Improved transformation efficiency in Mycoplasma hominis enables disruption of the MIB–MIP system targeting human immunoglobulins
GUIRAUD, Jennifer
Microbiologie Fondamentale et Pathogénicité [MFP]
Hospital University of Bordeaux, Bordeaux, France
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Microbiologie Fondamentale et Pathogénicité [MFP]
Hospital University of Bordeaux, Bordeaux, France
GUIRAUD, Jennifer
Microbiologie Fondamentale et Pathogénicité [MFP]
Hospital University of Bordeaux, Bordeaux, France
Microbiologie Fondamentale et Pathogénicité [MFP]
Hospital University of Bordeaux, Bordeaux, France
BÉBÉAR, Cécile
Microbiologie Fondamentale et Pathogénicité [MFP]
Hospital University of Bordeaux, Bordeaux, France
Microbiologie Fondamentale et Pathogénicité [MFP]
Hospital University of Bordeaux, Bordeaux, France
PEREYRE, Sabine
Microbiologie Fondamentale et Pathogénicité [MFP]
Hospital University of Bordeaux, Bordeaux, France
< Reduce
Microbiologie Fondamentale et Pathogénicité [MFP]
Hospital University of Bordeaux, Bordeaux, France
Language
en
Article de revue
This item was published in
Microbiology Spectrum. 2023-10-17, vol. 11, n° 5, p. e0187323
American Society for Microbiology
English Abstract
ABSTRACT 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 ...Read more >
ABSTRACT 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.Read less <
English Keywords
Mycoplasma hominis
SynMyco
mycoplasma immunoglobulin protease
transposon mutagenesis
Mycoplasma hominis transposon mutagenesis SynMyco mycoplasma immunoglobulin protease
Origin
Hal imported