M ETHODS IN MOLECULAR BIOLOGY™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Host-Fungus Interactions

Methods and Protocols

Edited by Alexandra C. Brand

School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK Donna M. MacCallum

School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK

Editors Alexandra C. Brand, Ph.D. Donna M. MacCallum, Ph.D. School of Medical Sciences School of Medical Sciences Institute of Medical Sciences Institute of Medical Sciences University of Aberdeen University of Aberdeen Foresterhill, Aberdeen, UK Foresterhill, Aberdeen, UK [email protected] [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-538-1 e-ISBN 978-1-61779-539-8 DOI 10.1007/978-1-61779-539-8 Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011945440

© Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

Cover illustration: Confocal microscopy of Candida albicans phagocytosis by J444.1 macrophages. Macrophage acidic organelles are stained with LysoTracker Red (Invitrogen), and the fungal cells with FITC (Sigma). The image was captured after 4 hours and shows C. albicans bound to and internalised within macrophages. Image courtesy of Leanne. E. Lewis.

Printed on acid-free paper

Humana Press is part of Springer Science+Business Media (www.springer.com) Preface

The incidence and profi le of fungal diseases affecting the human population has undergone considerable change within recent decades, refl ecting the availability of interventional medical care and an increase in the prevalence of immunodefi ciency syndromes. Microbiologists, medical mycologists, immunologists, and biochemists are increasingly working together to focus on the processes involved in the progression and treatment of fungal disease. Host– Fungus Interactions: Methods and Protocols is designed for research scientists who are involved in this work and interested in undertaking new or comparative studies of interac- tions between the mammalian host and clinically important fungal pathogens. The aim of this book is to combine approaches for reverse genetics in pathogenic fungi with methods for their application in in vitro and in vivo models of disease. The study of fungal pathogenesis is hugely enhanced by the ability of researchers to employ reverse genetics in the fungi of interest. The fi rst section of this book, therefore, provides methods for the culture and genetic manipulation of the primary fungal patho- gens, Histoplasma capsulatum, Coccidioides immitis , and Paracoccidioides brasiliensis , and the opportunistic pathogens, Aspergillus fumigatus, Cryptococcus neoformans , and Candida albicans ; the latter group emerging as a major cause of morbidity and mortality in countries where medical interventions are on the increase. Gene deletion, mutation, and regulatable expression are valuable molecular tools that can reveal key virulence determinants during interaction with the host. In addition, the use of fl uorescently tagged proteins for cellular localization and as expression reporters offers a wealth of information on fungal biology and spatial/temporal events during infection. Previously, such approaches have been ham- pered by the unavailability of genome sequences, lack of manipulatable sexual cycles, side effects of using auxotrophic selectable markers, low effi ciency of homologous reintegration, and diffi culty in fungal transformation. In light of the need to understand emerging fungal diseases, the past decade has seen signifi cant advances in the availability of methods designed to overcome these challenges in the study of pathogenic fungi, including genome availability with improved annotation, whole-genome screening vectors, auxotrophy-independent selectable markers, effi cient mRNA amplifi cation, RNAi knockdown, and the generation of Ku− strains to yield high rates of homologous recombination. The second section of this book focuses on methods for investigating host–fungus interactions in model systems. Methods are described to investigate direct interactions between host and fungal cells in vitro, using isolated host cells, cell lines, or tissue models to evaluate host cell recognition and response to fungal cells. Such experiments offer a valuable screening method to identify fungal mutants whose phenotypes warrant further investiga- tion in vivo in infection models or allow specifi c interactions to be further dissected. Key methods are also described for determining host and fungal responses occurring during infection, e.g., changes in gene expression. The ability to analyze transcriptional changes during initiation and progression of fungal infection in model systems is greatly increasing our understanding of these processes from the perspective of both host and fungus. Finally, protocols for fungal infection models are described. Considerable progress has been made

v vi Preface in implementing the 3Rs (Replacement, Refi nement, and Reduction) policy in host–fungus interactions, and this section describes validated models for virulence studies using mini- hosts, e.g., nematodes, fruit fl ies, and wax moth larvae. These models allow some analysis of host immune response; however, mammalian models are still required for more accurate modelling of human infection. Protocols for a number of rodent models of invasive and superfi cial fungal infection are described, which have been optimized for the study of dis- ease progression and response to antifungal agents. The rodent models described cover not only intravenous challenge but also inhalation models, which more accurately refl ect the infection route of many free-living pathogenic fungal species. Additional protocols describe refi ned infection models, such as the use of fl uorescent fungi to allow live imaging of infec- tion development, a catheter biofi lm infection model, and a model of concurrent vaginal and oral Candida albicans infection. The aim of this volume is to describe available molecular methods and fungal infection models in suffi cient detail to encourage researchers to try new approaches to investigating host–fungus interactions with confi dence.

Foresterhill, Aberdeen, UK Alexandra C. Brand, Ph.D. Donna M. MacCallum, Ph.D. Contents

Preface...... v Contributors...... xi

PART I GENE DISRUPTION

1 Gene Deletion in Candida albicans Wild-Type Strains Using the SAT1-Flipping Strategy ...... 3 Christoph Sasse and Joachim Morschhäuser 2 Mini-blaster-Mediated Targeted Gene Disruption and Marker Complementation in Candida albicans ...... 19 Shantanu Ganguly and Aaron P. Mitchell 3 Rapid Detection of Aneuploidy Following the Generation of Mutants in Candida albicans ...... 41 Megan D. Lenardon and André Nantel 4 Agrobacterium-Mediated Insertional Mutagenesis in Histoplasma capsulatum ...... 51 Olga Zemska and Chad A. Rappleye 5 Targeted Gene Disruption in Cryptococcus neoformans Using Double-Joint PCR with Split Dominant Selectable Markers...... 67 Min Su Kim, Seo-Young Kim, Kwang-Woo Jung, and Yong-Sun Bahn 6 Multiple Gene Deletion in Cryptococcus neoformans Using the Cre–lox System ...... 85 Lorina G. Baker and Jennifer K. Lodge 7 Gene Disruption in Aspergillus fumigatus Using a PCR-Based Strategy and In Vivo Recombination in Yeast ...... 99 Iran Malavazi and Gustavo Henrique Goldman 8 Targeted Gene Deletion in Aspergillus fumigatus Using the Hygromycin-Resistance Split-Marker Approach...... 119 Fabrice N. Gravelat, David S. Askew, and Donald C. Sheppard 9 Gene Disruption in Coccidioides Using Hygromycin or Phleomycin Resistance Markers...... 131 Chiung-Yu Hung, Hua Zhang Wise, and Garry T. Cole

PART II MODULATION OF GENE EXPRESSION: RNAI GENE KNOCKDOWN

10 RNAi-Based Gene Silencing Using a GFP Sentinel System in Histoplasma capsulatum ...... 151 Brian H. Youseff and Chad A. Rappleye

vii viii Contents

11 RNA Interference in Cryptococcus neoformans ...... 165 Michael L. Skowyra and Tamara L. Doering 12 Gene Knockdown in Paracoccidioides brasiliensis Using Antisense RNA ...... 187 João F. Menino, Agostinho J. Almeida, and Fernando Rodrigues

PART III MODULATION OF GENE EXPRESSION: REGULATABLE PROMOTERS

13 Tetracycline-Inducible Gene Expression in Candida albicans...... 201 Michael Weyler and Joachim Morschhäuser 14 Galactose-Inducible Promoters in Cryptococcus neoformans var. grubii ...... 211 Lorina G. Baker and Jennifer K. Lodge 15 Modular Gene Over-expression Strategies for Candida albicans ...... 227 Vitor Cabral, Murielle Chauvel, Arnaud Firon, Mélanie Legrand, Audrey Nesseir, Sophie Bachellier-Bassi, Yogesh Chaudhari, Carol A. Munro, and Christophe d’Enfert

PART IV HOST RESPONSES TO INFECTION IN VITRO

16 Interactions Between Macrophages and Cell Wall Oligosaccharides of Candida albicans ...... 247 Héctor M. Mora-Montes, Christopher McKenzie, Judith M. Bain, Leanne E. Lewis, Lars P. Erwig, and Neil A.R. Gow 17 Murine Bone Marrow-Derived Dendritic Cells and T-Cell Activation by Candida albicans ...... 261 Joanne Gibson, Neil A.R. Gow, and Simon Y.C. Wong 18 Phagocytosis and Intracellular Killing of Candida albicans by Murine Polymorphonuclear Neutrophils...... 277 Alieke G. Vonk, Mihai G. Netea, and Bart Jan Kullberg 19 Human Oral Keratinocytes: A Model System to Analyze Host–Pathogen Interactions ...... 289 Torsten Wöllert, Christiane Rollenhagen, George M. Langford, and Paula Sundstrom 20 Simple Assays for Measuring Innate Interactions with Fungi ...... 303 Ann M. Kerrigan, Maria da Glória Teixeira de Sousa, and Gordon D. Brown 21 Binding and Uptake of Candida albicans by Human Monocyte-Derived Dendritic Cells ...... 319 Annemiek B. van Spriel and Alessandra Cambi 22 Immune Responses to Candida albicans in Models of In Vitro Reconstituted Human Oral Epithelium ...... 333 Jeanette Wagener, Daniela Mailänder-Sanchez, and Martin Schaller 23 Analysis of Host-Cell Responses by Immunoblotting, ELISA, and Real-Time PCR ...... 345 David L. Moyes and Julian R. Naglik Contents ix

24 In Vitro Model of Invasive Pulmonary Aspergillosis in the Human Alveolus ...... 361 Lea Gregson, William W. Hope, and Susan J. Howard 25 Biofilm Formation Studies in Microtiter Plate Format ...... 369 Marta Riera, Emilia Moreno-Ruiz, Sophie Goyard, Christophe d’Enfert, and Guilhem Janbon

PART V FUNGAL RESPONSES DURING INFECTION

26 Transcript Profiling Using ESTs from Paracoccidioides brasiliensis in Models of Infection...... 381 Alexandre Melo Bailão, Maristela Pereira, Silvia Maria Salem-Izacc, Clayton Luiz Borges, and Célia Maria de Almeida Soares 27 Laser Capture Microdissection of Candida albicans from Host Tissue...... 397 Caroline Westwater and David A. Schofield 28 Isolation and Amplification of Fungal RNA for Microarray Analysis from Host Samples ...... 411 Anja Lüttich, Sascha Brunke, and Bernhard Hube

PART VI HOST RESPONSES TO INFECTION IN VIVO

29 Cytokine Measurement Using Cytometric Bead Arrays ...... 425 Luis Castillo and Donna M. MacCallum 30 Transcript Profiling of the Murine Immune Response to Invasive Aspergillosis...... 435 Zaneeta Dhesi, Susanne Herbst, and Darius Armstrong-James

PART VII NON-MAMMALIAN MODEL SYSTEMS FOR HOST–FUNGAL INTERACTIONS

31 Caenorhabditis elegans: A Nematode Infection Model for Pathogenic Fungi ...... 447 Maged Muhammed, Jeffrey J. Coleman, and Eleftherios Mylonakis 32 Drosophila melanogaster as a Model Organism for Invasive Aspergillosis...... 455 Michail S. Lionakis and Dimitrios P. Kontoyiannis 33 Galleria mellonella as a Model for Fungal Pathogenicity Testing ...... 469 John Fallon, Judy Kelly, and Kevin Kavanagh 34 Embryonated Chicken Eggs as Alternative Infection Model for Pathogenic Fungi ...... 487 Ilse D. Jacobsen, Katharina Große, and Bernhard Hube

PART VIII MAMMALIAN HOSTS AS INFECTION MODELS

35 Mouse Intravenous Challenge Models and Applications ...... 499 Donna M. MacCallum x Contents

36 A Nebulized Intra-tracheal Rat Model of Invasive Pulmonary Aspergillosis ...... 511 Guillaume Desoubeaux and Jacques Chandenier 37 Invasive Models of Histoplasmosis ...... 519 A. George Smulian 38 Murine Model of Concurrent Oral and Vaginal Candida albicans Colonisation ...... 527 Durdana Rahman, Mukesh Mistry, Selvam Thavaraj, Julian R. Naglik, and Stephen J. Challacombe 39 A Luciferase Reporter for Gene Expression Studies and Dynamic Imaging of Superficial Candida albicans Infections ...... 537 Donatella Pietrella, Brice Enjalbert, Ute Zeidler, Sadri Znaidi, Anna Rachini, Anna Vecchiarelli, and Christophe d’Enfert 40 Modeling of Fungal Biofilms Using a Rat Central Vein Catheter ...... 547 Jeniel E. Nett, Karen Marchillo, and David R. Andes 41 Orogastrointestinal Model of Mucosal and Disseminated Candidiasis ...... 557 Karl V. Clemons and David A. Stevens 42 A Nonlethal Murine Cutaneous Model of Invasive Aspergillosis ...... 569 Ronen Ben-Ami and Dimitrios P. Kontoyiannis

Index...... 583 Contributors

AGOSTINHO J. ALMEIDA • Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga , Portugal DAVID R. ANDES • Division of Infectious Diseases, Department of Medicine and Medical Microbiology and Immunology, University of Wisconsin, Madison , WI , USA DARIUS ARMSTRONG-JAMES • Department of Microbiology and Department of Infectious Diseases and Immunity , Imperial College, London , UK DAVID S. ASKEW • Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH , USA SOPHIE BACHELLIER-BASSI • Département Génomes et Génétique, Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France ; INRA USC2019, Paris , France YONG-SUN BAHN • Department of Biotechnology , College of Life Science and Biotechnology, Yonsei University, Seoul , Korea ALEXANDRE MELO BAILÃO • Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICBII, Campus II, Universidade Federal de Goiás, Goiás , Brazil JUDITH M. BAIN • School of Medicine and Dentistry , University of Aberdeen, Aberdeen, UK LORINA G. BAKER • School of Science and Technology, Georgia Gwinett College, Lawrenceville, GA , USA RONEN BEN-AMI • Infectious Disease Unit, Tel Aviv Sourasky Medical Center, Tel Aviv , Israel CLAYTON LUIZ BORGES • Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICBII, Campus II, Universidade Federal de Goiás, Goiás , Brazil GORDON D. BROWN • Section of Immunology and Infection, Institute of Medical Sci- ences, School of Medicine and Dentistry , University of Aberdeen, Aberdeen, UK SASCHA BRUNKE • Department of Microbial Pathogenicity Mechanisms , Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany ; Integrated Research and Treatment Center, Sepsis und Sepsisfolgen, Center for Sepsis Control and Care (CSCC) , Universitätsklinikum Jena, Jena , Germany VITOR CABRAL • Département Génomes et Génétique, Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France ; INRA USC2019, Paris , France ALESSANDRA CAMBI • Department of Tumor Immunology , Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands LUIS CASTILLO • Departamento de Biología, Facultad de Ciencias, Universidad de La Serena, La Serena, Elqui , Chile STEPHEN J. CHALLACOMBE • Department of Oral Medicine, King’s College London Dental Institute, London , UK

xi xii Contributors

JACQUES CHANDENIER • Parasitologie-Mycologie-Médecine tropicale , Centre Hospitalier Régional et Universitaire, Tours , France YOGESH CHAUDHARI • School of Medical Sciences, University of Aberdeen, Aberdeen, UK MURIELLE CHAUVEL • Département Génomes et Génétique, Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France ; INRA USC2019, Paris , France KARL V. CLEMONS • California Institute for Medical Research, San Jose, CA , USA ; Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA , USA ; Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford , CA , USA GARRY T. COLE • Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas, San Antonio, TX , USA JEFFREY J. COLEMAN • Division of Infectious Diseases, Massachusetts General Hospital, Boston , MA , USA CHRISTOPHE D’ENFERT • Département Génomes et Génétique, Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France ; INRA USC2019, Paris , France GUILLAUME DESOUBEAUX • Parasitologie-Mycologie-Médecine tropicale , Centre Hospitalier Régional et Universitaire, Tours , France ZANEETA DHESI • Department of Microbiology and Department of Infectious Diseases and Immunity, Imperial College, London , UK TAMARA L. DOERING • Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO , USA BRICE ENJALBERT • Institut National des Sciences Appliquées, Toulouse , France LARS P. ERWIG • School of Medicine and Dentistry , University of Aberdeen, Aberdeen, UK JOHN FALLON • Medical Mycology Unit, National University of Ireland, Maynooth, County Kildare, Ireland ARNAUD FIRON • Département Génomes et Génétique, Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France ; INRA USC2019, Paris , France SHANTANU GANGULY • Department of Biological Sciences, Carnegie Mellon University , Pittsburgh, PA , USA JOANNE GIBSON • School of Medicine and Dentistry , University of Aberdeen, Aberdeen, UK GUSTAVO HENRIQUE GOLDMAN • Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil NEIL A.R. GOW • School of Medical Sciences, University of Aberdeen, Aberdeen, UK SOPHIE GOYARD • Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France FABRICE N. GRAVELAT • Department of Microbiology and Immunology, McGill University, Montréal , QC , Canada LEA GREGSON • The University of Manchester, Manchester, UK Contributors xiii

KATHARINA GROßE • Department of Microbial Pathogenicity Mechanisms , Leibniz Institute for Natural Product Research and Infection Biology, Jena , Germany SUSANNE HERBST • Department of Microbiology and Department of Infectious Diseases and Immunity, Imperial College, London, UK WILLIAM W. HOPE • The University of Manchester, Manchester, UK SUSAN J. HOWARD • The University of Manchester, Manchester, UK BERNHARD HUBE • Department of Microbial Pathogenicity Mechanisms , Leibniz Institute for Natural Product Research and Infection Biology, Jena , Germany ; Friedrich-Schiller University , Jena , Germany CHIUNG-YU HUNG • Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas, San Antonio, TX , USA ILSE D. JACOBSEN • Department of Microbial Pathogenicity Mechanisms , Leibniz Institute for Natural Product Research and Infection Biology, Jena , Germany GUILHEM JANBON • Institut Pasteur, Unité de Aspergillus, Paris , France KWANG-WOO JUNG • Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul , Korea KEVIN KAVANAGH • Medical Mycology Unit, National University of Ireland, Maynooth , County Kildare, Ireland JUDY KELLY • Medical Mycology Unit, National University of Ireland, Maynooth , County Kildare, Ireland ANN M. KERRIGAN • Section of Immunology and Infection, Institute of Medical Sciences, School of Medicine and Dentistry , University of Aberdeen, Aberdeen, UK MIN SU KIM • Department of Biotechnology, College of Life Science and Biotechnology , Yonsei University, Seoul , Korea SEO-YOUNG KIM • Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul , Korea DIMITRIOS P. KONTOYIANNIS • The University of Texas MD Anderson Cancer Center, Houston, TX , USA BART JAN KULLBERG • Department of Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands GEORGE M. LANGFORD • Department of Biology, Syracuse University, Syracuse, NY , USA MÉLANIE LEGRAND • Département Génomes et Génétique, Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France ; INRA USC2019, Paris , France MEGAN D. LENARDON • School of Medical Sciences, University of Aberdeen, Aberdeen, UK LEANNE E. LEWIS • School of Medicine and Dentistry , University of Aberdeen, Aberdeen, UK MICHAIL S. LIONAKIS • Laboratory of Molecular Immunology , National Institute of Allergy and Infectious Diseases, Bethesda, MD , USA JENNIFER K. LODGE • Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO , USA ANJA LÜTTICH • Department of Microbial Pathogenicity Mechanisms , Leibniz Institute for Natural Product Research and Infection Biology, Jena , Germany xiv Contributors

DONNA M. MACCALLUM • School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK DANIELA MAILÄNDER-SANCHEZ • Department of Dermatology, Eberhard Karls University, Tuebingen, Germany IRAN MALAVAZI • Departamento de Genética e Evolução, Centro de Ciências Biológicas e da Saúde (CCBS), Universidade Federal de São Carlos (UFSCar), São Carlos , SP , Brazil KAREN MARCHILLO • Division of Infectious Diseases, Department of Medicine and Medical Microbiology and Immunology, University of Wisconsin, Madison , WI , USA CHRISTOPHER MCKENZIE • School of Medicine and Dentistry , University of Aberdeen, Aberdeen, UK JOÃO F. MENINO • Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga , Portugal ; ICVS/3B’s PT Government Associate Laboratory , Guimarães, Portugal MUKESH MISTRY • Department of Oral Immunology , King’s College London Dental Institute, London , UK AARON P. MITCHELL • Department of Biological Sciences, Carnegie Mellon University , Pittsburgh, PA , USA HÉCTOR M. MORA-MONTES • Departamento de Biología, Universidad de Guanajuato, Guanajuato, México EMILIA MORENO-RUIZ • Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France JOACHIM MORSCHHÄUSER • Institut für Molekulare Infektionsbiologie, Universität Würzburg, Würzburg, Germany DAVID L. MOYES • Department of Oral Immunology , King’s College London Dental Institute, London , UK MAGED MUHAMMED • Division of Infectious Diseases, Massachusetts General Hospital, Boston , MA , USA CAROL A. MUNRO • School of Medical Sciences, University of Aberdeen, Aberdeen, UK ELEFTHERIOS MYLONAKIS • Division of Infectious Diseases, Massachusetts General Hospital , Boston , MA , USA JULIAN R. NAGLIK • Department of Oral Immunology , King’s College London Dental Institute, London , UK ANDRÉ NANTEL • Biotechnology Research Institute, National Research Council of Canada, Montreal , QC , Canada AUDREY NESSEIR • Département Génomes et Génétique, Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France ; INRA USC2019, Paris , France MIHAI G. NETEA • Department of Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands JENIEL E. NETT • Division of Infectious Diseases, Department of Medicine and Medical Microbiology and Immunology, University of Wisconsin, Madison , WI , USA MARISTELA PEREIRA • Laboratório de Biologia Molecular, Instituto de Ciências Biológi- cas, ICBII, Campus II, Universidade Federal de Goiás , Goiás , Brazil DONATELLA PIETRELLA • Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy Contributors xv

ANNA RACHINI • Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy DURDANA RAHMAN • Department of Oral Immunology , King’s College London Dental Institute, London , UK CHAD A. RAPPLEYE • Department of Microbiology, Ohio State University, Columbus, OH , USA MARTA RIERA • Centre for Research in Agricultural Genomics (CRAG), Campus UAB Bellaterra, Cerdanyola del Valles, Barcelona, Spain FERNANDO RODRIGUES • Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga , Portugal ; ICVS/3B’s PT Government Associate Laboratory , Guimarães, Portugal CHRISTIANE ROLLENHAGEN • VA Medical Center, White River Junction, VT , USA SILVIA MARIA SALEM-IZACC • Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICBII, Campus II, Universidade Federal de Goiás, Goiás , Brazil CHRISTOPH SASSE • Institut für Molekulare Infektionsbiologie, Universität Würzburg, Würzburg, Germany MARTIN SCHALLER • Department of Dermatology, Eberhard Karls University, Tuebingen, Germany DAVID A. SCHOFIELD • Guild Associates Inc., Charleston, SC , USA DONALD C. SHEPPARD • Department of Microbiology and Immunology, McGill University, Montréal , QC , Canada MICHAEL L. SKOWYRA • Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO , USA A. GEORGE SMULIAN • Infectious Disease Division, Cincinnati VA Medical Center, University of Cincinnati, Cincinnati, OH , USA CÉLIA MARIA DE ALMEIDA SOARES • Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICBII, Campus II, Universidade Federal de Goiás, Goiás , Brazil MARIA DA GLÓRIA TEIXEIRA DE SOUSA • Laboratório de Micologia Médica, Instituto de Medicina Tropical, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil ANNEMIEK B. VAN SPRIEL • Department of Tumor Immunology , Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands DAVID A. STEVENS • California Institute for Medical Research, San Jose, CA , USA; Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA , USA; Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford , CA , USA PAULA SUNDSTROM • Microbiology and Molecular Pathogenesis Program, Dartmouth Medical School, Hanover , NH , USA SELVAM THAVARAJ • Department of Oral Pathology , King’s College London Dental Institute, London , UK ANNA VECCHIARELLI • Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy ALIEKE G. VONK • Department of Medical Microbiology and Infectious Diseases , University Medical Center Rotterdam, Rotterdam , The Netherlands JEANETTE WAGENER • Department of Dermatology, Eberhard Karls University, Tuebingen, Germany xvi Contributors

CAROLINE WESTWATER • Department of Craniofacial Biology , Medical University of South Carolina , Charleston, SC , USA MICHAEL WEYLER • Institut für Molekulare Infektionsbiologie, Universität Würzburg, Würzburg, Germany HUA ZHANG WISE • Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas, San Antonio, TX , USA TORSTEN WÖLLERT • Department of Biology, Syracuse University, Syracuse, NY , USA SIMON Y.C. WONG • Section of Immunology and Infection, School of Medicine and Dentistry , University of Aberdeen, Aberdeen, UK BRIAN H. YOUSEFF • Department of Microbiology, Ohio State University, Columbus, OH , USA UTE ZEIDLER • Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France OLGA ZEMSKA • Department of Microbiology, Ohio State University, Columbus, OH , USA SADRI ZNAIDI • Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris , France