Clonage et modification du génome de Mycoplasma hominis dans la levure Saccharomyces cerevisiae

Abstract : Mycoplasma hominis is an opportunistic human pathogen responsible for genital and neonatal infections. Genetically modifying this bacterium is necessary to understand the virulence and infection mechanisms of this pathogen. There is currently no effective molecular tool to engineer the genome of this bacterium, limiting research on its pathogenicity and its peculiar metabolism based on arginine.New technologies have recently emerged in the field of Synthetic Biology (BS), offering new perspectives for the study of mycoplasmas by allowing large scale genome modifications and the production of mutant strains. Work at the J. Craig Venter Institute (JCVI, USA) has shown that the genome of related mycoplasmas can be cloned and manipulated in yeast before being transplanted into a recipient cell. The yeast serves as a temporary host to modify the genome of the bacterium. This innovative approach opens many perspectives in the development of functional genomics in mycoplasmas for which there are few effective genetic tools. The goal of this thesis was to adapt a number of BS tools to M. hominis for the first time, in order to create mutants deficient for a given function. To achieve this goal, the genome of the M. hominis type strain PG21 (665 kb) was cloned into the yeast Saccharomyces cerevisiae by Transformation-Associated Recombination cloning (TAR-cloning). Two yeast clones (B3-2 and B3-4) possessing the complete genome of M. hominis were validated by simplex PCR, multiplex PCR and Pulsed Field Gel Electrophoresis (PFGE) analyses. These yeast clones were then propagated in a selective medium for 180 generations (30 passages) to evaluate the stability of the bacterial genome in its host. This experiment showed that (i) the size of the genome of M. hominis did not change during the first passages, it decreased progressively from the tenth passage (≈60 generations), and (ii) the enriched genome areas in repeated sequence were preferentially lost. Thus, the genome of M. hominis was modified in the B3-4 clone at early passages using the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) technology. Yeast clones with a complete M. hominis PG21 genome with a deleted vaa gene, encoding a major adhesion protein, were produced using this approach. The final step of this approach was to transplant the modified genome into a recipient cell of M. hominis or Mycoplasma arthritidis, the species phylogenetically closest to M. hominis. As no M. hominis transformation protocol was available at the beginning of our work, this step constituted a major obstacle in the implementation of BS tools in this species. This barrier has been partially lifted since a method of transformation of M. hominis based on polyethylene glycol (PEG) and involving the plasposon pMT85 (plasmid carrying a transposon conferring resistance to tetracycline) has been developed in the laboratory. This transformation technique, developed for the reference strain M. hominis M132 (745 kb) still remains not very efficient; it is nevertheless reproducible and allowed to obtain M. hominis mutants of interest. The Mhom132_2390 gene, encoding the precursor of the P75 protein, a putative adhesin of M. hominis, was effectively mutated in transformant No. 28-2. Complete genome sequencing of other transformants revealed the insertion of multiple copies of the transposon and the presence of duplication and inversion of large DNA fragments within at least two M. hominis genomes.In conclusion, this data has opened the way for the development and transposition of existing genetic modification approaches to M. hominis, previously considered as a genetically intractable bacterium.
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Fabien Rideau. Clonage et modification du génome de Mycoplasma hominis dans la levure Saccharomyces cerevisiae. Médecine humaine et pathologie. Université de Bordeaux, 2018. Français. ⟨NNT : 2018BORD0227⟩. ⟨tel-02138642⟩



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