A Serine/threonine kinase promotes resistance to antimicrobial coumpounds crucial during colonization in Clostridium difficile – DifKin

Clostridium difficile (CD) is the leading cause of intestinal nosocomial post-antibiotic infections. The impact of CD infection (CDI) in hospitals is considerable in terms of mortality, morbidity and disease management as well as for its financial burden. Due to the unsatisfactory nature of standard therapy for CDI, there is an urgent need to better understand the molecular mechanisms controlling key steps of CDI for the development of new strategies targeting one of the infection steps for preventive and/or curative therapies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Our objective aims to determine the role of the Ser/Thr-kinase PrkC of CD in the bacterial envelope homeostasis and in host colonization. We already demonstrated that PrkC controls events crucial for the first stages of CD infectious cycle such as germination of spores in the gut after ingestion, resistance to antibiotics promoting CDI like cephalosporin, resistance to antimicrobial peptides, a first line of defense inside the host as well as to bile salts that are toxic for CD vegetative cells. The molecular mechanisms of CD resistance to these compounds remain poorly characterized. We first plan to identify the compound(s) of CD envelope (peptidoglycan and/or polysaccharides) impacted (abundance, structure and/or modifications) in the absence of PrkC. To determine the involvement of the two PASTA domains and of the atypical C-terminal motif specific of PrkC of C. difficile in PrkC function, we will analyze their role using truncated PrkC proteins. To identify PrkC substrates, we then propose to use phosphoproteomics combined with a genetic strategy of isolation and identification by next generation sequencing of suppressors of the prkC mutation restoring resistance to antimicrobial compounds. PrkC and some substrates of particular interest will be analyzed using classical molecular biology, genetic and biochemistry to understand the physiological role of this kinase and of its targets in the control of envelop homeostasis and of the resistance to antimicrobial compounds. To determine the role of PrkC and of its substrates in gut colonization by CD and in adhesion to epithelial cells, perform adhesion test to intestinal epithelial cell lines and use adapted animal models. To determine the consequence of each phosphorylation event on CD physiology, strains carrying phosphoabblative and phosphomimetic mutations at the phosphorylation site mimicking 0 % or 100 % of phosphorylation of the target will be constructed and their phenotypes analyzed. In a nutshell, we expect to determine which kinase phosphorylates which substrate and the physiological consequences of these phosphorylations.
The aim of the project is to generate a comprehensive kinase-substrate network and to determine the PrkC regulatory role in the control of resistance to antimicrobial compounds and in CDI. This project could lead to far-reaching strategy on warfare against CD.