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      Metabolic and fitness determinants for in vitro growth and intestinal colonization of the bacterial pathogen Campylobacter jejuni

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          Abstract

          Campylobacter jejuni is one of the leading infectious causes of food-borne illness around the world. Its ability to persistently colonize the intestinal tract of a broad range of hosts, including food-producing animals, is central to its epidemiology since most infections are due to the consumption of contaminated food products. Using a highly saturated transposon insertion library combined with next-generation sequencing and a mouse model of infection, we have carried out a comprehensive genome-wide analysis of the fitness determinants for growth in vitro and in vivo of a highly pathogenic strain of C. jejuni. A comparison of the C. jejuni requirements to colonize the mouse intestine with those necessary to grow in different culture media in vitro, combined with isotopologue profiling and metabolic flow analysis, allowed us to identify its metabolic requirements to establish infection, including the ability to acquire certain nutrients, metabolize specific substrates, or maintain intracellular ion homeostasis. This comprehensive analysis has identified metabolic pathways that could provide the basis for the development of novel strategies to prevent C. jejuni colonization of food-producing animals or to treat human infections.

          Author summary

          There is accumulating evidence that in addition to canonical virulence factors such as toxins, adhesins, or invasins, bacterial pathogens utilize specific metabolic traits to colonize and proliferate within their hosts, a concept that is increasingly referred to as “nutritional virulence”. We have used transposon insertion mutagenesis combined with next-generation sequencing, a mouse model of infection, isotopologue profiling, and metabolic flow analysis to obtain a comprehensive view of the metabolic requirements for the intestinal colonization of C. jejuni, a leading cause of food-borne gastroenteritis in industrialized countries. This information could provide the basis to control C. jejuni colonization of food-producing animals or the human host.

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          Most cited references77

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          The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences.

          Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium-properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain-Barré syndrome. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.
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            Tn-seq; high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms

            Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how microorganisms cause disease. Here we present a method (Tn-seq) for accurately determining quantitative genetic interactions on a genome-wide scale in microorganisms. Tn-seq is based on the assembly of a saturated Mariner transposon insertion library. After library selection, changes in frequency of each insertion mutant are determined by sequencing of the flanking regions en masse. These changes are used to calculate each mutant’s fitness. Fitness was determined for each gene of the gram-positive bacterium Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. A genome-wide screen for genetic interactions identified both alleviating and aggravating interactions that could be further divided into seven distinct categories. Due to the wide activity of the Mariner transposon, Tn-seq has the potential to contribute to the exploration of complex pathways across many different species.
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              Identifying genetic determinants needed to establish a human gut symbiont in its habitat.

              The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. To identify microbial genes required for establishment of human symbionts in the gut, we developed an approach (insertion sequencing, or INSeq) based on a mutagenic transposon that allows capture of adjacent chromosomal DNA to define its genomic location. We used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon mutants of a saccharolytic human gut bacterium, Bacteroides thetaiotaomicron, as they established themselves in wild-type and immunodeficient gnotobiotic mice, in the presence or absence of other human gut commensals. In vivo selection transforms this population, revealing functions necessary for survival in the gut: we show how this selection is influenced by community composition and competition for nutrients (vitamin B(12)). INSeq provides a broadly applicable platform to explore microbial adaptation to the gut and other ecosystems.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Validation
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Validation
                Role: MethodologyRole: ValidationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Academic Editor
                Journal
                PLoS Biol
                PLoS Biol
                plos
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, CA USA )
                1544-9173
                1545-7885
                19 May 2017
                May 2017
                19 May 2017
                : 15
                : 5
                : e2001390
                Affiliations
                [1 ]Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America
                [2 ]Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany
                [3 ]Lehrstuhl für Biochemie, Technische Universität München, Garching, Germany
                [4 ]Microbial Sciences Institute, Yale University School of Medicine, New Haven, Connecticut, United States of America
                Brigham and Women's Hospital, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                [¤a]

                Current address: CAS Key Laboratory of Tropical Marine Bio Resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China

                [¤b]

                Current address: Klinik für Hämatologie und Internistische Onkologie, Friedrich-Schiller-Universität Jena, Jena, Germany

                Author information
                http://orcid.org/0000-0002-6531-0355
                Article
                pbio.2001390
                10.1371/journal.pbio.2001390
                5438104
                28542173
                66dcd7ad-e3ba-4f82-81de-66ebab5eb70b
                © 2017 Gao et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 20 October 2016
                : 24 April 2017
                Page count
                Figures: 7, Tables: 1, Pages: 37
                Funding
                The Ministry of Science and Culture of Lower Saxony (grant number Georg-Christoph-Lichtenberg Scholarship [to JM]). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Deutsche Forschungsgemeinschaft (grant number HO 4553/2-1 [to DH]). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Institute for Allergy and Infectious Diseases (grant number AI R21AI112810 [to JEG]). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
                Medicine and Health Sciences
                Anatomy
                Digestive System
                Gastrointestinal Tract
                Biology and Life Sciences
                Biochemistry
                Proteins
                Recombinant Proteins
                Biology and Life Sciences
                Biochemistry
                Proteins
                Serum Proteins
                Biology and Life Sciences
                Genetics
                Genetic Elements
                Mobile Genetic Elements
                Transposable Elements
                Biology and Life Sciences
                Genetics
                Genomics
                Mobile Genetic Elements
                Transposable Elements
                Biology and Life Sciences
                Biochemistry
                Biosynthesis
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Amino Acid Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Amino Acid Analysis
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Biology and Life Sciences
                Organisms
                Bacteria
                Campylobacter
                Campylobacter Jejuni
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Campylobacter
                Campylobacter Jejuni
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Campylobacter
                Campylobacter Jejuni
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

                Life sciences
                Life sciences

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