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      Gene Fitness Landscapes of Vibrio cholerae at Important Stages of Its Life Cycle

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          Abstract

          Vibrio cholerae has evolved to adeptly transition between the human small intestine and aquatic environments, leading to water-borne spread and transmission of the lethal diarrheal disease cholera. Using a host model that mimics the pathology of human cholera, we applied high density transposon mutagenesis combined with massively parallel sequencing (Tn-seq) to determine the fitness contribution of >90% of all non-essential genes of V. cholerae both during host infection and dissemination. Targeted mutagenesis and validation of 35 genes confirmed our results for the selective conditions with a total false positive rate of 4%. We identified 165 genes never before implicated for roles in dissemination that reside within pathways controlling many metabolic, catabolic and protective processes, from which a central role for glycogen metabolism was revealed. We additionally identified 76 new pathogenicity factors and 414 putatively essential genes for V. cholerae growth. Our results provide a comprehensive framework for understanding the biology of V. cholerae as it colonizes the small intestine, elicits profuse secretory diarrhea, and disseminates into the aquatic environment.

          Author Summary

          Cholera is a deadly diarrheal disease that spreads in explosive epidemics and is caused by the water-borne bacterium Vibrio cholerae. Pathogenic strains of V. cholerae can be found in both fresh and salt water estuaries in-between cholera outbreaks. Cholera infections are frequently derived from contaminated fresh water sources. In this study, we sought to determine on a genome-wide scale how V. cholerae is able to colonize and proliferate in the nutrient-rich environment of the small intestine, but then also survive dissemination and persist in the nutrient-limited aquatic environment. Using a host model that mimics the pathology of human cholera, we utilized genome-wide transposon mutagenesis and massively parallel sequencing of the insertion junctions to obtain the relative fitness of V. cholerae mutants during infection and dissemination. This extensive data set represents the first genetic screen of any kind to identify genes important for dissemination into the environment and has broad significance for understanding and controlling the spread and persistence of Vibrio cholerae and potentially other water-borne pathogens in the environment.

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          Most cited references39

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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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            Coordinating assembly of a bacterial macromolecular machine.

            The assembly of large and complex organelles, such as the bacterial flagellum, poses the formidable problem of coupling temporal gene expression to specific stages of the organelle-assembly process. The discovery that levels of the bacterial flagellar regulatory protein FlgM are controlled by its secretion from the cell in response to the completion of an intermediate flagellar structure (the hook-basal body) was only the first of several discoveries of unique mechanisms that coordinate flagellar gene expression with assembly. In this Review, we discuss this mechanism, together with others that also coordinate gene regulation and flagellar assembly in Gram-negative bacteria.
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              Cholera

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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                December 2013
                December 2013
                26 December 2013
                : 9
                : 12
                : e1003800
                Affiliations
                [1]Howard Hughes Medical Institute and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America
                University of Texas San Antonio, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: HDK DWL AC. Performed the experiments: HDK BPD DWL FWG AC. Analyzed the data: HDK DWL. Contributed reagents/materials/analysis tools: HDK DWL. Wrote the paper: HDK DWL AC.

                Article
                PPATHOGENS-D-13-02091
                10.1371/journal.ppat.1003800
                3873450
                24385900
                36ab2ab4-2273-414b-9e85-f1d5c56e5852
                Copyright @ 2013

                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
                : 3 August 2013
                : 14 October 2013
                Page count
                Pages: 11
                Funding
                This work was supported by US National Institutes of Health grant AI055058 and the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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