Methods I N Molecular Biology ™
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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