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The cell wall of Gram-positive bacteria is composed of proteins and peptidoglycan with the protruding secondary polysaccharides, which differ in the chemical composition and structure among various species. The cell wall secondary polysaccharides are covalently attached to the peptidoglycan and can account for more than a half of the total cell wall mass. They form a dense network of negative charges and impact cation homeostasis, membrane permeability, antibiotic susceptibility and survival in the host. In many species they also act as the anchors to which the outermost paracrystalline protein surface layer, called the S-layer, is non-covalently attached. We have already begun to elucidate the S-layer structure of Clostridium difficile. However, the mechanisms responsible for the organization of the S-layer structure and its attachment to the peptidoglycan remain poorly understood. C. difficile is a dangerous nosocomial pathogen. When the normal gut microbiota of a patient is compromised, C. difficile spores germinate and the infection, even when treated, can reoccur and lead to life-threatening complications. The wide-spread use of broad-spectrum antibiotics in treatment of C. difficile infections has already resulted in a number of (multiple) antibiotic resistant strains. Considering that the contact with the host takes place at the surface of the bacterium and that the S-layer of C. difficile is an essential virulence factor, it is worthwhile to study the associated processes. Gene manipulation studies of enzymes involved in biosynthesis of C. difficile secondary polysaccharides showed that their impairment leads to bacterial growth defects, diffused cell wall, defective anchoring, altered shedding and deposition of secondary polysaccharides, as well as defects in morphology and assembly of the S-layer, changes in biofilm formation and finally, changes in virulence. This prompts us to study the mechanisms of C. difficile S-layer assembly and in particular address the enzymes involved in the biosynthesis of secondary polysaccharides. Moreover, characterization of the respective enzymes might expand our understanding in the biogenesis of other bacterial cell wall polymers. With this proposal we plan to gain novel insight into the mechanisms underlying the biosynthesis of secondary polymers and their role in the S-layer assembly. We will study three groups of enzymes involved in biosynthesis of secondary polymers (enzymes involved in mannose conversion, glycosyltransferases, and enzymes attaching the secondary polysaccharides to the peptidoglycan). We will use complementary approaches of molecular biology, crystal structure analysis, enzyme activity measurements, structure-based virtual screening of ligands and classical inhibitor synthesis, biochemical and mass spectroscopy analysis, microscopic (optical, electronic and CryoEM) observations of bacteria or their fragments originating from wild type bacteria and those impaired by gene manipulations and inhibitors. We believe that research systematically addressing the whole group of the respective enzymes has the potential to deliver fundamental discoveries about their roles in the biosynthesis of secondary polymers and S-layer assembly. Hence, a successful outcome of this project will lay foundations for novel drug discovery programs that have the potential to improve human healthcare in life-threatening situations of C. difficile infections in a species-specific manner and thereby reduce risks of widely spreading antibiotic resistance.
