![4
mammalian models to study highly pathogenic bacteria is being
complicated: it requires specific laboratory structures (Animal
Biosafety Level 3 facility); it is time consuming, expensive and par-
ticularly raises ethical considerations. For these reasons, new infec-
tion models using insects have been developed (Drosophila
melanogaster, Galleria mellonella, silkworm larva) [1]. The larva of
the greater wax moth Galleria mellonella is one of the most used
insect models to study virulence, due to several advantages. These
larvae are cheap, do not need food or water in their last instar lar-
vae, and have a good size to permit precise injection of pathogens
or compounds and infection studies do not require an Animal
Biosafety laboratory. After infection, they can be kept at a wide
range of temperatures (up to 37 °C) which insures optimal expres-
sion of pathogen virulence factors and mimics temperature
conditions in mammalian hosts. G. mellonella can be infected by a
wide range of pathogens including fungi (Aspergillus fumigatus,
Cryptococcus neoformans, Candida albicans) [2–4] and bacteria
(including Legionella pneumophila, Listeria spp., Burkholderia cepa-
cia complex, Yersinia pseudotuberculosis) [5–8]. Even though
insects do not have an adaptive immune response like mammals,
they have a complex innate immune system with similarities to that
of mammals [9]. The Galleria innate immune system is composed
of hemocytes which can phagocytose and encapsulate pathogens
and a humoral response with the secretion of antimicrobial peptides,
melanization, and coagulation of the hemolymph. Furthermore, for
several pathogens good correlation between virulence in Galleria
and mammalian models has been shown [6, 10].
In summary, G. mellonella is a powerful good alternative to
mammalian models to study virulence factors of mammalian patho-
gens. Here, we present a protocol to analyze virulence of B. meli-
tensis, B. suis, F. tularensis, B. mallei and B. pseudomallei by
injection of bacterial suspension in the hemocoel.
2 Materials
1. Brucella melitensis 16 M T
ATCC 23456.
2. Brucella suis 1330T
ATCC 23444.
3. Francisella tularensis SCHUS4 (laboratory collection).
4. Burkholderia mallei ATCC 23344.
5. Burkholderia pseudomallei SID 4718 (clinical isolate).
6. Larvae of the great wax moth Galleria mellonella in last-instar
larvae (15–20 per strain tested).
1. Infusion pump KDS 100Y (Kd Scientific, Holliston, MA, USA)
remotely triggered by a foot switch (see Note 1).
2. 1 mL tuberculin syringe (Terumo, Tokyo, Japan).
2.1 Bacterial Strains
and Insects
2.2 Injection
Apparatus
and Equipment
Nicolas Sprynski et al.](https://image.slidesharecdn.com/58723-250319220738-639f2efd/85/Host-Bacteria-Interactions-Methods-and-Protocols-1st-Edition-Annette-C-Vergunst-22-320.jpg)
![7
4 Notes
1. For safety, we highly recommend the use of the Venofix®
A with
an infusion pump remotely triggered by a foot switch. This
system allows the manipulator to hold the caterpillar with one
hand, insert the needle with the other, and start the injection
with their foot. This is more comfortable and safer than other
described methods [11].
2. All operations with B. melitensis, B. suis, F. tularensis, B. mallei
and B. pseudomallei must be done in a Biosafety Level 3 labora-
tory in a microbiological safety cabinet.
3. Correlation of the OD600 with the CFU depends on the bacte-
rial strain, the age of the culture, the medium, and the spectro-
photometer used. We find that for an OD600 of 1, CFUs are
5.109
/mL for B. melitensis and B. suis, 1.109
/mL for F. tular-
ensis, and 2.109
/mL for B. mallei and B. pseudomallei. This has
to be determined empirically for each species and strain used.
4. Food privation has a direct implication in the immune
response. Larvae deprived of food have a reduced immune
response and thus an increased susceptibility to infection [12].
In order to compare different infection experiments, keep the
same time of fasting before infection. Here, we propose 24 h
as standard.
5. To remove the cocoon of the caterpillars, use small forceps to
carefully tear up the cocoon. Pieces of cocoon are light and
volatile. In order to avoid aspiration of pieces of cocoon by the
microbiological safety cabinet, put the pieces of cocoon on a
tissue soaked in ethanol.
6. Washes, resuspension in PBS of the bacterial culture, and disinfec-
tion of the larvae are important steps to avoid humoral response
of the caterpillars that are not specific to the pathogen tested. This
nonspecific activation of G. mellonella humoral response can lead
to an important decrease in virulence in the assay.
7. For the injection, insert 2–3 mm of the needle in the hindmost
left proleg of the larvae in the direction of the head (see Fig. 1).
Activate the injection with the foot switch. At the end of the
injection, gently remove the needle. Sometimes, a small
amount of hemolymph bleeds from the injection point. This
phenomenon does not affect the infection assay. If the needle
passes through the larva, exclude the caterpillar from the
experiment.
8. The group of PBS-injected larvae are essential to show
pathogen-specific death of caterpillars. Do not use the data if
one of the control-injected larva dies due to injection injury.
9. Sick larvae make fewer or no cocoons.
Galleria mellonella as an Infection Model](https://image.slidesharecdn.com/58723-250319220738-639f2efd/85/Host-Bacteria-Interactions-Methods-and-Protocols-1st-Edition-Annette-C-Vergunst-25-320.jpg)
Host Bacteria Interactions Methods and Protocols 1st Edition Annette C. Vergunst
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- 5. Host Bacteria Interactions Methods and Protocols 1st Edition Annette C. Vergunst Digital Instant Download Author(s): Annette C. Vergunst, David O'Callaghan (eds.) ISBN(s): 9781493912605, 1493912607 Edition: 1 File Details: PDF, 9.43 MB Year: 2014 Language: english
- 6. Host-Bacteria Interactions Annette C.Vergunst David O‘Callaghan Editors Methods and Protocols Methods in Molecular Biology 1197
- 7. ME T H O D S I N MO L E C U L A R BI O LO G Y 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
- 9. Host-Bacteria Interactions Methods and Protocols Edited by Annette C. Vergunst and David O’Callaghan INSERM,U1047,Nîmes,FranceandUniversitéMontpellier1,UFRMédecine,Nîmes,France
- 10. ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-1260-5 ISBN 978-1-4939-1261-2 (eBook) DOI 10.1007/978-1-4939-1261-2 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014945547 © Springer Science+Business Media New York 2014 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Editors Annette C. Vergunst INSERM, U1047 Nîmes, France and Université Montpellier 1 UFR Médecine Nîmes, France David O’Callaghan INSERM, U1047 Nîmes, France and Université Montpellier 1 UFR Médecine Nîmes, France
- 11. v Bacterial infections are a complex interplay between host and pathogen. Over the last 20 years, there have been great advances in our understanding of the pathogenesis of infec- tious disease by integrating detailed knowledge of bacterial genetics, cell biology, immunol- ogy, and host physiology. This has led to the development of the new field of “cellular microbiology.” Early studies began with well-studied organisms such as Salmonella, Shigella, and Listeria and used simple cell culture models such as HeLa cells; however, this rapidly extended to other pathogens and to a wide array of cell types. Cellular microbiology has been instrumental in the identification of bacterial virulence factors required for interaction with the host, their cellular targets, and how the interactions can modulate host cell biology in favor of the pathogen. Recent advances now make it possible to study in great detail the infection in vivo, at the cellular, tissue, and whole animal level. In this volume we have brought together a set of cutting edge protocols that cover aspects of the investigation of host–bacteria interactions using mammalian and novel non- mammalian infection models, cell biology, OMICS, and bacterial genetics. Our aim is to provide a pathway through the techniques that can be used to investigate different aspects of the physiopathology of bacterial infections, from the whole animal to tissue, cellular, and molecular levels. The pathogens used in the protocols are mainly, but not exclusively facul- tative and obligate intracellular bacteria, for which we are trying to decipher how the intra- cellular stages of a pathogen contribute to disease. However, the protocols are generally applicable to most other pathogens. Since the principal goal of the book is to provide researchers with a comprehensive account of the practical steps necessary for carrying out each protocol successfully, the Methods section contains detailed step-by-step descriptions of every protocol. The Notes section complements the Methods with tips based on the authors first-hand experience explaining the “tricks of the trade” and the best ways to deal with any problem or difficulty that might arise. From the earliest times, infection models have been instrumental in understanding infectious disease. Over recent years, there has been a move away from classical models using mammals and mammalian cells to nonvertebrate systems. In this volume, chapters in Part I will describe how to use Galleria (wax moth) larvae (Chapter 1) or Drosophila as infection models (Chapter 2), whereas the non-animal models using amoeba (Chapter 9) or plants (Chapters 6 and 11) are included in Part II. The zebrafish has recently emerged as a model where we can exploit both the genetic tractability and optical transparency of developing embryos to follow the infection and assess the role of both host and pathogen factors in real time at the cellular and whole animal level (Chapter 3). Advances in live imag- ing techniques have also allowed the development of mammalian systems where lumines- cent bacteria or cells can be seen in the body using highly sensitive cameras (Chapter 4). Two photon microscopy now allows the observation of events at the cellular level in living tissue (Chapter 5). To fully understand bacterial virulence, it is essential to investigate the host–pathogen interaction at the cellular and molecular level (Part II). Using plant or yeast cells as a surrogate model, we can identify and characterize the bacterial proteins, or effectors, Preface
- 12. vi translocated into host cells through bacterial secretion systems to understand how the bacterium tries to manipulate host defense mechanisms to create its own niche (Chapter 6). We present a protocol that can be used to identify the host targets of a bacterial virulence factor, either when exposed on the pathogen surface or injected into the cell (Chapter 7). We also present protocols to show how pathogens modulate key host cell processes including protein degradation through the proteasome (Chapter 8), phosphoinositide dynamics (Chapter 9), and apoptosis (Chapter 10). Using bimolecular fluorescence com- plementation, in vivo interactions between host and bacterial proteins can be identified (Chapter 11). There is also a protocol to examine how bacterial pathogens can modulate innate immune signaling through the TLR pathway (Chapter 12). Technological advances have led to an explosion in the quantity and complexity of OMICS data that can be generated (Part III). A Drosophila cell line can be used for siRNA screens (Chapter 13) to identify host factors required for the infection. Two chapters describe the purification of bacteria for proteomic or RNAseq from infected cells (Chapter 14), and the isolation of host phagocytes for RNAseq from zebra fish embryos using FACS sort- ing (Chapter 15). We also include a protocol for rapid sample production for high- throughput proteomic analysis and data extraction (Chapter 16). Exploiting the masses of data generated in these studies requires powerful bioinformatics support. Chapter 17 describes PATRIC, an NIH-funded database dedicated to OMICS data from pathogens. Genetic manipulation of the bacterial pathogen is crucial to elucidate the molecular basis of bacteria–host interactions (Part IV). We include three protocols (Chapters 18–20) describing techniques to manipulate bacteria that are either highly recalcitrant or obligate intracellular. We would like to thank all the contributors, who are leading researchers in the field and have either developed, or are expert users of the presented methods, for providing their comprehensive protocols and tips for this volume. We would like to take the opportunity to thank Dr. John Walker, the Editor-in-Chief of the Methods in Molecular Biology series, for giving us the opportunity to edit this volume and his constant support. We hope you enjoy this volume of Methods in Molecular Biology. Nîmes, France Annette C. Vergunst David O’Callaghan Preface Preface
- 13. vii Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I INFECTION MODELS TO STUDY BACTERIAL VIRULENCE 1 Galleria mellonella as an Infection Model for Select Agents . . . . . . . . . . . . . . . 3 Nicolas Sprynski, Eric Valade, and Fabienne Neulat-Ripoll 2 Drosophila as a Model for Intestinal Infections. . . . . . . . . . . . . . . . . . . . . . . . . 11 Matthieu Lestradet, Kwang-Zin Lee, and Dominique Ferrandon 3 Zebrafish Embryos as a Model to Study Bacterial Virulence. . . . . . . . . . . . . . . 41 Jennifer Mesureur and Annette C. Vergunst 4 Studying Host-Pathogen Interaction Events in Living Mice Visualized in Real Time Using Biophotonic Imaging. . . . . . . . . . . . . . . . . . . . 67 Gary Splitter, Jerome Harms, Erik Petersen, Diogo Magnani, Marina Durward, Gireesh Rajashekara, and Girish Radhakrishnan 5 Intravital Two-Photon Imaging to Understand Bacterial Infections of the Mammalian Host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Ferdinand X. Choong and Agneta Richter-Dahlfors PART II CELLULAR ASPECTS OF HOST-BACTERIA INTERACTIONS 6 Cre Reporter Assay for Translocation (CRAfT): A Tool for the Study of Protein Translocation into Host Cells. . . . . . . . . . . . . . . . . . . 103 Amke den Dulk-Ras, Annette C. Vergunst, and Paul J.J. Hooykaas 7 Detection of the Interaction Between Host and Bacterial Proteins: Eukaryotic Nucleolin Interacts with Francisella Elongation Factor Tu . . . . . . . 123 Monique Barel and Alain Charbit 8 Hijacking the Host Proteasome for the Temporal Degradation of Bacterial Effectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Tomoko Kubori, Andree M. Hubber, and Hiroki Nagai 9 Live Cell Imaging of Phosphoinositide Dynamics During Legionella Infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Stephen Weber and Hubert Hilbi 10 Investigating Interference with Apoptosis Induction by Bacterial Proteins . . . . 169 Hua Niu and Yasuko Rikihisa 11 Bimolecular Fluorescence Complementation for Imaging Protein Interactions in Plant Hosts of Microbial Pathogens . . . . . . . . . . . . . . . 185 Lan-Ying Lee and Stanton B. Gelvin Contents
- 14. viii 12 Investigating TLR Signaling Responses in Murine Dendritic Cells Upon Bacterial Infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 Suzana Pinto Salcedo and Lena Alexopoulou PART III OMICS AND LARGE SCALE SCREENING 13 siRNA Screens Using Drosophila Cells to Identify Host Factors Required for Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 Aseem Pandey, Sheng Li Ding, Thomas A. Ficht, and Paul de Figueiredo 14 Purification of Intracellular Bacteria: Isolation of Viable Brucella abortus from Host Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Esteban Chaves-Olarte, Pamela Altamirano-Silva, Caterina Guzmán-Verri, and Edgardo Moreno 15 RNA Sequencing of FACS-Sorted Immune Cell Populations from Zebrafish Infection Models to Identify Cell Specific Responses to Intracellular Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 Julien Rougeot, Ania Zakrzewska, Zakia Kanwal, Hans J. Jansen, Herman P. Spaink, and Annemarie H. Meijer 16 Taking the Shortcut for High-Throughput Shotgun Proteomic Analysis of Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 Erica Marie Hartmann, François Allain, Jean-Charles Gaillard, Olivier Pible, and Jean Armengaud 17 Comparative Genomic Analysis at the PATRIC, A Bioinformatic Resource Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Alice R. Wattam, Joseph L. Gabbard, Maulik Shukla, and Bruno W. Sobral PART IV APPROACHES FOR DIFFICULT BACTERIA 18 A Markerless Deletion Method for Genetic Manipulation of Burkholderia cenocepacia and Other Multidrug-Resistant Gram-Negative Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 Daniel F. Aubert, Mohamad A. Hamad, and Miguel A. Valvano 19 Gene Inactivation in Coxiella burnetii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 Paul A. Beare and Robert A. Heinzen 20 A Chemical Mutagenesis Approach to Identify Virulence Determinants in the Obligate Intracellular Pathogen Chlamydia trachomatis. . . . . . . . . . . . . 347 Bidong Nguyen and Raphael Valdivia Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 Contents Contents
- 15. ix LENA ALEXOPOULOU • Centre d’Immunologie de Marseille-Luminy, Aix-Marseille Université UM 2, Marseille, France FRANÇOIS ALLAIN • CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze, France; Bertin Technologies, Montigny-le-Bretonneu, France PAMELA ALTAMIRANO-SILVA • Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica; Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica; Centro de Investigación en Estructuras Microscópicas, Universidad de Costa Rica, San José, Costa Rica JEAN ARMENGAUD • CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze, France DANIEL F. AUBERT • Centre for Human Immunology, University of Western Ontario, London, ON, Canada; Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada MONIQUE BAREL • INSERM U1002, Unité de Pathogénie des Infections Systémiques, Université Paris Descartes, Paris, France PAUL A. BEARE • Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA ALAIN CHARBIT • INSERM U1002, Unité de Pathogénie des Infections Systémiques, Université Paris Descartes, Paris, France ESTEBAN CHAVES-OLARTE • Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica; Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica; Centro de Investigación en Estructuras Microscópicas, Universidad de Costa Rica, San José, Costa Rica FERDINAND X. CHOONG • Department of Neuroscience, Swedish Medical Nanoscience Center, Karolinska Institutet, Stockholm, Sweden SHENG LI DING • Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX, USA; Norman Borlaug Center, Texas A&M University, College Station, TX, USA; Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX, USA AMKE DEN DULK-RAS • Sylvius Laboratory, Institute of Biology Leiden (IBL), Leiden University, Leiden, The Netherlands MARINA DURWARD • Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI, USA DOMINIQUE FERRANDON • UPR9022 du CNRS, Université de Strasbourg, Strasbourg, France THOMAS A. FICHT • Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, USA Contributors
- 16. x PAUL DE FIGUEIREDO • Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, USA; Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX, USA; Norman Borlaug Center, Texas A&M University, College Station, TX, USA; Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M Health Science Center, College Station, TX, USA JOSEPH L. GABBARD • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA JEAN-CHARLES GAILLARD • CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze, France STANTON B. GELVIN • Department of Biological Sciences, Purdue University, West Lafayette, IN, USA CATERINA GUZMÁN-VERRI • Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica MOHAMAD A. HAMAD • Centre for Human Immunology, University of Western Ontario, London, ON, Canada; Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada JEROME HARMS • Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI, USA ERICA MARIE HARTMANN • CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze, France ROBERT A. HEINZEN • Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA HUBERT HILBI • Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich, Germany PAUL J.J. HOOYKAAS • Sylvius Laboratory, Institute of Biology Leiden (IBL), Leiden University, Leiden, The Netherlands ANDREE M. HUBBER • Research Institute for Microbial Diseases, Osaka University, Osaka, Japan HANS J. JANSEN • Institute of Biology, Leiden University, Leiden, The Netherlands ZAKIA KANWAL • Institute of Biology, Leiden University, Leiden, The Netherlands TOMOKO KUBORI • Research Institute for Microbial Diseases, Osaka University, Osaka, Japan KWANG-ZIN LEE • UPR9022 du CNRS, Université de Strasbourg, Strasbourg, France LAN-YING LEE • Department of Biological Sciences, Purdue University, West Lafayette, IN, USA MATTHIEU LESTRADET • UPR9022 du CNRS, Université de Strasbourg, Strasbourg, France DIOGO MAGNANI • University of Miami, Miami, FL, USA ANNEMARIE H. MEIJER • Institute of Biology, Leiden University, Leiden, The Netherlands JENNIFER MESUREUR • INSERM, U1047, Nîmes, France and Université Montpellier 1, UFR Médecine, Nîmes, France EDGARDO MORENO • Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica; Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica HIROKI NAGAI • Research Institute for Microbial Diseases, Osaka University, Osaka, Japan FABIENNE NEULAT-RIPOLL • Unité de Bactériologie, Institut de Recherche Biomédicale des Armées, La Tronche, France; UMR_MD-1, Facultés de Médecine et de Pharmacie, IRBA, Aix-Marseille Université, Marseille, France Contributors Contributors
- 17. xi BIDONG NGUYEN • Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA HUA NIU • Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, USA ASEEM PANDEY • Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, USA ERIK PETERSEN • Department of Medicine, University of Washington, Seattle, WA, USA OLIVIER PIBLE • CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze, France GIRISH RADHAKRISHNAN • National Institute of Animal Biotechnology, University of Hyderabad, Hyderabad, India GIREESH RAJASHEKARA • Ohio State University, Wooster, OH, USA AGNETA RICHTER-DAHLFORS • Department of Neuroscience, Swedish Medical Nanoscience Center, Karolinska Institutet, Stockholm, Sweden YASUKO RIKIHISA • Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, USA JULIEN ROUGEOT • Institute of Biology, Leiden University, Leiden, The Netherlands SUZANA PINTO SALCEDO • Bases Moléculaires et Structurales des Systèmes Infectieux, CNRS UMR 5086, Institut de Biologie et Chimie des Protéines, IBCP, Université Lyon 1, Lyon, France MAULIK SHUKLA • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA BRUNO W. SOBRAL • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA HERMAN P. SPAINK • Institute of Biology, Leiden University, Leiden, The Netherlands GARY SPLITTER • Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI, USA NICOLAS SPRYNSKI • Unité de Bactériologie, Institut de Recherche Biomédicale des Armées, La Tronche, France; UMR_MD-1, Facultés de Médecine et de Pharmacie, IRBA, Aix-Marseille Université, Marseille, France; Antabio, Labège, France ERIC VALADE • Unité de Bactériologie, Institut de Recherche Biomédicale des Armées, La Tronche, France; UMR_MD-1, Facultés de Médecine et de Pharmacie, IRBA, Aix-Marseille Université, Marseille, France; Ecole du Val-de-Grâce, Paris, France RAPHAEL VALDIVIA • Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA MIGUEL A. VALVANO • Centre for Human Immunology, University of Western Ontario, London, ON, Canada; Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada; Centre for Infection and Immunity, Queen’s University Belfast, Belfast, UK ANNETTE C. VERGUNST • INSERM, U1047, Nîmes, France and Université Montpellier 1, UFR Médecine, Nîmes, France ALICE R. WATTAM • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA STEPHEN WEBER • Max von Pettenkofer Institute, Ludwig-Maximilians University, Munich, Germany ANIA ZAKRZEWSKA • Institute of Biology, Leiden University, Leiden, The Netherlands Contributors Contributors
- 19. Part I Infection Models to Study Bacterial Virulence
- 21. 3 Annette C. Vergunst and David O’Callaghan (eds.), Host-Bacteria Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 1197, DOI 10.1007/978-1-4939-1261-2_1, © Springer Science+Business Media New York 2014 Chapter 1 Galleria mellonella as an Infection Model for Select Agents Nicolas Sprynski, Eric Valade, and Fabienne Neulat-Ripoll Abstract The use of animal models is a key step to better understand bacterial virulence factors and their roles in host/pathogen interactions. To avoid the ethical and cost problems of mammalian models in bacterial virulence research, several insect models have been developed. One of these models, the larvae of the greater wax moth Galleria mellonella, has been shown to be relevant for several fungal and bacterial mam- malian pathogens. Here, we describe the use G. mellonella to study virulence of the highly virulent faculta- tive intracellular bacterial pathogens: Brucella suis, Brucella melitensis, Francisella tularensis, Burkholderia mallei, and Burkholderia pseudomallei. Key words Galleria mellonella, Infection model, Insect model, Brucella, Burkholderia, Francisella Abbreviations TS Trypticase soy BHI Brain–heart infusion CFU Colony-forming units MOI Multiplicity of infection PBS Phosphate-buffered saline 1 Introduction Francisellatularensis, Brucellasuis,Brucellamelitensis,Burkholderia mallei, and Burkholderia pseudomallei are highly pathogenic bacteria that have been classified A or B bioterrorism agents by the CDC (Center for Disease Control and Prevention). Study of these pathogens is crucial to better understand their virulence and to develop new therapies. In addition to in vitro approaches, infection models are necessary to understand the role of virulence factors at the whole organism level. Mammalian models are the paradigm for infectious disease studies due to the close relationship to the natural host (human or other mammals). Nevertheless, the use of
- 22. 4 mammalian models to study highly pathogenic bacteria is being complicated: it requires specific laboratory structures (Animal Biosafety Level 3 facility); it is time consuming, expensive and par- ticularly raises ethical considerations. For these reasons, new infec- tion models using insects have been developed (Drosophila melanogaster, Galleria mellonella, silkworm larva) [1]. The larva of the greater wax moth Galleria mellonella is one of the most used insect models to study virulence, due to several advantages. These larvae are cheap, do not need food or water in their last instar lar- vae, and have a good size to permit precise injection of pathogens or compounds and infection studies do not require an Animal Biosafety laboratory. After infection, they can be kept at a wide range of temperatures (up to 37 °C) which insures optimal expres- sion of pathogen virulence factors and mimics temperature conditions in mammalian hosts. G. mellonella can be infected by a wide range of pathogens including fungi (Aspergillus fumigatus, Cryptococcus neoformans, Candida albicans) [2–4] and bacteria (including Legionella pneumophila, Listeria spp., Burkholderia cepa- cia complex, Yersinia pseudotuberculosis) [5–8]. Even though insects do not have an adaptive immune response like mammals, they have a complex innate immune system with similarities to that of mammals [9]. The Galleria innate immune system is composed of hemocytes which can phagocytose and encapsulate pathogens and a humoral response with the secretion of antimicrobial peptides, melanization, and coagulation of the hemolymph. Furthermore, for several pathogens good correlation between virulence in Galleria and mammalian models has been shown [6, 10]. In summary, G. mellonella is a powerful good alternative to mammalian models to study virulence factors of mammalian patho- gens. Here, we present a protocol to analyze virulence of B. meli- tensis, B. suis, F. tularensis, B. mallei and B. pseudomallei by injection of bacterial suspension in the hemocoel. 2 Materials 1. Brucella melitensis 16 M T ATCC 23456. 2. Brucella suis 1330T ATCC 23444. 3. Francisella tularensis SCHUS4 (laboratory collection). 4. Burkholderia mallei ATCC 23344. 5. Burkholderia pseudomallei SID 4718 (clinical isolate). 6. Larvae of the great wax moth Galleria mellonella in last-instar larvae (15–20 per strain tested). 1. Infusion pump KDS 100Y (Kd Scientific, Holliston, MA, USA) remotely triggered by a foot switch (see Note 1). 2. 1 mL tuberculin syringe (Terumo, Tokyo, Japan). 2.1 Bacterial Strains and Insects 2.2 Injection Apparatus and Equipment Nicolas Sprynski et al.
- 23. 5 3. Venofix® A 27G (Braun, Melsungen, Germany) (see Note 1). 4. Disposable sterile fields. 5. Petri dishes. 6. Microfuge. 7. Pliers. 8. Cotton swabs. 9. 70 % (v/v) Ethanol for disinfection. 10. 37 °C incubator. 11. PSM. 12. Fully equipped BLS3 laboratory. 13. Labeling tape. 1. Tryptic soy (TS) broth and agar: Prepare 1.6 % TS agar plates. 2. IsoVitalex™ (Becton Dickinson): Reconstitute with supplied diluent as described by the manufacturer. After reconstitution, use immediately, or store at 2–8 °C and use within 2 weeks. 3. Brain–heart infusion broth and agar (1.6 %) containing 1 % IsoVitalex™: IsoVitalex™ should be added after autoclaving in a precooled medium (approximately 50 °C). 4. Phosphate-buffered saline (D-PBS) 1×. 3 Methods 1. Brucella and Burkholderia are streaked from a frozen stock to a TS plate and then grown in a 37 °C incubator for 3 and 2 days, respectively. Francisella tularensis is streaked from a frozen stock to a BHI agar plate supplemented with 1 % IsoVitalex™ and grown in a 37 °C incubator for 3 days (see Note 2). 2. The day before G. mellonella infection, a single bacterial colony is inoculated and grown overnight in 5 mL of the appropriate liquid medium culture (TS for B. melitensis, B. suis, B. mallei, and B. pseudomallei; BHI supplemented with 1 % IsoVitalex™ for F. tularensis) in a 37 °C shaking incubator. 3. Harvest the bacteria by centrifugation (3 min at 5,500 g in a benchtop centrifuge), wash in PBS, and dilute in PBS to 5×109 colony-forming units (CFU)/mL for B. melitensis and B. suis, 1×109 CFU/mL for F. tularensis, and 1×104 CFU/mL for B. mallei and B. pseudomallei (see Note 3). 4. Plate several dilutions of bacterial solutions on TS agar plates for Brucella and Burkholderia on BHI with 1 % IsoVitalex™ plates for F. tularensis to verify the CFU of the different solutions. 2.3 Solutions for Bacterial Growth and Preparation 3.1 Bacterial Preparation Galleria mellonella as an Infection Model
- 24. 6 1. The day before infection, remove the cocoons from the G. mellonella larvae (see Note 4) and 15 larvae are distributed in each Petri dish without food. The larvae are stored until infection in the dark at room temperature. 2. On the day of infection, the cocoons of the larvae are removed (see Note 5). 3. Set up the infusion pump: Syringe type (Terumo 1 mL), volume of injection to 10 μL, flow rate to 3.6 mL/h. 4. Transfer the bacterial suspension into the syringe and put it on the infusion pump. Plug the Venofix® A into the syringe and purge the Venofix® into a sterile tube. 5. Take out one larva and put it on a disposable sterile field. The larva injection area (the hindmost left proleg) is disinfected before inoculation using a cotton swab impregnated with 70 % ethanol (see Note 6). Wedge the head of the larva with your index, thumb, and middle finger on the bench. In reaction, the larva will relax itself. Inject 10 μL of the bacterial suspension in the hindmost left proleg (see Note 7) (Fig. 1). Transfer the injected larva in a clean Petri dish. As negative control, inject a group of 15 larvae with PBS solution (see Note 8; Fig. 1). 6. After injection of the larvae group, close the Petri dish with adhesive tape. Incubate the Petri dish in the dark in a 37ºC incubator. 7. Every 24 h remove the cocoon from the larvae (see Note 9) and check the mortality. Caterpillars are considered dead when they display no movement in response to touch (see Note 10). 8. Stop the experiment when all the caterpillars are dead or when caterpillars begin metamorphosis (see Note 11). 3.2 Preparation and Infection of G. mellonella Fig. 1 G. mellonella injection: The caterpillar is wedged on the bench with the thumb, index, and middle finger. Prior to injection, disinfect the injection site on the larva with ethanol. Inject 10 μL of the bacterial suspension in the hindmost left proleg (indicated with an arrow) Nicolas Sprynski et al.
- 25. 7 4 Notes 1. For safety, we highly recommend the use of the Venofix® A with an infusion pump remotely triggered by a foot switch. This system allows the manipulator to hold the caterpillar with one hand, insert the needle with the other, and start the injection with their foot. This is more comfortable and safer than other described methods [11]. 2. All operations with B. melitensis, B. suis, F. tularensis, B. mallei and B. pseudomallei must be done in a Biosafety Level 3 labora- tory in a microbiological safety cabinet. 3. Correlation of the OD600 with the CFU depends on the bacte- rial strain, the age of the culture, the medium, and the spectro- photometer used. We find that for an OD600 of 1, CFUs are 5.109 /mL for B. melitensis and B. suis, 1.109 /mL for F. tular- ensis, and 2.109 /mL for B. mallei and B. pseudomallei. This has to be determined empirically for each species and strain used. 4. Food privation has a direct implication in the immune response. Larvae deprived of food have a reduced immune response and thus an increased susceptibility to infection [12]. In order to compare different infection experiments, keep the same time of fasting before infection. Here, we propose 24 h as standard. 5. To remove the cocoon of the caterpillars, use small forceps to carefully tear up the cocoon. Pieces of cocoon are light and volatile. In order to avoid aspiration of pieces of cocoon by the microbiological safety cabinet, put the pieces of cocoon on a tissue soaked in ethanol. 6. Washes, resuspension in PBS of the bacterial culture, and disinfec- tion of the larvae are important steps to avoid humoral response of the caterpillars that are not specific to the pathogen tested. This nonspecific activation of G. mellonella humoral response can lead to an important decrease in virulence in the assay. 7. For the injection, insert 2–3 mm of the needle in the hindmost left proleg of the larvae in the direction of the head (see Fig. 1). Activate the injection with the foot switch. At the end of the injection, gently remove the needle. Sometimes, a small amount of hemolymph bleeds from the injection point. This phenomenon does not affect the infection assay. If the needle passes through the larva, exclude the caterpillar from the experiment. 8. The group of PBS-injected larvae are essential to show pathogen-specific death of caterpillars. Do not use the data if one of the control-injected larva dies due to injection injury. 9. Sick larvae make fewer or no cocoons. Galleria mellonella as an Infection Model
- 26. Other documents randomly have different content
- 27. FOOTNOTES: 1 For example, in the New Jersey State Penitentiary, the plan of which was inspected by the English and French commissioners, the warden’s department is in immediate connection with the observatory. 2 See our April Number, pp. 84, 85. 3 In those States where it is usual to transfer insane convicts to a Lunatic Asylum, the boon would certainly have been extended to two of our convicts, and thus the institution in which the mental disease originated would not have had to account for its physical termination. This fact should be remembered, when comparing our state of health with similar establishments, as it shows that the Eastern Penitentiary has always had a double mortality to account for, that which is due to it as a penal institution, as well as that which properly belongs to an Insane Asylum. 4 Published by Acting Committee of the Philadelphia Society. Transcriber’s Note: Page ii, continuation of paragraph from Page iii joined to start of paragraph.
- 28. Page 96, erratum incorporated into text. Obvious printer errors corrected silently. Inconsistent spelling and hyphenation are as in the original.
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