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Improved transformation efficiency in Mycoplasma hominis enables disruption of the MIB–MIP system targeting human immunoglobulins

hal.structure.identifierMicrobiologie Fondamentale et Pathogénicité [MFP]
hal.structure.identifierHospital University of Bordeaux, Bordeaux, France
dc.contributor.authorGUIRAUD, Jennifer
hal.structure.identifierMicrobiologie Fondamentale et Pathogénicité [MFP]
dc.contributor.authorLE ROY, Chloé
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorRIDEAU, Fabien
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorSIRAND-PUGNET, Pascal
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorLARTIGUE, Carole
hal.structure.identifierMicrobiologie Fondamentale et Pathogénicité [MFP]
hal.structure.identifierHospital University of Bordeaux, Bordeaux, France
dc.contributor.authorBÉBÉAR, Cécile
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorARFI, Yonathan
hal.structure.identifierMicrobiologie Fondamentale et Pathogénicité [MFP]
hal.structure.identifierHospital University of Bordeaux, Bordeaux, France
dc.contributor.authorPEREYRE, Sabine
dc.date.issued2023-10-17
dc.identifier.issn2165-0497
dc.description.abstractEnABSTRACT The pathogenicity of Mycoplasma hominis is poorly understood, mainly due to the absence of efficient genetic tools. A polyethylene glycol-mediated transformation protocol was recently developed for the M. hominis reference strain M132 using the pMT85-Tet plasmid. The transformation efficiency remained low, hampering generation of a large mutant library. In this study, we improved transformation efficiency by designing M. hominis -specific pMT85 derivatives. Using the Gibson Assembly, the Enterococcus -derived tet (M) gene of the pMT85-Tet plasmid was replaced by that of a M. hominis clinical isolate. Next, the Spiroplasma -derived spiralin gene promoter driving tet (M) expression was substituted by one of three putative regulatory regions (RRs): the M. hominis arginine deiminase RR, the M. hominis elongation factor Tu RR, or the 68 bp SynMyco synthetic RR. SynMyco-based construction led to a 100-fold increase in transformation efficiency in M. hominis M132. This construct was also transformed into the M. hominis PG21 reference strain and three other clinical isolates. The transposon insertion locus was determined for 128 M132-transformants. The majority of the impacted coding sequences encoded lipoproteins and proteins involved in DNA repair or in gene transfer. One transposon integration site was in the mycoplasma immunoglobulin protease gene. Phenotypic characterization of the mutant showed complete disruption of the human antibody cleavage ability of the transformant. These results demonstrate that our M. hominis -optimized plasmid can be used to generate large random transposon insertion libraries, enabling future studies of the pathogenicity of M. hominis . IMPORTANCE Mycoplasma hominis is an opportunistic human pathogen, whose physiopathology is poorly understood and for which genetic tools for transposition mutagenesis have been unavailable for years. A PEG-mediated transformation protocol was developed using the pMT85-Tet plasmid, but the transformation efficiency remained low. We designed a modified pMT85-Tet plasmid suitable for M. hominis . The use of a synthetic regulatory region upstream of the antibiotic resistance marker led to a 100-fold increase in the transformation efficiency. The generation and characterization of large transposon mutagenesis mutant libraries will provide insight into M. hominis pathogenesis. We selected a transformant in which the transposon was integrated in the locus encoding the immunoglobulin cleavage system MIB–MIP. Phenotypic characterization showed that the wild-type strain has a functional MIB–MIP system, whereas the mutant strain had lost the ability to cleave human immunoglobulins.
dc.language.isoen
dc.publisherAmerican Society for Microbiology
dc.rights.urihttp://creativecommons.org/licenses/by/
dc.subject.enMycoplasma hominis
dc.subject.enSynMyco
dc.subject.enmycoplasma immunoglobulin protease
dc.subject.entransposon mutagenesis
dc.subject.enMycoplasma hominis transposon mutagenesis SynMyco mycoplasma immunoglobulin protease
dc.title.enImproved transformation efficiency in Mycoplasma hominis enables disruption of the MIB–MIP system targeting human immunoglobulins
dc.typeArticle de revue
dc.identifier.doi10.1128/spectrum.01873-23
dc.subject.halSciences du Vivant [q-bio]
dc.subject.halSciences du Vivant [q-bio]/Médecine humaine et pathologie
bordeaux.journalMicrobiology Spectrum
bordeaux.pagee0187323
bordeaux.volume11
bordeaux.issue5
bordeaux.institutionCNRS
bordeaux.peerReviewedoui
hal.identifierhal-04296187
hal.version1
hal.popularnon
hal.audienceInternationale
hal.origin.linkhttps://hal.archives-ouvertes.fr//hal-04296187v1
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