. Automatic laser fluorescence analysis (ALFA) [454] provides an automated detection and analysis of fluorescently labeled samples. The AFLP has the power of PCR and the solidity of RFLP analysis. As the AFLP is highly reproducible and has a deep enough resolution due to the number of estimable detected fragments; this technique allows us to analyze the strains at the intraspecies level. With this technique we can carry out epidemiological studies or can generate a database for routine identification. We proved that its application is very useful in the determination of the route of an infection or in the correct identification of pathogenic yeasts. Figure 12 shows a digital electrophoresis image of ALFA-AFLP fragments of Candida guilliermondii and C. glabrata strains, run on an automatic DNA sequencer. A high degree of similarity can be clearly recognized among the strains belonging to the same species. They share several (supposedly species- specific) bands in the same position, while differentiation of some of the isolates can be performed according to the noncommon fragments. Completely identical patterns can be seen, e.g. lanes 12 and 13, indicating that these isolates are probably in epidemiological relation. Proof of the identity, however, requires the performance of AFLP by an additional adapter and primer set. Separation of the isolates belonging to the two different species is clear by a simple visual evaluation of the patterns. Recently we have shown that it is not possible to identify ascomycetous and basidiomycetous yeasts unequivocally using the classical phenotypic approach [208]. The score of correct identification using the phenotypic approach is rather low especially in basidiomycetous yeasts. Partial sequences of the 18S and 26S rDNA have become a reliable tool to identify yeast species correctly [148, 452]. Meanwhile, partial sequences of the D1/D2 region of the large subunit rDNA are available for all known ascomycetous [23] and basidiomycetous [24] yeasts in GenBank. This offers the possibility of a reliable genotypic identification using the D1/D2 region. According to our experience

Systematics of the Ascomycota and Basidiomycota 273 1 2 3 4 5 6 7 12 13 16 25 34

Fig. 12. Digital electrophoresis image of AFLP fragments of Candida strains run on an automatic DNA sequencer. Markers: lanes 1, 16, 25, 34; Candida guilliermondii: clinical isolates, lanes 2–6; Candida glabrata: type strain CBS 138 lane 7, clinical isolates: others. it seems worthwhile to corroborate the identification data obtained with a RAPD-PCR analysis with the respective type strain. For the identification of new yeast species from nature we use a new polyphasic approach [452]: (1) Species delimitation using RAPD-PCR or

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 274 AFLP; (2) Species identification using partial sequences of the 18S or 26S rDNA and corroboration of the identification data using RAPD-PCR and the respective type strain; (3) Ubiquinone system according to Messner et al. [340], and (4) Qualitative and quantitative monosaccharide pattern of purified and hydrolyzed cell walls [449]. Just as for the identification of yeasts, partial sequences of the 18S and 26S rDNA are also useful for identifying filamentous microfungi. In contrast to yeasts, however, reliable identification [16] is not always possible due to a lack of sequences in Genbank.

Acknowledgments

We would like to thank Dr. D. Begerow, Dr. T. Boekhout, Prof. M. Breitenbach, Dr. T.D. Bruns, Emeritus Prof. F. Ehrendorfer, Prof. O.E. Eriksson, Emeritus Prof. K. Esser, Prof. J.W. Fell, Dr. M. Fischer, Emeritus Prof. W. Gams, Mag. G. Hagedorn, Dr. M. Hamamoto, Dr. D.S. Hibbett, Dr. G. Himmler, Prof. G.S. de Hoog, Dr. P. Hoffmann, Dr. J. Kämper, Prof. C. Kubicek, Dr. U. Kües, Dr. C.P. Kurtzman, Dr. S. Landvik, Dr. J. Loidl, Prof. M. Melkonian, DI. Dr. R. Messner, Prof. U.G. Mueller, Dr. T. Nakase, Prof. L. Sigler, Emeritus Prof. J. Sugiyama, Dr. M. Suzuki, Dr. E. Swann, Emeritus Prof. Y. Yamada for sending interesting strains, valuable literature, critical comments or unpublished sequences. My wife J. Prillinger and Ms. I. Mondl improved figures 2, 6 and 7. Ing. S. Huss prepared a CD version of all figures. Prof. G.S. de Hoog informed the first author that his excellent Atlas of Clinical Fungi is scheduled in a new, fully revised and greatly expanded edition for early 2000. Finally the first author is grateful to Prof. F. Oberwinkler of the University Tübingen for his excellent introduction into mycology, sending interesting strains and many valuable comments on the manuscript.

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Systematics of the Ascomycota and Basidiomycota 293 452 Prillinger H, Kraepelin G, Lopandic K, Schweigkofler W, Molnár O, Weigang F, Dreyfuss MM: New species of Fellomyces isolated from epiphytic lichen species. Syst Appl Microbiol 1997; 20:572–584. 453 Vos P, Hogers R, Bleeker M, Reijaus M, van de Lee T, Hornes M, Frijtens A, Pot J, Peleman J, Kuiper M, Zabeau M: AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res 1995;23:4407–4414. 454 Aarts H, Keijer J: Genomic Fingerprinting of Micro-Organisms by Automatic Laser Fluorescence Analysis (ALFA) of Amplified Fragment Length Polymorphism (AFLP™); in Akkermans ADL, van Elsas JD, de Bruijn FJ (eds): Molecular Microbial Ecology Manual 3.4.9. Dordrecht, Kluwer Academic, 1999. 455 Laaser G, Möller E, Jahnke KD, Bahnweg G, Prillinger H, Prell HH: Ribosomal DNA restriction fragment analysis as a taxonomic tool in separating physiologically similar Basidiomycetous yeasts. Syst Appl Microbiol 1989;11:170–175.

Notes added in proof

The 2nd edition of the Atlas of Clinical Fungi [De Hoog et al., 2000] includes 65 additional clinical fungi. It often presents small subunit, large subunit or ITS restriction maps for genotypic identification. According to the data of Binder and Hibbett [2002], the Schizophyllales can be included among the euagarics. A publication by Sterflinger and Prillinger [2001] presents molecular evidence that the genera Phoma and Epicoccum can be included among the Pleosporales. According to Sugita et al. [2000], C. laurentii is a genetically heterogeneous species; this must be taken into consideration when identifying C. laurentii clinical isolates. Data by Sugita et al. [in press] suggest that the IGS region has a powerful capacity of distinguishing between phylogenetically closely related strains and that there may be a geographic substructure among T. ashai clinical isolates. O’Donnell et al. [2001] and Voigt and Wöstemeyer [2001] present overviews on the phylogenetic relationship among the Zygomycota. Sterflinger et al. [1999] describe the RFLP technique used in the new edition of the Atlas of Clinical Fungi [Hoog et al., 2000]. Dr. C. Kurtzman kindly showed us, with strongly deleted files, that the alignment in the Ascomycota becomes much better. With these files the Archiascomycetes sensu Nishida and Sugiyama move into a basal position. Sipiczki [2001] presents molecular data on the Archiascomycetes; however, he did not find many similarities between the Archiascomycetes and the Basidiomycota (e.g. 5S rDNA of Taphrina, enteroblastic budding, carotin pigments in Saitoella). New notes on Ascomycota can be obtained from Myconet, vol. 6, 2001, edited by O.E. Eriksson.

References

Binder M, Hibbett DS: Higher-level phylogenetic relationships of the Homobasidiomycetes (mushroom- forming fungi) inferred from four rDNA regions. Mol Phylogenet Evol 2002;22:76–90. De Hoog GS, Guarro J, Gené J, Figueras MJ (eds): Atlas of Clinical Fungi, ed 2. Centraalbureau voor Schimmelcultures, Utrecht/Universitat Rovira i Virgili, Reus, 2000. O’Donnell K, Lutzoni F, Ward TJ, Benny GL: Evolutionary relationships among mucoralean fungi (Zygomycota): Evidence for family polyphyly on a large scale. Mycologia 2001;93:286–296. Sipiczki M: Identification of Schizosaccharomyces pombe genes that encode putative homologues of Saccharomyces cerevisiae mediator complex subunits. Acta Microbiol Immunol Hung 2001;48:519–531.

Prillinger/Lopandic/Schweigkofler/Deak/Aarts/Bauer/Sterflinger/Kraus/Maraz 294 Sterflinger K, de Hoog GS, Haase G: Phylogeny and ecology of meristematic ascomycetes. Stud Mycol 1999;43:5–22. Sterflinger K, Prillinger H: Molecular taxonomy and biodiversity of rock fungal communities in an urban environment (Vienna, Austria). Antonie van Leeuwenhoek 2001;80:275–286. Sugita T, Nakajima M, Ikeda R, Matsushima T, Shinoda T: Molecular analysis of intergenic spacer 1 region of the rRNA gene of Trichosporon species. J Clin Microbiol, in press. Sugita T, Takashima M, Ideka R, Nakase T, Shinoda T: Intraspecies diversity of Cryptococcus laurentii as revealed by sequences of internal transcribed spacer regions and 28S rDNA gene and taxonomic positions of C. laurentii clinical isolates. J Clin Microbiol 2000;38:1468–1471. Voigt K, Wöstemeyer J: Phylogeny and origin of 82 zygomycetes from all 54 genera of the Mucorales and Mortierellales based on combined analysis of actin and translation elongation factor EF-1␣ genes. Gene 2001;270:113–120

Prof. DI. Dr. Hansjörg Prillinger, Universität für Bodenkultur, Institut für Angewandte Mikrobiologie, Arbeitsgruppe Mykologie und Bodenmikrobiologie, Muthgasse 18, A–1190 Wien (Austria) Tel. ϩ43 1 36006 6207, Fax ϩ43 1 3697615, E-Mail [email protected]

Systematics of the Ascomycota and Basidiomycota 295 Glossary

ABPA anthropophilic sites of a hypha; allergic broncho- fungus prefers to grow infectious parasitic form pulmonary in humans in tinea aspergillosis apomictic propagation Arthrospores adiaspore propagation without a spore derived from the a spherical spore with a karyogamy and meiosis disarticulation of hypha rigid cell wall apomixis ascocarp (chlamydospore) development of sexual an ascus-containing produced in the lungs by cells into spores without morphological structure the enlargement of an being fertilized (ascoma) inhaled conidium of apothecial ascoma Emmonsia a cup- or saucer-like see ascocarp aecidium ascoma in which the ascospore vegetative fruiting body hymenium is exposed at meiotic spore of the of the Uredinales at the maturity Ascomycota end of the haploid stage arbuscular mycorrhiza ascus containing dikaryotic a symbiotic, non- meiotic sac-like cell aeciospores pathogenic or feebly within the ascoma of the agaric pathogenic endoinfection Ascomycota fungus belonging to the formed by zygomycetes aspergilloma Agaricales of the order Glomales. a ‘fungus ball’ of hyphae anamorph The penetrating hyphae of Aspergillus, found in structure of the asexual produce finely branched the upper lobe of the cycle and name of the haustorial structures lung fungus within the (arbuscules) or coils and auricularioid artificial commonly vesicles as transversely septate Deuteromycota well (commonly four) anisogamy arthroconidia cylindrical copulation of gametes segments developed by meiosporangium of the with unlike morphology breaking up at the septal Basidiomycota

296 autodiploidization cell separates from the conidiophore self-inducing fusion of parent cell leaving a bud specialized nuclei scar conidiogenous hyphal autogamy carpophore structure self-fertilization; fusion (1) stalk of sporocarp; conidiospore of nuclei without cell (2) basidiocarp an exogenous, nonfusion chitinous fungi motile vegetative spore bacteriophage fungi with chitin in their conidium virus that infects cell walls asexual spore bacteria chlamydospore corticioid ballistoconidium thick-walled conidium, flat fruiting body of the forcibly discharged often formed as a resting Basidiomycota which vegetative spore form in unfavorable develops directly on the ballistospore conditions; depending substrate with the forcibly discharged on the site on a hypha, hymenium on the outer basidiospore chlamydospores are side basidia (pl.) called laterales (on the crozier meiosporangium of the side), terminales (on the hook of an Basidiomycota the tip), or intercalares ascogenous hypha before basidiocarp (centrally located) ascus development meiotic fruiting body chloroplast cystidium (basidioma) of the photosynthetic organelle a sterile cell, frequently Basidiomycota with chlorophyll a and b of distinctive shape, basidiolichen choanoflagellate between basidia of the symbiosis between an unicellular flagellates Basidiomycota alga and a basidiomycete with phylogenetic dematiaceous basidiospore relationships to animals fungi with brown or meiospore of the and fungi black pigment in the cell Basidiomycota clade wall, thus appearing basidium monophyletic group of brown or black micro- meiosporangium of the organisms and macroscopically Basidiomycota, organ clavarioid denticle diagnostic for the club- or coral-like a small tooth-like Basidiomycota fruiting structure projection especially one Bioaerosol cleistothecium on which a spore is biological airborne a closed fruiting body produced particle(s), for instance having no predefined dicotyledonous plants fungal spores, pollen etc. opening within the plants with two seed blastospore Ascomycota leaves a conidium formed by coenocytic dikaryotic budding non-septate a cell or hyphal body trama multinucleate mass of compartment having two hyphal layer within protoplasm genetically distinct fruiting bodies collarette haploid nuclei budding a cup-shaped structure at dimitic process of reproduction the apex of a fruiting body of by which the daughter conidiogenous cell Basidiomycota having

Glossary 297 two types of hyphae endophyte germ tube (generative hyphae an organism that lives a hypha growing out of a commonly with clamps, within a plant spore skeletal hyphae endothrix gleba chambers commonly thick-walled, fungus growing inside hymenial cavities within aseptate, and of limited the hair shaft a fruiting body where length) eukaryotic gastroid basidia are dimorphic cells having a produced having two forms; in membrane-bound true gloeoplerous mycology often meaning nucleus hypha with hyaline or the yeast (often exoskeleton yellowish and highly saprophytic) and the outer skeleton found in refractile fluid hyphal (often parasitic; insects and other halotolerant e.g. Ustilago maydis) arthropods tolerant of higher salt stage filamentous phage concentrations dolipore septa virus infecting a haplophase septa of higher bacterial cell with a the part of the life cycle Basidiomycota with a variable length of the tail where the cells are barrel-shaped structure fissitunicate haploid in the middle portion asci with two functional haustorium ectomycorrhizal fungus wall layers (bitunicate), a special hyphal branch fungus with a hyphal splitting at discharge which extends in the sheath on the surface of flagellum living cell of the host, the roots of trees. cylindrical extension of for absorption of food Hyphae extend outward an eukaryotic cell hemiascomycetous into the soil and inward responsible for active fungi which belong to between outer cortical movement, bound by a the class of the cells forming a ‘Hartig plasma membrane Hemiascomycetes net’ fungi imperfecti hetero-bifactorial ectothrix fungi without known a mating system with two fungus growing on the sexual reproduction different factors. One outside of a hair shaft, (Deuteromycota) codes for pheromones destroying the cuticula fungus ball and their receptors and endocytobiotic theory ball-like hyphal the other one for DNA- see endosymbiotic aggregates of binding proteins theory Aspergillus, found in the heterokont flagellae endosymbiotic theory upper lobe of the lung two different types of theory stating that gametangiogamy flagellae which differ in mitochondria and fusion of sexually length, type of motion chloroplasts have been differentiated hyphae and external appendages once free living bacteria gastroid heterothallism and became basidia which do not condition of sexual symbiotically included actively discharge their reproduction in which in the cytoplasm of the basidiospores conjugation is possible host cell thus leading to geophilic fungus only through the the origin of the fungus growing on or in interaction of different eukaryotes soil mating types

Glossary 298 heterotrophic karyogamy monoecious organisms using organic fusion of genotypically male and female sex compounds as primary different nuclei organs on the same sources of energy lysotroph mycelium holobasidium to obtain food by monokaryon a basidium which is extracellular enzymes hyphal compartments not divided by primary (absorbtive nutrition) with a single haploid septa macroconidium nucleus, in homothallism larger asexual Basidiomycota often the a condition where sexual extracellular spore mycelium of a reproduction occurs meiospore basidiospore without the interaction a spore produced in the monophyletic of two different mating meiosis a group composed of a types meristem collection of organisms, hydnoid tissue at the tip of a including the most producing basidia on growing plant structure, recent common ancestor spines or tooth-like where cell division is of all those organisms projections most active, true mycelium hymenium meristems are absent in mass of hyphae meiospore-bearing layer fungi mycetoma of a fruiting body mesophilic fungi a fungal disease of the hypha fungi growing between foot or other parts of the septate or nonseptate 10 and 40°C human body, especially vegetative filament microconidium in the tropics hypogeous smaller asexual spore mycophagy having subterranean mitochondrial crista the use of fungi as food fruiting bodies structure of the inner oligokaryotic hyphomycete mitochondrial hyphal compartment mitosporic fungus membrane where the with 3–10 nuclei forming a mycelium respiratory chain oogamy with or without pigment complexes are located heterogamy with a non- isogamy mitosporangium motile female egg and a conjugation of a sporangium where motile male sperm morphologically similar spore formation occurs opportunistic pathogens gametes after mitosis pathogens that convert isogenized ramarioid mitosporic from a saprophytic to a inbreeding strains spore formation after parasitic form in a haploid offspring which mitosis predisposed host becomes monadal paraphysis morphologically similar primitive type of a sterile upward growing (Ramaria-like) after morphological hyphal element in the crossing of organisation in algae or hymenium of the morphologically similar unicellular flagellates Ascomycota parental strains monocotylendonous parenthesome isoprene unit plants a curved double chemical building block plants with one seed membrane (which may containing 5 C-atoms leaf be perforate, continuous

Glossary 299 or vesiculate) on each polyphyletic origin septate basidium side of a dolipore a collection of a basidium having septum in the organisms in which the transverse or cruciate Basidiomycota, most recent common septa septal pore cap ancestor of all the shikimate-chorismate perithecium included organisms is pathway a round or flask-like not included the most common fruiting body of the pseudohypha biosynthetic pathway Ascomycota with an hypha-like structure leading to aromatic opening at the top formed by budding compounds phagemid yeasts, totally siphonal a bacterial plasmid that separated by septa hyphal compartments can be propagated both without cytoplasmic without septa having as a plasmid and a exchange many nuclei bacteriophage pseudoparenchyma solopathogenicity phialoconidium see plectenchyma a pathogenic a conidium produced on ramarioid strains monosporidial line a special conidiogenous haploid or dikaryotic (e.g. Ustilago maydis) cell (phialide) fruiting bodies which somatogamous autogamy phragmobasidium resemble a Ramaria karyogamy without a basidium which is (coralloid basidiocarp) plasmogamy in divided by primary resupinate vegetative cells septa, usually transverse flat fruiting body of somatogamy or cruciate Basidiomycota directly fusion of somatic cells phragmoplast on the substrate with the or hyphae involving cell division structure hymenium on the outer plasmogamy but not occurring only in higher side karyogamy plants rhizopodial spermatium pileate type of morphological non-motile male stalked fruiting body organisation forming gamete with a pileus (hymenium- amoeboid cells which spermogonium supporting part of the lack a rigid cell wall a walled structure in basidioma, cap) saprobe which spermatia are pileitrama an organism using produced hyphae within the cap of dead organic material as spindle pole body a basidiomycete food, and commonly organelle for the division plectenchyma causing its of nuclei in Zygo-, fruiting body of firmly decomposition Asco- and interwoven hyphae scolytid Basidiomycota looking like a fungus associated with spore parenchyma of plants beetles (Scolytidae, sexually or asexually polygenic beetle family) produced reproductive genetically controlled by secotioid unit many genes the margin of the pileus stichic polykariotic does not break free from horizontal orientation of hyphal compartment the stipe, gastroid the spindle of nuclei in with more than ten nuclei basidiospores the basidium

Glossary 300 symbiosis morphological structure unifactorial association between of the sexual cycle heterothallism unlike organisms, which thallus a system in which the is advantageous for both body of mold colony sexual propagation to the organisms consisting of vegetative mycelia is controlled by sympodial hyphae two different mating spore formation tremelloid basidium types (e.g. ϩ and Ϫ or a characterized by cruciate septate basidium and ␣) continued growth, after of the Tremellales vegetative hypha the main axis has trichal form fungal thread without produced a terminal septate filamentous mitotic or meiotic spore, by the growth form fruiting bodies development of a trichogyne xerophilic fungi succession of apices the receptive hypha of favouring habitats in each of which originates the female sexual organ which water is scarce below and to one side of trimitic zoophilic fungus the previous apex fruiting body having fungus which prefers synanamorph three kinds of hyphae: animals for growth two or more anamorphs generative (often with zoospore of the same teleomorph clamps), skeletal (often a motile spore having teleomorph thick-walled) and one or more flagellae the perfect (sexual) form binding hyphae (thin- of an anamorph; walled)

Glossary 301 Author Index

Aarts, H.J.M. 207 Hawranek, T. 129 Schweigkofler, W. 207 Helbling, A. 28 Simon-Nobbe, B. 48 Bauer, R. 207 Horner, W.E. 10, 28 Sterflinger, K. 207 Bhatnagar, D. 167 Borg-von Zepelin, M. 114 Kauffman, H.F. 94 Tomee, J.F.C. 94 Brander, K.A. 28 Kraus, G.F. 207 Breitenbach, M. 5, 48 Yu, J. 167 Lehrer, S.B. 5, 28 Chiu, A.M. 1 Levetin, E. 10 Crameri, R. 5, 73 Lopandic, K. 207

Deak, R. 207 Maraz, A. 207 Monod, M. 114 Ehrlich, K.C. 167 Prillinger, H. 207 Fink, J.N. 1

302 Subject Index

Aerobiology environmental factors in exposure 1 biochemical assays 14, 15 extract reproducibility problems with culture analysis 13, 14 molds 49–51 epidemiological studies 22–24 recombinant allergens for testing 3 historical perspective 10, 11 Allergic bronchopulmonary aspergillosis indoor mold exposure 23, 24 allergens and serology 83–87 measurement problems clinical features 97 counting methods for Burkard spore diagnosis 73, 74, 76, 77, 97 traps 19–21 histopathology 106, 107 culture-related errors 21, 22 hypersensitivity reaction types 97 reporting lag 21 microinvasive processes 98 microscopic analysis 14 prevalence 76, 77, 97 passive sampling of spores 11, 12 Allergy sampling equipment 10–13 prevalence 1 variation patterns respiratory, due to fungi 2 altitude effects 18 Alternaria alternata ascospores 17 allergens atmospheric variability 18, 19 Alt a 1 basidospores 16, 17 characterization 57, 58 diurnal variation 15, 16 purification 66 rain effects 17 Alt a 2 63 seasonal effects 17, 18 Alt a 3 63 wind effects 17 enolase Aflatoxins allergen activity 59, 60 carcinogenesis 177, 179 cross-reactivity 60, 61 structures 177, 178 purification 66, 67 toxicity 177 extract reproducibility problems with types 177 molds 49–51 Agaricales, phylogenetic relationships gene cloning and expression in 270–272 Escherichia coli 54–57 Air sampling, see Aerobiology growth conditions and expression 50 Allergens, see also individual species mass spectrometry 56

303 Alternaria alternata (continued) Aspergillus fumigatus allergens (continued) allergens skin testing 67, 68 Asp f 1 78 table of allergens 64 characterization 78–80 asthma patient sensitivity 22 cloning 78–81 exposure routes 48, 49 enzymes 81 sensitization rates 49 phage display and screening 78, 79, specific immunotherapy 51–54 82, 86, 87 Alternaria toxins, types and features 190, purification 77, 78 191 skin challenge testing of recombinant Amorolfine, cutaneous mycoses proteins 82, 83 management 162 table 79 Amphotericin B, cutaneous mycoses diagnosis of allergy 2, 73 management 161 diseases, see also Allergic Amplified fragment length polymorphism, bronchopulmonary aspergillosis genotypic identification 272–275 aspergilloma 96 Antileukoprotease, Aspergillus fumigatus atopic diseases and asthma 75, 76, mucosal defense 100, 102 95–97 Ascomycota, see also individual species hypersensitivity pneumonitis 98, 99 ascospore variation patterns in air 17 invasive aspergillosis 75, 99 phylogenetic relationships proteases in pathology 106–108 Chaetothyriales 234, 235 saprophytic colonization 75, 95, 96 class interpretation 227, 228 exposure routes 48, 49 Dothideales 244–246 genome sequencing 5–8 Euascomycetes 233, 234 host defense Eurotiales 235–237 adaptive immunity 105–108 Hemiascomycetes 228–231 hyphal forms 103, 104 Hypocreales 239–242 immunosuppressive therapy effects Leotiales 248, 249 108, 109 Microascales 244 innate defense of airway mucosa 99, Onygenales 237, 239 100, 102–105 Ophiostomatales 242 overview 74, 75, 94 Pezizales 249 pathogenicity 74 Phyllachorales 242, 243 species 74, 95 phylogenetic tree based on 18S rRNA virulence factor identification 74 sequence 214, 226 Asthma Pleosporales 246–248 air quality effects on symptoms 22, 23 Protomycetes 231–233 Alternaria sensitivity 22 Sordariales 243 Aspergillus fumigatus 75, 76, 95–97 Aspartate proteases, Candida albicans Athlete’s foot, see Tinea pedis adherence role 121–123 deep-seated candidiasis role 123, 124 Basidomycetes, see also individual species secretion 120, 121 allergens Aspergillosis Boletus edulis 35 deep mycosis features and management Calvatia 35, 36 155 Coprinus 36 neurotropic mycotoxins 193 Coprinus comatus

Subject Index 304 Cop c 1 41, 43 sampling 28–30 Cop c 2 44 variation patterns in atmosphere 16, phage display library screening 40 17 potential allergens 40, 44 taxonomic distribution 28, 29 cross-reactivity 39, 40 Black piedra, features 133, 134 Ganoderma 37 Blastomycosis, deep mycosis features and Pleurotus 37, 38 management 153 Psilocybe 38, 39 Boletales, phylogenetic relationships 268, atopic eczema 32 269 bronchial and nasal challenge response Boletus edulis 32 allergens 35 contact dermatitis 33 food allergy 33 food allergy 33 nasal challenge response 32 invasive mycosis 34 sensitization rates 31 phylogenetic relationships Agaricales 270–272 Calvatia, allergens 35, 36 Boletales 268, 269 Candida albicans, see also Candidiasis Cantharellales 263, 264 adherence and adhesion molecules cell wall sugar analysis 251–254 118, 119 class overview 249–252 dimorphism and switching system Cystobasidales 255 116, 117 Georgefischerales 258, 259 genome sequencing 5–8, 115 Gomphales 264 opportunistic diseases 114 Hymenochaetales 267, 268 prototrophy 114, 115 Hymenomycetes 260, 261 site-directed mutagenesis 115 Malasseziales 258 virulence factors Microbotryales 255, 256 aspartate proteases Microstromatales 259 adherence role 121–123 phylogenetic tree based on 18S rRNA deep-seated candidiasis role sequence 216 123, 124 Polyporales 265–267 secretion 120, 121 Russulales 268 identification 115, 116 Schizophyllales 269, 270 phospholipases 119, 120 Thelephorales 264 Candida balantitis, features 146 Tremellales 261–263 Candidiasis Uredinales 256 budding meiosis 230 Urediniomycetes 253, 254 chronic mucocutaneous 148 Ustilaginales 259, 260 congenital 147, 148 Ustilaginomycetes 257, 258 deep mycosis features and management respiratory allergy 31, 32 153, 154 sensitization rates genital 146, 147 skin test reactivity prevalence 28, 29 interdigital 148, 149 species differences 30, 31 management of superficial disease 145 source materials for allergy testing 34, 35 opportunistic infection 153, 154 spores oral 145, 146 abundance in atmosphere 28–30 paronychia and onychomycosis 147 indoor spores 30 predisposing factors 144, 145

Subject Index 305 Cantharellales, phylogenetic relationships Culture 263, 264 air spore samples 13, 14, 21, 22 Chaetothyriales, phylogenetic relationships cutaneous mycology diagnostics 158, 234, 235 159 Chromoblastomycosis, management 149, Cutaneous mycology 150 candidiasis Chytridiomycota, phylogenetic chronic mucocutaneous candidiasis relationships 224–226 148 Citrinin, toxin features 182, 183 congenital candidiasis 147, 148 Cladosporium herbarum genital candidiasis 146, 147 allergens interdigital candidiasis 148, 149 Cla h 1 characterization 59 management of superficial disease Cla h 2 63, 64 145 enolase oral candidiasis 145, 146 allergen activity 59, 60 paronychia and onychomycosis 147 cross-reactivity 60, 61 predisposing factors 144, 145 epitope mapping 61, 62 cutaneous mycoses extract reproducibility problems with dermatophytes 134–137 molds 49–51 onychomycosis 142–144 gene cloning and expression in tinea barbae 141 Escherichia coli 54–57 tinea capitis 138–141 growth conditions and expression tinea corporis 134, 138 50 tinea incognito 144 phage display and screening 65, 66 tinea inguinalis 138 table 64 tinea manuum 142 exposure routes 48, 49 tinea pedis 141, 142 sensitization rates 49 deep mycosis specific immunotherapy 51–54 opportunistic fungi 153–155 Coccidioidomycosis, deep mycosis features pathogenic fungi diseases 151–153 and management 152 host defense 132 Collectins, Aspergillus fumigatus mucosal laboratory diagnosis defense 102 culture 158, 159 Complement, Aspergillus fumigatus dye-conjugated antibody assays 158 mucosal defense 102, 103 histopathology 159 Coprinus, allergens 36 mold identification 159, 163 Coprinus comatus potassium hydroxide test 157, 158 atopic eczema 32 sample collection 157 Cop c 1 41, 43 serology 159 Cop c 2 44 yeast identification 159 phage display library screening for pathogenesis of cutaneous fungal allergens 40 infection 130, 132 potential allergens 40, 44 rare mycoses 155–157 sensitization rates 31 subcutaneous mycoses Cryptococcosis, opportunistic infection chromoblastomycosis 149, 150 154 entomophthoromycosis 150 Cryptococcus neoformans, genome lobomycosis 151 sequencing 5–8 mycetoma 150

Subject Index 306 rhinosporidiosis 151 phylogenetic tree of eukaryotic sporotrichosis 149 organisms based on 18S rRNA zygomycosis 150 sequence 207–210 superficial mycoses Eurotiales, phylogenetic relationships black piedra 133, 134 235–237 pityriasis versicolor 123, 133 tinea nigra 134 Fluconazole, cutaneous mycoses white piedra 133 management 160 treatment agents and principles 160–163 Flucytosin, cutaneous mycoses Cyclopiazonic acid, toxin features 181 management 162 Cyclopiroxolamine, cutaneous mycoses Fumonisins management 162 biosynthesis 186 Cystobasidales, phylogenetic relationships food contamination 184, 185 255 fumonisin B1 186 types and structures 184, 185 Defensins, Aspergillus fumigatus mucosal Fungi defense 100, 102 air sampling, see Aerobiology Dermatophytes, types and characteristics clinical classificaton of infections 130, 134–137 131 Dothideales, phylogenetic relationships extracts in allergy testing 3 244–246 kingdom, see Mycobionta molds vs yeasts 129, 130 Econazole, cutaneous mycoses management 160 Ganoderma, allergens 37 Enolase Georgefischerales, phylogenetic allergen activity in molds 60 relationships 258, 259 cross-reactivity between Alternaria ␤-Glucans alternata and Cladosporium herbarum air sample analysis 14 60, 61 indoor respiratory complaint role 23 epitope mapping of Cladosporium Gomphales, phylogenetic relationships 264 herbarum protein 61, 62 Griseofulvin function 59 cutaneous mycoses management 161 purification of recombinant Alternaria tinea capitis management 140 alternata protein 66, 67 Entomophthoromycosis, management 150 Hemiascomycetes, phylogenetic Epithelium, Aspergillus fumigatus mucosal relationships 228–231 defense 104, 105, 110 Histoplasmosis, deep mycosis features and Ergosterol, air sample analysis 14 management 152, 153 Ergot alkaloids Hortaea (Phaeoannelomyces) werneckii, biosynthesis 192 tinea nigra 134 classification 191, 192 Hyalohyphomycosis, features 156 history of epidemics 191 Hymenochaetales, phylogenetic physiological effects 192 relationships 267, 268 Euascomycetes, phylogenetic relationships Hymenomycetes 233, 234 cell wall sugar analysis 254 Eukarya phylogenetic relationships 260, 261 origins 209 Hyphomycosis, features 155, 156

Subject Index 307 Hypocreales, phylogenetic relationships classification 173 239–242 contamination management preharvest control 193, 194 Immunoglobulin A, Aspergillus fumigatus postharvest control 194–196 mucosal defense 103 dietary considerations 196 Impactor samplers, types and features 12, removal 195 13 inactivation 195, 196 Itraconazole, cutaneous mycoses definition 167 management 160 detection and screening 176 economic impact and regulations 173–176 Keratinases, cutaneous mycology 130 indoor respiratory complaint role 23, 24 Ketoconazole, cutaneous mycoses mycotoxicology management 160 definition 170 historical perspective 171–173 Lentinus edodes mycotoxicosis 168 food allergy 33 natural occurrence 170, 171 respiratory allergy 31 neurotropic mycotoxins 191–193 Leotiales, phylogenetic relationships 248, table of examples 169 249 Lobomycosis, management 151 Naftifine, cutaneous mycoses management 160 Macrophage, Aspergillus fumigatus Natamycin, cutaneous mycoses mucosal defense 103, 104 management 161 Malasseziales, phylogenetic relationships Nystatin, cutaneous mycoses management 258 161 Merulius lacrymans, respiratory allergy 31 Miconazole, cutaneous mycoses Ochratoxins management 160 food contamination 179, 181 Microascales, phylogenetic relationships mechanism of action 180 244 structures 179, 180 Microbotryales, phylogenetic relationships Onychomycosis 255, 256 fungal nail infections and management Microstromatales, phylogenetic 142, 143 relationships 259 mold infections 143, 144 Morphological differentiation, Mycobionta Onygenales, phylogenetic relationships 212, 213, 215 237, 239 Mycetoma, management 150 Ophiostomatales, phylogenetic Mycobionta relationships 242 features of kingdom 1, 2, 209 morphological differentiation 212, 213, Paracoccidioidomycosis, deep mycosis 215 features and management 152 sexual differentiation 215, 217–219, Patulin, toxin features 181, 182 221, 223, 224 Penicillic acid, toxin features 182 taxonomic overview 210–212 Penicillium marneffei, infection 156, Mycotoxin, see also specific toxins 157 biosynthesis 167, 168, 174 Pezizales, phylogenetic relationships 249

Subject Index 308 Phage display eukaryotic organisms 207–210 Aspergillus fumigatus allergen screening pathogenic yeasts and fungi 238 78, 79, 82, 86, 87 Russulales, phylogenetic relationships 268 Cladosporium herbarum allergen screening 65, 66 Saccharomyces cerevisiae, genome features Coprinus comatus allergen screening 40 5, 6, 8 Pheohyphomycosis, features 156 Schizophyllales, phylogenetic relationships Phospholipases, Candida albicans 119, 269, 270 120 Scrotal candidiasis, features 147 Phyllachorales, phylogenetic relationships Sexual differentiation, Mycobionta 215, 242, 243 217–219, 221, 223, 224 Phylogenetic tree, see Ribosomal RNA Sick building syndrome, clinical features 2 genes Sordariales, phylogenetic relationships 243 Piedraia hortai, black piedra 133 Spores Pityriasis versicolor, features 132, 133 air sampling, see Aerobiology Pityrosporum folliculitis, features 133 basidomycetes, see Basidomycetes Pleosporales, phylogenetic relationships Sporotrichosis, management 149 246–248 Stachybotrys atra, pulmonary hemorrhage Pleurotus, allergens 37, 38 and hemosiderosis 2 Pleurotus ostreatus, respiratory allergy 31 Sterigmatocystin, toxin features 183 Pleurotus pulmonarius atopic eczema 32 T helper cell, Aspergillus fumigatus defense sensitization rates 31 105, 106 Polymerase chain reaction Terbinafine, cutaneous mycoses air sample analysis 14, 15 management 161 genotypic identification techniques Thelephorales, phylogenetic relationships 272–275 264 Polyporales, phylogenetic relationships Thrush 265–267 erythematous candidiasis 146 Potassium hydroxide, cutaneous mycology leukoplakia 146 testing 157, 158 management 145 Protomycetes, phylogenetic relationships opportunistic infection 153, 154 231–233 pseudomembranous candidiasis 146 Protothecosis, features 156 Tinea barbae, features 141 Pseudallescheriasis, deep mycosis features Tinea capitis and management 155 clinical presentation 138, 139 Psilocybe, allergens 38, 39 diagnosis 139, 140 management 140, 141 RAPD-PCR, genotypic identification Tinea corporis, features 134, 138 272–275 Tinea incognito, features 144 Rhinosporidiosis, management 151 Tinea inguinalis, features 138 Ribosomal RNA genes Tinea manuum, features 142 genotypic identification 272–275 Tinea nigra, features 134 phylogenetic trees based on 18S rRNA Tinea pedis, features 141, 142 sequences Tolnaftate, cutaneous mycoses management Ascomycota 214, 226 162 Basidomycota 216 Toxin, definition and classification 167

Subject Index 309 Tremellales, phylogenetic relationships Ustilaginomycetes 261–263 cell wall sugar analysis 253 Trichosporon, white piedra 133 phylogenetic relationships 257, 258 Trichothecenes biosynthesis 187 Vaginal candidiasis diseases 189, 190 clinical features 146 food contamination 187, 189 management 147 mechanism of action 190 structures 187, 188 types 187, 189 White piedra, features 133

Uredinales, phylogenetic relationships 256 Zearalenone, toxin features 183, 184 Urediniomycetes Zygomycosis cell wall sugar analysis 252 superficial disease management 150 phylogenetic relationships 253–254 systemic disease 154, 155 Ustilaginales, phylogenetic relationships Zygomycota, phylogenetic relationships 259, 260 224–226

Subject Index 310