Characterisation of lipoprotieins of Clostridium difficile and their role in virulence

Abstract

Antibiotic-associated diarrhoea (AAD) and colitis, with the causative agent being the Gram-positive anaerobe, Clostridium difficile, are some of the most important hospital-acquired infections and significant burdens to healthcare services worldwide. Treatment of the infection is often ineffective and currently no vaccine is available against C. difficile infection (CDI). Research to identify novel virulence factors potentially leads to the development of new therapeutic and prophylactic drugs. As lipoproteins have been shown to play key roles in the virulence of several pathogens, the aim of this project was to investigate whether lipoproteins are involved in the virulence of C. difficile. Lipoproteins are anchored to the extracellular side of the cytoplasmic membrane in Gram-positive bacteria. Two enzymes are involved in the biosynthesis of lipoproteins: lipoprotein diacylglycerol transferase (Lgt) attaches lipoproteins to the membrane, and lipoprotein signal peptidase (Lsp) cleaves the signal peptide from the amino-terminus of lipoproteins. In order to study lipoprotein processing in C. difficile, lgt and lsp mutants of the C. difficie 630Δerm strain were generated using the ClosTron system. Antibody reactivity of 14 C. difficile lipoproteins was also investigated. It was shown in this study that lgt mutation caused changes in the lipoproteome of C. difficile. Therefore, inactivation of the lgt gene allowed investigation of the global contribution of lipoproteins to bacterial processes. The physiology and virulence of the lgt mutant was studied in vitro and in vivo. Surprisingly, many of the assayed phenotypes were not significantly affected by disruption of the lgt gene. Nevertheless, the ability of the lgt mutant to adhere to Caco-2 cells was markedly reduced. In addition, the phenotype of the lgt mutant observed in mice suggests that the faecal shedding of C. difficile is affected by Lgt inactivation. In further studies, the CD0873 lipoprotein as a potential adhesin of C. difficile was identified by in silico approach. Contribution of the CD0873 lipoprotein to the adherence of C. difficle was investigated by several different assays and the results strongly suggest that the CD0873 lipoprotein is directly involved in adhesion