Études de perturbations de l’enveloppe de Vibrio cholerae et Escherichia coli : mécanismes de vulnérabilités ou de résistances

The increased incidence of bacterial infections by resistant, multiresistant and even extensively drug-resistant strain, combined with the crisis of new molecules discovery since the 1960s and the chronic underinvestment of certain states in public research, could cost 10 million human lives per year by 2050. The overwhelming majority of resistant bacteria are the Gram-negative one. This is mainly due to the intrinsic composition of their envelope, allowing them to be insensitive to many molecules yet lethal for other types of bacteria. The study of envelope components and biosynthetic mechanisms, which are essential for the maintenance of bacterial integrity and could be involved in their virulence, should lead to the identification of new therapeutic targets. The first chapter gave an overview of the different elements composing Gram-negative bacteria envelope, mechanisms of resistance and the threat that Gram-negative bacteria represent. The presentation of my work, divided in four articles from chapter two to chapter five, was preceded by a contextualization of the research projects specific to both laboratories, on biofilm and motility of Vibrio cholerae on one hand and on Escherichia coli envelope from the other hand. In the first paper, we determined the mechanism by which a sub-inhibitory concentration of polymyxin B affects biofilm formation in Vibrio cholerae. We observed that polymyxin B mainly affected the flagellum by a mechanical action. Since biofilm formation requires the flagellum in the early stages of its formation, we concluded that the action of polymyxin B prevented this change in Vibrio cholerae lifestyle. In the second paper, we designed an experimental evolution protocol which allowed us to identify mutations in different genes that increase Vibrio cholerae motility in the presence of sub-inhibitory concentration of polymyxin B. We then identified that different mutations had altered ihfA, dacB, vacJ (mlaA) and mlaF genes in different mutants. These mutations have probably induced reduction or loss of function of the proteins for which they respectively code. Based on literature, we hypothesize that the loss function of these proteins inducing increase in mobility in the presence of polymyxin B could be explained by three   7 mechanisms involving envelope stability, secretion of membrane vesicles or altered expression of various genes. In the third paper, we investigated the involvement of oxidative stress in the fatal peptidoglycan synthesis arrest occurring in the ∆elyC mutant of Escherichia coli. We then demonstrated that lysis of the ∆elyC mutant is caused by the overproduction of hydroxyl radicals in its envelope. This molecule causes multiple and sufficiently important damages in the envelope to stop the synthesis of the wall. The mechanism causing this overproduction of toxic radicals has yet to be determined. By studying the function of the ElyC factor, we have discovered a new weakness in envelope homeostasis that we are eager to exploit. In the last paper, based on a previously formulated hypothesis, we investigated the importance of the enterobacterial common antigen synthesis pathway in the lethal process occurring in the absence of ElyC. We found that the ∆elyC mutant accumulates common element to the cell wall and enterobacterial common antigen synthesis pathways, undecaprenyl pyrophosphate. We also observed that the ∆elyC mutant lysis phenotype could be suppressed by increasing undecaprenyl pyrophosphate recycling via the overexpression of the gene coding for the phosphatase PgpB. We propose that the absence of ElyC induces a misallocation of undecaprenyl phosphate between the cell wall and enterobacterial common antigen synthesis pathways, in favors the latter pathway. Overactivation of the enterobacterial common antigen pathway would induce undecaprenyl phosphate accumulation, causing inhibition of Pbp1b, thus blocking addition of new subunits to the cell wall and leading to its lysis. Ultimately, all the original data generated by my research project has led to a better understanding of envelope vulnerabilities of Vibrio cholerae and Escherichia coli, to clarify certain mechanisms but also to glimpse the role of various factors involved in the homeostasis of their envelope.

Keyphrases: Vibrio cholerae, Escherichia coli, biofilm, virulence, Vibrio cholerae, Escherichia coli, motility, virulence, antimicrobial peptides, cell wall, envelope homeostasis and targets