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      Tn-Seq reveals hidden complexity in the utilization of host-derived glutathione in Francisella tularensis

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Host-derived glutathione (GSH) is an essential source of cysteine for the intracellular pathogen Francisella tularensis. In a comprehensive transposon insertion sequencing screen, we identified several F. tularensis genes that play central and previously unappreciated roles in the utilization of GSH during the growth of the bacterium in macrophages. We show that one of these, a gene we named dptA, encodes a proton-dependent oligopeptide transporter that enables growth of the organism on the dipeptide Cys-Gly, a key breakdown product of GSH generated by the enzyme γ-glutamyltranspeptidase (GGT). Although GGT was thought to be the principal enzyme involved in GSH breakdown in F. tularensis, our screen identified a second enzyme, referred to as ChaC, that is also involved in the utilization of exogenous GSH. However, unlike GGT and DptA, we show that the importance of ChaC in supporting intramacrophage growth extends beyond cysteine acquisition. Taken together, our findings provide a compendium of F. tularensis genes required for intracellular growth and identify new players in the metabolism of GSH that could be attractive targets for therapeutic intervention.

          Author summary

          Prominent amongst the host-derived nutrients Francisella tularensis requires for intracellular growth is glutathione (GSH), a tripeptide which serves as an essential source of cysteine. Here we comprehensively identify those genes F. tularensis requires for intramacrophage growth and characterize two that play previously unrecognized roles in GSH utilization. One of these encodes a transporter of the dipeptide Cys-Gly which is a breakdown product of GSH, while the other encodes a member of the ChaC family of proteins which we show plays a role in GSH breakdown. Our findings uncover a critical role for a member of the proton-dependent oligopeptide transporter family in Francisella intramacrophage growth and provide the first example of a ChaC family enzyme acting to catabolize GSH in bacteria.

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          Most cited references 70

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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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            Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice.

            The proportion of the human gut bacterial community that is recalcitrant to culture remains poorly defined. In this report, we combine high-throughput anaerobic culturing techniques with gnotobiotic animal husbandry and metagenomics to show that the human fecal microbiota consists largely of taxa and predicted functions that are represented in its readily cultured members. When transplanted into gnotobiotic mice, complete and cultured communities exhibit similar colonization dynamics, biogeographical distribution, and responses to dietary perturbations. Moreover, gnotobiotic mice can be used to shape these personalized culture collections to enrich for taxa suited to specific diets. We also demonstrate that thousands of isolates from a single donor can be clonally archived and taxonomically mapped in multiwell format to create personalized microbiota collections. Retrieving components of a microbiota that have coexisted in single donors who have physiologic or disease phenotypes of interest and reuniting them in various combinations in gnotobiotic mice should facilitate preclinical studies designed to determine the degree to which tractable bacterial taxa are able to transmit donor traits or influence host biology.
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              Glutathione: overview of its protective roles, measurement, and biosynthesis.

              This review is the introduction to a special issue concerning, glutathione (GSH), the most abundant low molecular weight thiol compound synthesized in cells. GSH plays critical roles in protecting cells from oxidative damage and the toxicity of xenobiotic electrophiles, and maintaining redox homeostasis. Here, the functions and GSH and the sources of oxidants and electrophiles, the elimination of oxidants by reduction and electrophiles by conjugation with GSH are briefly described. Methods of assessing GSH status in the cells are also described. GSH synthesis and its regulation are addressed along with therapeutic approaches for manipulating GSH content that have been proposed. The purpose here is to provide a brief overview of some of the important aspects of glutathione metabolism as part of this special issue that will provide a more comprehensive review of the state of knowledge regarding this essential molecule.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: Writing – original draftRole: Writing – review & editing
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: ConceptualizationRole: Writing – review & editing
                Role: Funding acquisitionRole: SupervisionRole: Writing – original draft
                Role: ConceptualizationRole: Funding acquisitionRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                3 June 2020
                June 2020
                : 16
                : 6
                Affiliations
                [1 ] Division of Infectious Diseases, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America
                [2 ] Departments of Cell and Molecular Biology and Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, Rhode Island, United States of America
                [3 ] Department of Microbiology, University of Washington School of Medicine, Seattle, Washington, United States of America
                [4 ] Division of Pulmonary, Critical Care and Sleep Medicine, Harborview Medical Center, University of Washington, Seattle, Washington, United States of America
                [5 ] Howard Hughes Medical Institute, University of Washington, Seattle, Washington, United States of America
                Stanford University School of Medicine, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Article
                PPATHOGENS-D-20-00447
                10.1371/journal.ppat.1008566
                7340319
                32492066
                © 2020 Ramsey 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.

                Page count
                Figures: 5, Tables: 0, Pages: 24
                Product
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: AI081693
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: AI145954
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: AI145954
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: AI130798
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: HD055148-08
                Award Recipient :
                Funded by: NIH
                Award ID: S10OD026741
                Award Recipient :
                This work was funded by grants AI081693 (to SLD), AI145954 (to JDM and SLD), AI130798 (to SJS), HD055148-08 (in support of KMR) from the National Institutes of Health (NIH), and research reported in this publication was supported by the Office of the Director, NIH under Award Number S10OD026741. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Amino Acids
                Sulfur Containing Amino Acids
                Cysteine
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Amino Acids
                Sulfur Containing Amino Acids
                Cysteine
                Biology and Life Sciences
                Biochemistry
                Proteins
                Amino Acids
                Sulfur Containing Amino Acids
                Cysteine
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Francisella Tularensis
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Francisella Tularensis
                Biology and Life Sciences
                Organisms
                Bacteria
                Francisella
                Francisella Tularensis
                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
                Cell Biology
                Cellular Types
                Animal Cells
                Blood Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Immune Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Macrophages
                Medicine and Health Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Library Screening
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Library Screening
                Biology and Life Sciences
                Organisms
                Bacteria
                Francisella
                Biology and Life Sciences
                Genetics
                Gene Identification and Analysis
                Genetic Screens
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cytoplasm
                Custom metadata
                vor-update-to-uncorrected-proof
                2020-07-07
                All Tn-Seq data files are available from the GEO repository under accession number GSE138658.

                Infectious disease & Microbiology

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