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      Mycoplasmas under experimental antimicrobial selection: The unpredicted contribution of horizontal chromosomal transfer

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          Abstract

          Horizontal Gene Transfer was long thought to be marginal in Mycoplasma a large group of wall-less bacteria often portrayed as minimal cells because of their reduced genomes (ca. 0.5 to 2.0 Mb) and their limited metabolic pathways. This view was recently challenged by the discovery of conjugative exchanges of large chromosomal fragments that equally affected all parts of the chromosome via an unconventional mechanism, so that the whole mycoplasma genome is potentially mobile. By combining next generation sequencing to classical mating and evolutionary experiments, the current study further explored the contribution and impact of this phenomenon on mycoplasma evolution and adaptation using the fluoroquinolone enrofloxacin (Enro), for selective pressure and the ruminant pathogen Mycoplasma agalactiae, as a model organism. For this purpose, we generated isogenic lineages that displayed different combination of spontaneous mutations in Enro target genes ( gyrA, gyrB, parC and parE) in association to gradual level of resistance to Enro. We then tested whether these mutations can be acquired by a susceptible population via conjugative chromosomal transfer knowing that, in our model organism, the 4 target genes are scattered in three distinct and distant loci. Our data show that under antibiotic selective pressure, the time scale of the mutational pathway leading to high-level of Enro resistance can be readily compressed into a single conjugative step, in which several Enro R alleles were transferred from resistant to susceptible mycoplasma cells. In addition to acting as an accelerator for antimicrobial dissemination, mycoplasma chromosomal transfer reshuffled genomes beyond expectations and created a mosaic of resistant sub-populations with unpredicted and unrelated features. Our findings provide insights into the process that may drive evolution and adaptability of several pathogenic Mycoplasma spp. via an unconventional conjugative mechanism.

          Author summary

          Genome downsizing is often viewed as a degenerative process of evolution. Such erosion has left current mycoplasmas with a minimal genome: for some species its size barely exceeds the amount of information needed for sustaining autonomous life. Despite such limitations, these simple bacteria showcase a baffling capacity for adaptation to complex environments such as that provided by the animal host. By using the enrofloxacin antibiotic as selective pressure, we performed a genome scale analysis of macro- and micro-events leading to antimicrobial resistance in mycoplasmas. Sexually competent cells were found to shortcut this process by using an unconventional mechanism of chromosomal transfer driving massive exchanges of DNA materials. Remarkably, this powerful mechanism was associated with a profound genomic reorganization that reshuffled parental features and created mosaicism. This finding emphasizes the extraordinary adaptability of some pathogenic Mycoplasma spp. and provides major insights into the processes that contribute to shaping the evolution of their minimal genome. While unconventional conjugative mechanisms are being documented in more complex bacteria, the reduced mycoplasma genome may provide a simplified model to study mosaicism and its role in bacterial evolution.

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          Most cited references57

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          Artemis: sequence visualization and annotation

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            Molecular biology and pathogenicity of mycoplasmas.

            The recent sequencing of the entire genomes of Mycoplasma genitalium and M. pneumoniae has attracted considerable attention to the molecular biology of mycoplasmas, the smallest self-replicating organisms. It appears that we are now much closer to the goal of defining, in molecular terms, the entire machinery of a self-replicating cell. Comparative genomics based on comparison of the genomic makeup of mycoplasmal genomes with those of other bacteria, has opened new ways of looking at the evolutionary history of the mycoplasmas. There is now solid genetic support for the hypothesis that mycoplasmas have evolved as a branch of gram-positive bacteria by a process of reductive evolution. During this process, the mycoplasmas lost considerable portions of their ancestors' chromosomes but retained the genes essential for life. Thus, the mycoplasmal genomes carry a high percentage of conserved genes, greatly facilitating gene annotation. The significant genome compaction that occurred in mycoplasmas was made possible by adopting a parasitic mode of life. The supply of nutrients from their hosts apparently enabled mycoplasmas to lose, during evolution, the genes for many assimilative processes. During their evolution and adaptation to a parasitic mode of life, the mycoplasmas have developed various genetic systems providing a highly plastic set of variable surface proteins to evade the host immune system. The uniqueness of the mycoplasmal systems is manifested by the presence of highly mutable modules combined with an ability to expand the antigenic repertoire by generating structural alternatives, all compressed into limited genomic sequences. In the absence of a cell wall and a periplasmic space, the majority of surface variable antigens in mycoplasmas are lipoproteins. Apart from providing specific antimycoplasmal defense, the host immune system is also involved in the development of pathogenic lesions and exacerbation of mycoplasma induced diseases. Mycoplasmas are able to stimulate as well as suppress lymphocytes in a nonspecific, polyclonal manner, both in vitro and in vivo. As well as to affecting various subsets of lymphocytes, mycoplasmas and mycoplasma-derived cell components modulate the activities of monocytes/macrophages and NK cells and trigger the production of a wide variety of up-regulating and down-regulating cytokines and chemokines. Mycoplasma-mediated secretion of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-1 (IL-1), and IL-6, by macrophages and of up-regulating cytokines by mitogenically stimulated lymphocytes plays a major role in mycoplasma-induced immune system modulation and inflammatory responses.
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              The competitive cost of antibiotic resistance in Mycobacterium tuberculosis.

              Mathematical models predict that the future of the multidrug-resistant tuberculosis epidemic will depend on the fitness cost of drug resistance. We show that in laboratory-derived mutants of Mycobacterium tuberculosis, rifampin resistance is universally associated with a competitive fitness cost and that this cost is determined by the specific resistance mutation and strain genetic background. In contrast, we demonstrate that prolonged patient treatment can result in multidrug-resistant strains with no fitness defect and that strains with low- or no-cost resistance mutations are also the most frequent among clinical isolates.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: ResourcesRole: Validation
                Role: MethodologyRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Validation
                Role: InvestigationRole: MethodologyRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                22 January 2019
                January 2019
                : 15
                : 1
                : e1007910
                Affiliations
                [1 ] IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France
                [2 ] UMR Mycoplasmoses of ruminants, ANSES, VetAgro Sup, University of Lyon, Lyon, France
                [3 ] Asia-Pacific Centre for Animal Health, University of Melbourne, Melbourne, Victoria, Australia
                University of Geneva Medical School, SWITZERLAND
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-3789-4418
                http://orcid.org/0000-0002-6315-5670
                http://orcid.org/0000-0003-3968-4801
                http://orcid.org/0000-0003-4754-6003
                Article
                PGENETICS-D-18-01630
                10.1371/journal.pgen.1007910
                6358093
                30668569
                4ae5c346-f2ed-4ffe-b316-a334e3081250
                © 2019 Faucher 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
                : 13 August 2018
                : 19 December 2018
                Page count
                Figures: 5, Tables: 0, Pages: 23
                Funding
                This work was supported by National Institute for Agricultural Research (INRA; institut.inra.fr), National Veterinary School of Toulouse (ENVT; www.envt.fr) and French Agency for Food, Environmental and Occupational Health & Safety (ANSES; www.anses.fr). The funders 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
                Organisms
                Bacteria
                Mollicutes
                Mycoplasma
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Processes
                Horizontal Gene Transfer
                Biology and Life Sciences
                Genetics
                Gene Transfer
                Horizontal Gene Transfer
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Cloning
                Research and Analysis Methods
                Molecular Biology Techniques
                Cloning
                Biology and Life Sciences
                Computational Biology
                Genome Complexity
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Complexity
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Biology and Life Sciences
                Mycology
                Fungal Evolution
                Biology and Life Sciences
                Genetics
                Genetic Loci
                Biology and Life Sciences
                Computational Biology
                Comparative Genomics
                Biology and Life Sciences
                Genetics
                Genomics
                Comparative Genomics
                Custom metadata
                vor-update-to-uncorrected-proof
                2019-02-01
                All sequence files are available from the European Nucleotide Archive database (ENA; www.ebi.ac.uk/ena), under study accession numbers PRJEB28036 and PRJEB27571. The Galaxy workflows are available in json format at these URL: https://usegalaxy.org/u/xnouvel/w/transfered-fragments-imported-from-uploaded-file and https://usegalaxy.org/u/xnouvel/w/mutation-analysis-imported-from-uploaded-file.

                Genetics
                Genetics

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