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Molecular and its Application to Medical NATO ASI Series Advanced Science Institutes Series A series presenting the results of activities sponsored by the NA TO Science Committee, which aims at the dissemination of advanced scientific and technological knowledge, with a view to strengthening links between scientific communities. The Series is published by an international board of publishers in conjunction with the NATO Scientific Affairs

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Series H: , Vol. 69 and its Application to Medical Mycology

Edited by Bruno Maresca International Institute of and Via Marconi 12, 80125 Naples, Italy George S. Kobayashi Washington University School of Medicine Division of Infectious Diseases St. Louis, Missouri 63110, USA Hideyo Yamaguchi Research Center for Medical Mycology Teikyo University School of Medicine 359 Otsuka, Hachioji-shi, Tokyo 192-03,

Springer-Verlag Berlin Heidelberg New York London Paris Tokyo Hong Kong Barcelona Budapest Published in cooperation with NATO Scientific Affairs Division Proceedings of the NATO Advanced Research Workshop on Molecular Biology and its Application to Medical Mycology, held at Taormina, Italy, January 6-8, 1992

Book design by Donatella Capone, Secretary 11GB Press, Naples, Italy

ISBN -13 :978-3-642-84627 -4 e-ISBN-13 :978-3-642-84625-0 DOl: 10.1007/978-3-642-84625-0

Library of Congress Cataloglng-In-Publicatlon Data Molecular biology and Its applicatIOn to medical mycology / edited by Bruno Maresca, and George S Kobayashi, and Hldeyo Yamaguchi (NATO ASI series Series H, Cell biology, vol 69) "Proceedings of the NATO Advanced Research Workshop on Molecular Biology and Its Application to Medical Mycology, held at Taormina, Italy, January 6-8, 1992" ~ Tp verso Includes bibliographical references and Index

1 Medical mycology~Congresses 2 Molecular blology~Congresses 3 Mycoses~Molecular aspects-Con• gresses I Maresca, B (Bruno), 1950- II Kobayashi, GeorgeS, 1927- III Yamaguchi, Hldeyo, 1934- IV North Atlantic Treaty OrganizatIOn SCientifiC Affairs DIvIsion V NATO Advanced Research Workshop on Molecular Biology and Its Application to Medical Mycology (1992 Taormina, Italy) VI Series [DNLM 1 Fungl~genetlcs~congresses 2 Molecular Blology-congresses 3 Morphogenesls-congresses 4 Mycology-congresses 5 ~genetlcs~congresses OW 180 M718 1992] OR245 M645 1993616' 015- dc20

This work IS subject to copYright All rights are reserved, whether the whole or part of the material IS concerned, speCifically the rights of translation, reprinting, reuse of Illustrations, recitation, broadcasting, on microfilm or In any other way, and storage In data banks DuplicatIOn of this publication or parts thereof IS permitted only under the provIsions of the German CopYright Law of September 9, 1965, In ItS current verSion, and permission for use must always be obtained from Springer-Verlag Violations are liable for prosecution under the German COPYright Law

© Springer-Verlag Berlin Heidelberg 1993 Softcover reprint of the hardcover 1 st edition 1993

Typesetting Camera ready by author Book design Donatella Capone, Secretary 11GB Press, Naples, Italy 31/3145 - 5 4 3 2 1 0 - Printed on aCid-free paper Preface

With a spectacular view of a grumbling and lava spewing mount Etna in the background, the NATO sponsored workshop "Molecular Biology and its Application to Medical Mycology" provided a stimulatingforumfor a group of researchers who work with fungi pathogenic for humans to interact and discuss problems with molecular who use non-pathogenic fungi primarily as laboratory models for investigation. The workshop was held January 6-8, 1992 in the Congress Center of the San Domenico Palace Hotel, aformer monastery dating back to 1430 located in Taormina, situated high above the coastline of the Mediterranean sea overlooking Isola Bella on the island of Sicily. In this setting the two groups were encouraged to participate in active discussions and exchange of ideas. Thirty invited papers and one poster session were presented in the three day workshop that was divided into six sessions covering: (1) the molecular biology of yeasts; (2) the molecular biology offilamentous fungi; (3) fungal morphogenesis; and (4) the use and application of the newer molecular biology tools in the study of medically importantfungL Eachsession was designed to include studies using pathogenic and non-pathogenic fungi as laboratory models.

The opening presentation by Sergio Moreno from Paul Nurse's group at OJiford University set the standardfor theformal presentations. In his paper "Cell cycle regulation in flSsion ", he showed that the cdc2+ gene of Schizosaccharomyces pombe encodes a 34kDa phosphoprotein with serine/threonine kinase activity. He described current molecular biology studies on its role in during ceU cycle of this yeast. This talk was followed by Claudina Roudrigues-Pousadafrom Oeiras, Portugal who described her studies on acquisition ofthermo tolerance in Saccharomyces cerevisiae using 1, 1 O-phenantroline an iron chelating compound that induces arrest ofgrowth at "start", during which time they acquire thermotolerance without producing a full set of heat shock . Bridging the gap between studies on non• pathogenic fungi and pathogens was the paper given by Gerald Fink of the Whitehead Institute in Boston, Massachusetts. He described his thought provoking studies with nutritionally deprived diploid strains ofS. cerevisiae that undergo changes in ceU shape and pattern of cell division that results in filamentous growth. These observations implied some very important implications for pathogenesis in that fllamentation in yeasts of the Candida is often correlated with the process of pathogenicity. The difficulty of using clasSical genetic technics to investigate yeast belonging to the genus Candida was emphasized by Masamichi Takagifrom the University VI of Tokyo with his strategy of reverse genetics by using recombinant DNA technology. Using technics of molecular cloning, Richard Calderone of Georgetown University School of Medicine described the isolation of a gene fragment from C. albicans which shows homology with human complement receptor (CR2) gene. Closing the.first session was Jacob Reiserfrom the Swiss Federal Institute of Technology of Zurich, Switzerland who described his molecular biological studies with the genus Trichosporon, which include yeast• like fungi that have been implicated as opportunistic pathogens of humans.

The contributions presented in the two sessions onftlamentousfungifocused mainly on studies using Aspergillus nidulans and Neurospora crassa. These studies clearly illustrated the advantages these fungi offer over those that cause disease in man and particularly in the area of molecular genetics. Based on advances made in this area, Yuzuru Iimura described his studies on the development ofmolecular genetics systemsfor several industrial including A. niger and A. oryzae. Studies from Geojfrey Turner's laboratory on expression of genes for the biosynthesis of further support the contention that rapid basic and molecular biological advances are made when the problem involves an industrially important metabolite particularly when it is produced by a non-pathogen. The exception, however is in the area of phytopathology where Flora Banuett described her elegant studies on Ustilago maydis. Her work and those ofothers who work with pathogens clearly point out the deficiencies and paucity of information that exists when it comes to Similar studies on the molecular biology involvingfungi that cause disease of humans and animals.

In the second session, molecular biological studies with yeast, it became evident that great inroads are being made in Similar areas with medicaUy importantfungL Several investigators have been motivated to study Candida albicans and Cryptococcus neoformans because ofimportant clinical questions that remain unanswered concerning host-parasite interaction and the paucity of information on its virulence, and pathogenesis. Jim Cutler described his studies on the characterization ofC. albicans-speciftc DNAfragment that may be useful diagnostically. Interest in and studies with C. neoformans are rapidly increasing and a great deal ofprogress has been made in our knowledge ofthe genetics and of this opportunistic pathogen. Jejfrey Edman reviewed work on the of DNA-mediated transformation and the usefulness of this in studies of host-parasite interaction.

William Timberlake discussed transcriptional regulators of morphogenetic genes in Aspergillus and emphasized that genetic and molecular biological studies with this group have been facilitated because these fungi have well defmed sexual phases, stable mutants are available and easy to generate, effiCient transformation systems exist. and the biochemistry ofthese has been extensively studied.. While research using non-pathogenicjilamentous fungi and yeast as experimental models have contributed a greatly to our knowledge ofmolecular genetics and biology. none of these organisms cause disease. A great deal of basic information was presented on Histoplasma capsulatum and Wangiella dermatitidis in the seSSion on morphogenesis. VII

Bruno Maresca of11GB, Naples, emphasized that transition ofmycelia to yeast is necessaryforpathogenesis and showed thatwhenH. capsulatumgoesfrom an ambient environment in nature to 3'rC of the host heat shock genes are turned on. Furthermore he provided data to show that the phospholipid composition differs in strain ofR. capsulatum that have different susceptibilities to temperature and virulence and the difference unsaturated fatty acids in these strains could explain why each strain has a different capacity to adapt to the new environment during host invasion. In contrast to the dimorphic nature of H. capsulatum, the dematiaceous fungus W. dennatitidis is polymorphic. it can grow as a budding yeast, a ftlamentous , or as a multicellular mass. Thefmdings that chitin content increases and deposition is delocalized during yeast-to-multicellular form transition in W. dennatitidis and that S. cerevisiae possessed several chitin synthases led Paul Szaniszlo from the University of Texas, Austin, to determine whether W. dennatitidis possesses multiple chitin synthases. These studies were described in the session on morphogenesis.

It became apparent in the session on prospectives for medical mycology that the newer technologies of molecular biology are being applied to the study of fungi pathogenic for humans and animals. The studies of Antonio Cassone from lstituto Superiore di Sanitd, Rome, and those of Somay Murayama of Teikyo University of Tokyo underscored the current needs for tools that can rapidly and with specificity diagnose the life threateningjungal infections. In addition to the needfor these tests, it was apparentfrom the presentation of Gerald Medoff that the current available chemotherapeutic agents used to treat these infections are inadequate and that newer strategies need to be consideredfor development ofmore effective . Hugo Vanden Bossche, Steven Kelly reviewed the current approach and targets that are being emphasized and Hideyo Yamaguchi described newerfungal targets that need to be exploited.

The workshop was concluded with a round table discussion on the topic of integrating the technics ofmolecular biology to the needs ofmedical mycology. Audience participation was active, with constructive dialogue between those with expertise in molecular biology and researchers who study medically importantjungi. During this session, it became evident that many molecular biological technics described in standard handbooks are not directly applicable to the study of pathogeniC fungi and that a need exists to focus on the uniqueness offungal pathogens ofhumans and animals. This NATO sponsored workshop provided the proper forum to address these problems by bringing together groups ofscientist who studyfungi with disparate interests to discuss the shortcomings and needs ofclinically relevant problems involving pathogenic fungi. In summary, the participants were unanimous in their opinion that the workshop achieved its goals and recommended thatfuture meetings be held every other year in order to update interested parties on research progress and advance in newer technics that have been developed.

Bruno Maresca, George Kobayashi and Hideyo Yamaguchi First row from left: Bill Timberlake. Judith Medoff. Sergio Moreno. Bruno Maresca. Geny Fink. Second row from left: Robert Brambl. Paul Szaniszlo. Ray Dixon. Richard Calderone. Jeny Medoff. Third row.from left: 11GB group of Naples. Hugo Vanden Bossche. George Kobayashi. Hideyo Yamaguchi. The NATO Workshop was also supported by

Consiglio Nazionale delle Ricerche. Italy

Regione Siciliana. Italy

Istituto Italiano per gli Studi Filosoflci. Naples. Italy

PfIZer Pharmaceuticals Inc .. Tokyo. Japan

Tsumura & Co .. Tokyo. Japan

Bayer Yakuhin. Ltd .. Osaka. Japan

Nichinichi Foods Co .. Ltd., Mie-ken, Japan

Sandoz Pharmaceuticals. Ltd .. Tokyo. Japan

Boehring-pfIZer. U.S.A.

Monsanto. U.S.A.

Abbott Labs. U.S.A. Contents

Molecular Biology of Yeast

Cell Cycle Regulation in Fission Yeast S. Moreno, P. Nurse...... '" ...... 3 Yeast Genes Overcoming Growth Arrest Induced by 1.10-Phenanthroline P. Bossier. L. Fernandes, C. Rodrigues-Pousada...... 7 Reverse Genetics in a Non-Conventional Yeast. Candida maltosa M. Takagi...... 13 The Complement C3D-Binding Receptor (CR2) of Candida albicans: Cloning and Sequence Analysis of a Gene Fragment Homologous with a Human CR2 cDNA Clone R.A. Calderone, S. Southard. C. Hale...... 23 Biology. . Biochemistry and Molecular Genetics of Trichosporon Yeasts J. Reiser, V. Glumolf, M. Kellin, U. Ochsner, A. Fiechter...... 31 Physiological Functions of Vacuoles in Yeast: Mechanism of Sequestration of Metabolites and Proteins into Vacuoles Y. OhsumL...... 39 Characterization of a Candida albicans-Specific DNA Fragment P.M. Glee, J.E. Cutler...... 47 Recent Advances in the Molecular Biology of Cryptococcus neojormans J.C. Edman ...... 53 Molecular Biology of Kluyveromyces lactis L. Frontali, M.M. Bianchi, L. Fabiani, C. Falcone, C. Mazzoni. A. Ragnini. M. SaliDla, C. Wilson...... 63 Gene Regulatory Circuits in S. cerevisiae as a Tool for the Identification of Heterologous Eukaryotic Regulatory Elements J.F. Pulitzer, A. Pollice...... 75

Molecular Biology of Filamentous Fungi

Molecular Breeding in Filamentous Fungi with Emphasis on Aspergilli Y. Iimura, K. GomL...... 87 TubB a-Tubulin is Essential for Sexual Development in Aspergillus nidulans N.R. Morris, K.E. Kirk...... 97 The Heat Shock Response of Neurospora crassa N. Plesojsky-Vig, R. Brambl...... 105 Blue Light Regulated Expression of Geranylgeranyl Pyrophosphate Synthetase (Albino-3) Gene in Neurospora crassa G. Macino, S. Baima, A. Carattoli, G. Morelli, E.M. Valle...... 117 XII

Expression of Genes for the Biosynthesis of Penicillin G. Thrner, P.E. Browne, AA. Brakhage...... 125

New Tools and Prospectives for Medical Mycology

Imaging of the Yeast Killer Phenomenon L. Polonelli, S. Contt, M. Gerlont, W. Magliant, G. Morace, J.C. Caillez...... 141 cDNA Cloning of Candida albicans Aspartic Proteinase and Its Diagnostic Application S.Y. Murayama...... 149 Molecular Biotyping of Candida in Experimental and Clinical Studies A. Cassone, F. De Bernardis, E. Pontieri, G. Carruba...... 159 Homoserine Dehydrogenase as a Selective Target for Antifungal Action H. Yamaguchi...... 169 Molecular Mechanisms of Antifungal Activi1y and Fungal Resistence: Focus on Inhibitors of Ergosterol Biosynthesis H. Vanden Bossche, P. Marichal, H. Moereels...... 179 Molecular Studies on Azole Sensitivi1y in Fungi S.L. Kelly, D.E. Kelly ...... 199 Antifungal Chemotherapy and Drug Strategies: Projected Clinical Needs G. Medojf...... 215 Funding Medical Mycology: Strategies for Attracting the Private Sector J.N. Galgiani...... 221

Fungal Morphogenesis

Chromosomal Organization of Histoplasma capsulatum P.E. Steele, R.L. Himes...... '" ...... 227 Chitin. Chitin Synthase and Chitin Synthase Conserved Region Homologues in Wangiella dermatitidis P.J. Szaniszlo, M. Momany...... 229 Heat Shock Response and during Morphogenesis in Histoplasma capsulatwn B. Maresca, G. Di Lallo, C.L. Pardini, G. Petrella, L. Carratu...... 243

Subject Index...... 251 Contributors

The numbers in brackets are the opening page numbers of the contributors' articles.

RobertBrambL University ofMinnesota, Dept. ofPlant Biology, 220 Biological Sciences, 1445 Gartner Ave., St. Paul, 55108-1095 MN USA, (105) Richard Calderone, Dept. of Georgetown University School of Medicine, Washington D.C. 20007 USA, (23) Antonio Cassone, Istituto Superiore di Sanita, Divisione di Micologia Medica, Viale Regina Elena, 299, Roma 00161 Italy, (159) JimE. Cutler, Department ofMicrobiology, Montana State University, Bozeman 59717, Montana USA, (47) Jeifrey C. Edman. Hormone Res. Institute, University of California School of Medicine San Francisco 94143-0534 CA USA, (53) Laura Frontali, Universita di Roma "La Sapienza" Dip. di BioI Cell e Sviluppo• Sez. di Scienze Fisiol., Piazzale AIdo Moro, Roma 00195 Italy, (63) John Galgiani, University of Arizona Medical Center, Sec. of Infectious Diseases, Tucson 85723, Arizona USA, (221) Yuzuru Iimura, National Research of Brewing, 2-6-30 Takinogawa, Kita-ku Tokyo 114 Japan, (87) StevenL. Kelly, The University ofSheffield Dept. ofMol. Biol. & , P.O. Box 594 Firth Court Western Bank, Sheffield S 10 2UH U.K., (199) Giuseppe Macino, Dept. ofBiopathology ,Policlinico, University of Rome "La Sapienza", Viale Regina Elena, 324, Rome 00161 Italy, (117) Bruno Maresca, International Institute of Genetics and Biophysics, Via MarCOni, 12, Naples 80125 Italy, (243) Gerald Medoff, Department of Infectious Deseases, Washington University, Box 8051, St. Louis 63110, MO USA, (215) Sergio Moreno, Dept. of Biochemistry University of Oxford , South Parks Road, Oxford OX1 3QU U.K., (3) N. Ronald Morris, Dept. ofPharmacology, UMDNJ -RWJMS, Piscataway 08854- 5635, NJ USA, (97) Somay Y. Murayama, Dept. of Microbiology, Teikyo University School of Medicine, 2-11-1 Kaga Itabashi-ku Tokyo 173 Japan, (149) Yoshinori Ohsumi, Dept. of Biology, College of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku Tokyo 153 Japan, (39) Luciano Polonelli, Istituto di Microbiologia Facolta di Medicina e Chirurgia, Via Gramsci, 14, Parma 40300 Italy, (141) XIV

JolmPulitzer, International Institute of Genetics and Biophysics, Via Marconi, 12, Naples 80125 Italy, (75) Jakob Reiser, Institut fUr Biotechnologie ETH -Honggerberg, Zurich CH-8093 Switzerland, (31) Claudina Rodrigues-Pousada, Instituto Gulbenkian de Ciencia, Rua da QUinta Grande Apartado 14, Oeiras Codex 2781 Portugal, (7) PaulE. Steele, Dept. ofPathology School ofMedicine, UniversityofCincinnatti, 231 Bethesda Ave., Cincinnatti 45267-0529, OH USA, (227) PaulJ. Szaniszlo, Dept. of Microbio}ogy, University of Texas Austin, Austin 78712, TX USA, (229) Masamichi Takagi, Dept. ofAgricultural Chemistry, The University ofTokyo , Yayoi, Bunkyo-ku Tokyo 113 Japan, (13) Geoffrey furner, The University of Sheffield, Dept. of Molecular Biology and Biotech., Sheffield SlO 2TN U.K., (125) Hugo Vanden Bossche, Dept. of Comparative Biochem., Janssen Res. Foundation, Turnhoutseweg 30, Beerse B-2340 Belgium, (179) Hideyo Yamaguchi, Res. Center for Medical Mycology, Teikyo University School of Medicine, 359 Otsuka, Tokyo 192-03 HachiojiJapan, (169)