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Pubmed Article
The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens.
PLoS ONE
Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis.
Authors: Bidong D. Nguyen, Raphael H. Valdivia.
Published: 10-23-2013
ABSTRACT
Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype–phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.
18 Related JoVE Articles!
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Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches
Authors: Nicolas Jacquier, Sébastien Aeby, Julia Lienard, Gilbert Greub.
Institutions: University Hospital Center and University of Lausanne.
Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.
Immunology, Issue 80, Environmental Microbiology, Soil Microbiology, Water Microbiology, Amoebae, microorganisms, coculture, obligate intracellular bacteria
51055
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The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques
Authors: Annette Prohl, Carola Ostermann, Markus Lohr, Petra Reinhold.
Institutions: Friedrich-Loeffler-Institut.
There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals. The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions. Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.
Medicine, Issue 89, translational medicine, respiratory models, bovine lung, bronchoscopy, transbronchial lung biopsy, bronchoalveolar lavage, bronchial brushing, cytology brush
51557
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A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions
Authors: Mandy Muller, Patricia Cassonnet, Michel Favre, Yves Jacob, Caroline Demeret.
Institutions: Institut Pasteur , Université Sorbonne Paris Cité, Dana Farber Cancer Institute.
Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors. This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells. A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.
Immunology, Issue 77, Genetics, Microbiology, Biochemistry, Molecular Biology, Cellular Biology, Biomedical Engineering, Infection, Cancer Biology, Virology, Medicine, Host-Pathogen Interactions, Host-Pathogen Interactions, Protein-protein interaction, High-throughput screening, Luminescence, Yeast two-hybrid, HT-GPCA, Network, protein, yeast, cell, culture
50404
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Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes
Authors: Andreas Kühbacher, Edith Gouin, Jason Mercer, Mario Emmenlauer, Christoph Dehio, Pascale Cossart, Javier Pizarro-Cerdá.
Institutions: Pasteur Institute, INSERM U604, Institut National de la Recherche Agronomique (INRA), USC2020, ETH Zürich, University of Basel.
Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.
Immunology, Issue 79, HeLa Cells, Listeria monocytogenes, Gram-positive Bacterial Infections, Fluorescence, High-Throughput Screening Assays, RNA Interference, Listeria monocytogenes, Infection, microscopy, small interfering RNA
51043
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Isolation of Lymphocytes from Mouse Genital Tract Mucosa
Authors: Janina Jiang, Kathleen A. Kelly.
Institutions: University of California, Los Angeles , California NanoSystems.
Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse. 
Immunology, Issue 67, Mucosal immunity, sexually transmitted diseases, genital tract lymphocytes, lymphocyte isolation, flow cytometry, FACS
4391
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Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model
Authors: Neveda Naz, Dominic C. Mills, Brendan W. Wren, Nick Dorrell.
Institutions: London School of Hygiene & Tropical Medicine.
The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.
Infection, Issue 80, Gram-Negative Bacteria, Bacterial Infections, Gastrointestinal Diseases, Campylobacter jejuni, bacterial invasion, intestinal epithelial cells, models of infection
50741
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A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium
Authors: Jiuli Zhang, Kailiang Jia.
Institutions: Florida Atlantic University.
In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.
Immunology, Issue 88, C. elegans, Salmonella typhimurium, autophagy, infection, pathogen, host, RNAi
51703
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A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Authors: George Papadopoulos, Carolyn D. Kramer, Connie S. Slocum, Ellen O. Weinberg, Ning Hua, Cynthia V. Gudino, James A. Hamilton, Caroline A. Genco.
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation. Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90, Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
51556
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In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Authors: Mark E. Kusek, Michael A. Pazos, Waheed Pirzai, Bryan P. Hurley.
Institutions: Harvard Medical School, MGH for Children, Massachusetts General Hospital.
Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state.  Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).  The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.
Infection, Issue 83, Cellular Biology, Epithelium, Neutrophils, Pseudomonas aeruginosa, Respiratory Tract Diseases, Neutrophils, epithelial barriers, pathogens, transmigration
50823
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Aseptic Laboratory Techniques: Plating Methods
Authors: Erin R. Sanders.
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: ● Perform plating procedures without contaminating media. ● Isolate single bacterial colonies by the streak-plating method. ● Use pour-plating and spread-plating methods to determine the concentration of bacteria. ● Perform soft agar overlays when working with phage. ● Transfer bacterial cells from one plate to another using the replica-plating procedure. ● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
3064
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A Human Fallopian Tube Model for Investigation of C. trachomatis Infections
Authors: Stefan Jerchel, Gudrun Knebel, Peter König, Michael K. Bohlmann, Jan Rupp.
Institutions: University of Lübeck, University of Lübeck, University of Lübeck, University of Lübeck.
Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues 1,2. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4. Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell- culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5. Further limitations are given by the fact that central signaling cascades (e.g. IFN-γ mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7. We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9. For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.
Medicine, Issue 66, Infection, Microbiology, Physiology, Chlamydia trachomatis, human fallopian tube, tissue model, scanning electron microscopy, transmission electron microscopy
4036
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Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Authors: Maria J. Mazon Moya, Emma Colucci-Guyon, Serge Mostowy.
Institutions: Imperial College London, Institut Pasteur, Unité Macrophages et Développement de l'Immunité.
Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.
Infection, Issue 91, ATG8/LC3, autophagy, cytoskeleton, HeLa cells, p62, septin, Shigella, zebrafish
51601
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Tractable Mammalian Cell Infections with Protozoan-primed Bacteria
Authors: Samuel L. Drennan, Amrita Lama, Ben Doron, Eric D. Cambronne.
Institutions: Oregon Health & Science University.
Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.
Infection, Issue 74, Immunology, Microbiology, Infectious Diseases, Medicine, Cellular Biology, Bacteria, Bacterial Infections, Mycoses, Legionella, amoeba, macrophage, priming, intracellular pathogen, fluorescence microscopy, flow cytometry, cell
50300
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Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Authors: Leah M. Rommereim, Miryam A. Hortua Triana, Alejandra Falla, Kiah L. Sanders, Rebekah B. Guevara, David J. Bzik, Barbara A. Fox.
Institutions: The Geisel School of Medicine at Dartmouth.
Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination. Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4. Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).
Infectious Diseases, Issue 77, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
50598
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Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages
Authors: Maruti Nandan Rai, Sapan Borah, Gaurav Bairwa, Sriram Balusu, Neelima Gorityala, Rupinder Kaur.
Institutions: Centre for DNA Fingerprinting and Diagnostics, Andhra Pradesh, India, Fiers-Schell-Van Montagu Building, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium.
A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.
Immunology, Issue 82, Candida glabrata, THP-1 macrophages, colony forming unit (CFU) assay, fluorescence microscopy, signature-tagged mutagenesis
50625
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A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
Authors: Christophe. J Queval, Ok-Ryul Song, Vincent Delorme, Raffaella Iantomasi, Romain Veyron-Churlet, Nathalie Deboosère, Valérie Landry, Alain Baulard, Priscille Brodin.
Institutions: Université de Lille.
Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read out inferring on the pathogenesis of Mtb within host cells.
Infection, Issue 83, Mycobacterium tuberculosis, High-content/High-throughput screening, chemogenomics, Drug Discovery, siRNA library, automated confocal microscopy, image-based analysis
51114
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Population Replacement Strategies for Controlling Vector Populations and the Use of Wolbachia pipientis for Genetic Drive
Authors: Jason Rasgon.
Institutions: Johns Hopkins University.
In this video, Jason Rasgon discusses population replacement strategies to control vector-borne diseases such as malaria and dengue. "Population replacement" is the replacement of wild vector populations (that are competent to transmit pathogens) with those that are not competent to transmit pathogens. There are several theoretical strategies to accomplish this. One is to exploit the maternally-inherited symbiotic bacteria Wolbachia pipientis. Wolbachia is a widespread reproductive parasite that spreads in a selfish manner at the extent of its host's fitness. Jason Rasgon discusses, in detail, the basic biology of this bacterial symbiont and various ways to use it for control of vector-borne diseases.
Cellular Biology, Issue 5, mosquito, malaria, genetics, infectious disease, Wolbachia
225
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Isolation of Genomic DNA from Mouse Tails
Authors: Tony Zangala.
Institutions: University of California, Irvine (UCI).
Basic Protocols, Issue 6, genomic, DNA, genotyping, mouse
246
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