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Generation of <em>Mycoplasma hominis</em> gene-targeted mutants by targeting-induced local lesions in genomes (TILLING).

hal.structure.identifierUSC IHMC - Infections humaines à mycoplasmes et à chlamydiae
dc.contributor.authorPEREYRE, Sabine
hal.structure.identifierUSC IHMC - Infections humaines à mycoplasmes et à chlamydiae
dc.contributor.authorBÉNARD, Clémence
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorBRES, Cécile
hal.structure.identifierInstitut National de la Recherche Agronomique [INRA]
dc.contributor.authorLE ROY, Chloé
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorMAUXION, Jean-Philippe
hal.structure.identifierUSC IHMC - Infections humaines à mycoplasmes et à chlamydiae
dc.contributor.authorRIDEAU, Fabien
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorSIRAND-PUGNET, Pascal
hal.structure.identifierInstitute of Medical Microbiology and Hospital Hygiene [University of Düsseldorf]
dc.contributor.authorHENRICH, B.
hal.structure.identifierUSC IHMC - Infections humaines à mycoplasmes et à chlamydiae
dc.contributor.authorBEBEAR, Cécile
dc.date.issued2018
dc.identifier.issn1471-2164
dc.description.abstractEn<strong>Background</strong>: Mycoplasma hominis is a human urogenital pathogen involved in gynaecological, neonatal and extragenital infections. However, no versatile genetic tools are currently available to study the pathogenicity of this bacterium. Targeting-Induced Local Lesions IN Genomes (TILLING) is a reverse-genetic method that combines point mutations induced by chemical mutagenesis with a DNA screening technique. We used ethyl methanesulfonate (EMS) that introduces C-G to T-A transition mutations to generate a library of M. hominis mutants. As a proof of concept, mutagenized organisms were screened for mutations in two target genes previously associated with the mycoplasma pathogenicity, the vaa gene encoding an adhesin lipoprotein and the oppA gene encoding the main ectoATPase of the bacterium. The resulting mutants were evaluated using functional assays, an adhesion to HeLa cell assay for vaa-mutants and an ATPase activity test for oppA-mutants. <strong>Results</strong>: A 1200-clone library was generated by exposing M. hominis PG21 to 9 mg/mL EMS for 3 h. To identify mutants of interest, targeted gene fragments were amplified, heat-denatured, slowly reannealed and digested with the mismatch-specific endonuclease ENDO1. If multiple alleles were present in the PCR amplicons, these alleles formed heteroduplexes during reannealing that were specifically cleaved by ENDO1 at mismatching positions. A total of four vaa-mutants and two oppA-mutants harbouring missense mutations were obtained and fully sequenced. Zero to eight additional mutations were identified in the genomes of each mutant. The vaa-mutants were tested for adhesion to immobilized HeLa cells but their adhesion was not significantly different from the adhesion of M. hominis PG21. One of the two oppA-mutants that were tested for ATPase activity presented a higher affinity for its ATP substrate than the parental strain. <strong>Conclusion</strong>: For the first time, we demonstrated that M. hominis gene-targeted mutants could be successfully obtained using this TILLING strategy. In the absence of robust genetic tools for studying M. hominis, the TILLING strategy that can target any gene of the genome could help to elucidate gene functions and to better understand the pathogenesis of this human pathogenic species.
dc.language.isoen
dc.publisherBioMed Central
dc.rights.urihttp://creativecommons.org/licenses/by/
dc.subject.enTILLING
dc.subject.enchemical mutagenesis
dc.subject.enethyl methanesulfonate
dc.subject.enmycoplasma hominis
dc.title.enGeneration of <em>Mycoplasma hominis</em> gene-targeted mutants by targeting-induced local lesions in genomes (TILLING).
dc.typeArticle de revue
dc.identifier.doi10.1186/s12864-018-4917-1
dc.subject.halSciences du Vivant [q-bio]/Médecine humaine et pathologie
bordeaux.journalBMC Genomics
bordeaux.page1-9
bordeaux.volume19
bordeaux.peerReviewedoui
hal.identifierhal-02621895
hal.version1
hal.popularnon
hal.audienceInternationale
hal.origin.linkhttps://hal.archives-ouvertes.fr//hal-02621895v1
bordeaux.COinSctx_ver=Z39.88-2004&amp;rft_val_fmt=info:ofi/fmt:kev:mtx:journal&amp;rft.jtitle=BMC%20Genomics&amp;rft.date=2018&amp;rft.volume=19&amp;rft.spage=1-9&amp;rft.epage=1-9&amp;rft.eissn=1471-2164&amp;rft.issn=1471-2164&amp;rft.au=PEREYRE,%20Sabine&amp;B%C3%89NARD,%20Cl%C3%A9mence&amp;BRES,%20C%C3%A9cile&amp;LE%20ROY,%20Chlo%C3%A9&amp;MAUXION,%20Jean-Philippe&amp;rft.genre=article


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