Phenotypic Characterisation of Clostridium Difficile Strains Defective in Lipoprotein Biosynthesis

Abstract

Clostridium difficile is regarded as the primary etiological agent of antibiotic - associated diarrhoea, posing a significant challenge to healthcare facilities. The changing nature of C. difficile infection is causing an increase in associated disease occurrence outside of the healthcare setting and a gradual move away from the historical association with antimicrobial treatment. Adhesio n of spores and vegetative cells to host gut epithelium is thought to be a key aspect of C. difficile virulence; disruption of this process may significantly reduce the impact of an infection and the likelihood of infection spread. Lipoproteins are involve d in adhesion of C. difficile to host tissues and may have roles in other key aspects of virulence. Lipoproteins undergo a specific biosynthesis process within the bacterial cell involving addition of an acyl - glyceryl moiety by lipoprotein glyceryl transfe rase (Lgt) followed by signal peptide cleavage by lipoprotein signal peptidase (LspA); disruption of this process may cause attenuation of virulence and a reduction in adhesion to host tissue . C. difficile has been shown to encode two functional and homolo gous lipoprotein signal peptidases: LspA and LspA2. The novel antimicrobials globomycin and myxovirescin directly target lipoprotein signal peptidases and therefore may have potential for use in treatment of C. difficile infection. Evaluation of their effi cacy against LspA and LspA2 can be determined by protection assays using Escherichia coli strains expressing LspA or LspA2 from C. difficile . In this study, both LspA and LspA2 from C. difficile are shown to contain the consensus sequences, domains and in silico predicted tertiary structure expected of lipoprotein signal peptidases. Characteris ation of C. difficile strains with silencing mutations in either lspA or lspA2 , in comparison to a wild type , reveals that the absence of either lipoprotein signal pe ptidase causes an increased survivability in hydrogen peroxide and may affect protein localis ation within the bacterium. Finally, successful cloning of C. difficile lspA and lspA2 and subsequent expression of LspA and LspA2 via auto - induction in E. coli is reported, paving the way for further investigation into the effect of globomycin or myxovirescin treatment.