Étude du mécanisme d'action d'une bactériocine à deux peptides ciblant une protéine impliquée dans la synthèse du peptidoglycane chez Enterococcus faecalis

Enterococcus faecalis is an opportunistic pathogenic bacterium frequently responsible for serious nosocomial infections, and its natural resistance to β-lactams makes it particularly difficult to treat. The World Health Organization includes it among the ESKAPE bacteria for which alternatives to conventional antibiotics are required. One potential alternative could rely on the use of bacteriocins, which are natural antibacterial peptides that exhibit strong cytotoxicity against targeted species. A wide variety of bacteriocins have been identified to date, but knowledge of their mechanism of action is still largely lacking. It is crucial to understand these mechanisms in order to use them in medicine or to potentiate their activity. During my thesis, I studied Enterocin C (EntC), which targets enterococci and consists of two peptides that act synergistically, whereas the peptides are inactive individually. Research on homologous bacteriocins to EntC has shown that their action depends on the undecaprenyl phosphate phosphatase, BacA, which is responsible for recycling of the lipid carrier of bacterial cell wall subunits across the plasma membrane. We demonstrated that BacA is also essential for EntC activity by generating an E. faecalis strain deleted for the bacA gene, which showed resistance, suggesting that BacA is the target of EntC and related bacteriocins. I then studied the mechanism of EntC by characterising its binding mode to BacA and its consequences using a combination of approaches. After purifying BacA, I reconstituted it into artificial liposome membranes and demonstrated that the peptides induced membrane permeabilisation in a BacA-dependent manner. I also observed that one of the peptides inhibited BacA's enzymatic activity. Moreover, chemical cross-linking experiments demonstrated peptide binding to BacA. Together, these data established that BacA is indeed the direct target of EntC and that it may suffice for its action by enabling membrane permeabilisation. The binding mode of the peptides was then detailed using biophysical approaches. I revealed a sequential binding of the peptides to BacA, with the first peptide, EntC1, binding to the protein and inhibiting its activity, followed by the binding of EntC2 to the BacA/EntC1 pre-complex with high affinity. A tripartite complex model was generated using AlphaFold with a high degree of confidence, correlating with experimental data. The EntC1 peptide binds directly to BacA's catalytic pocket open towards the bacterial surface, thus blocking substrate access, while the EntC2 peptide binds to both BacA and EntC1 more at the surface of the complex. The peptides are thus positioned parallel to the membrane plane, via amphipathic α-helices embedded in the lipid bilayer. This binding likely disturbs locally the membrane integrity, leading to permeabilisation. This binding model was validated by mutagenesis of several BacA residues potentially involved in complex formation, followed by cytotoxicity and interaction experiments. A second therapeutic strategy could involve the development of small molecules that inhibit one of the critical steps in bacterial cell wall biosynthesis, such as BacA's recycling of undecaprenyl phosphate. I participated in a study aimed at identifying small BacA inhibitors through high-throughput screening of a compound library. Overall, my work has demonstrated BacA's high potential as an antibacterial target and paved the way for bacteriocin engineering and optimisation of inhibitory molecules.