ED Sciences de la Vie et de la Santé
Expression conditions and transfer capacity of integrative and conjugative elements in Mycoplasma hominis, a human genital pathogen
by Bachir BOUREIMA ABDOU (Microbiologie fondamentale et Pathogénicité)
The defense will take place at 14h30 - Amphithéâtre du bâtiment BBS 2 rue du Dr Hoffmann Martinot, 33076 Bordeaux cedex
in front of the jury composed of
- Sabine PEREYRE-WASSNER - Professeure des universités - praticienne hospitalière - Université de Bordeaux - Directeur de these
- Christine CITTI - Directrice de recherche - Ecole Nationale Vétérinaire de Toulouse - Rapporteur
- Florence TARDY - Directrice de recherche - Laboratoire Anses Ploufragan - Rapporteur
- Pascal SIRAND-PUGNET - Professeur - Université de Bordeaux - Examinateur
- Olivier BARRAUD - Professeur des universités - praticien hospitalier - Université de Limoges - Examinateur
Integrative and Conjugative Elements (ICEs) are self-transmissible mobile genetic elements that play a key role in bacterial genome evolution by mediating horizontal gene transfer (HGT). Unlike plasmids, ICEs integrate into the host genome, excise to form circular intermediates, and are then transferred to recipient cells via conjugation before reintegrating into the chromosome. These elements significantly contribute to bacterial adaptability by promoting the acquisition of antibiotic resistance genes, virulence factors, and metabolic traits. A novel family of ICEs, referred to as MICE (Mycoplasma Integrative and Conjugative Elements), was identified in several Mollicutes species. In Mycoplasma hominis, a human urogenital pathogen, approximately 45% of clinical isolates carry ICEs randomly inserted into the chromosome. These elements span 27 to 30 kbp and contain between 25 and 30 CDSs. In this thesis, we first aimed to assess the circularization capacities of M. hominis 4788 ICE (ICEHo-4788) under different environmental conditions and investigate the transcription of the ICE CDSs. The second part of our study focused on transfer capacities of M. hominis ICE through mating experiments. Using qPCR, RT-qPCR, and LC-MS/MS, we evaluated ICEHo-4788 circularization, gene transcription, and protein expression under different conditions: axenic growth phases, exposure to mitomycin C (MMC), to thermal stress (cold and heat shocks), and co-culture with HeLa cells. Under axenic conditions, a peak of circularization was observed at 12 h of growth, with a 9.4-fold increase compared to the baseline. MMC exposure and cold shock led to 3.3- and 3.0-fold increases in circular forms, respectively. In co-culture with HeLa cells, circularization was strongly enhanced, with peaks at 72 h and 7 days post-inoculation, showing 10- and 23-fold increases, respectively. RT-qPCR analysis showed that all ICEHo-4788 CDSs were transcribed. Additionally, RT-PCR targeting all intergenic regions yielded amplicons, suggesting that the element may be transcribed as a single polycistronic mRNA. No major transcriptional changes were observed in axenic culture or under MMC and cold shock conditions. However, in HeLa co-culture, a marked transcriptional upregulation was detected at 7 days post-inoculation, with 5- to 23-fold increases depending on the CDS. Proteomic analyses revealed that four ICEHo4788-associated proteins were significantly upregulated at 12 h compared to 19 h in axenic culture, while no significant changes in protein expression were detected in response to thermal stress or MMC. Given that co-culture with HeLa cells strongly promoted ICE circularization and transcription, we then used this condition to investigate the conjugative properties of ICE in M. hominis. Mating experiments were conducted by co-incubating HeLa cells for 7 days with a donor M. hominis strain carrying a tetracycline resistance gene inserted within an intergenic region of the ICE, and two recipient strains harboring a different antibiotic resistance marker (gentamicin or ofloxacin). Whole genome sequencing and SNP polymorphism analyses revealed two distinct HGT mechanisms. While the ICE was transferred from the donor to the recipient strain, a Mycoplasma Chromosomal Transfer (MCT) occurred in the opposite direction, from the recipient to the donor cell, independently of ICE mobilization and involved the exchange of large chromosomal regions. Together, these findings demonstrate that environmental stimuli, particularly host-like conditions, modulate the circularization and transcriptional activity of M. hominis ICEs. Moreover, our results provide the first experimental evidence of two HGT mechanisms in M. hominis, ICE transfer and MCT, with MCT acting as a potent driver of extensive genomic recombination. These complementary mechanisms underline the efficiency of HGT in M. hominis, contributing to its genome plasticity and adaptability.