Characterisation of CapC, a novel strain-specific autotransporter in campylobacter species.

Campylobacter jejuni and Campylobacter coli are recognised as the principal causative agents of bacterial gastroenteritis in the developed world. However, despite the identification of factors integral to infection, characterisation of the virulence strategies employed by Campylobacter remains a significant challenge. Bacterial autotransporter proteins comprise the largest and most diverse class of secretory proteins in Gram-negative bacteria; notably almost all previously characterised autotransporter proteins contribute to bacterial virulence to some extent. The aim of this study was to characterise CapC, a newly identified, strain-specific gene predicted to encode an autotransporter protein, and to examine the contribution of this factor to the virulence of Campylobacter jejuni. The capC gene was initially confirmed as being encoded by approximately 60% of C. jejuni and C. coli human clinical and poultry associated isolates. Moreover, CapC was confirmed as a member of the autotransporter family through the use of bioinformatic prediction tools and the localisation site of this protein was determined as the outer membrane of C. jejuni. Targeted mutagenesis of the capC gene in C. jejuni 81116 and C. jejuni M1 and subsequent comparison of wild-type and isogenic mutant strains demonstrated that CapC contributes directly to virulence in the Galleria mellonella invertebrate model (p=0.00017; p=0.002323). Furthermore, tissue culture assays using non-polarised, partially differentiated Caco-2 and T84 intestinal epithelial cells indicate that deletion of CapC resulted in significantly decreased adhesion and invasion efficiency. Additional analyses indicated that CapC primarily contributes to adhesion to intestinal epithelial cells. Additional assays showed that deletion of the capC gene has no significant phenotypic effect on cytotoxicity in a Caco-2 cell model. A secondary aim of this study was to examine the distribution of capC amongst campylobacters and to establish any potential genetic associations of this virulence determinant. Using publically vi available genome sequences, capC was established to be highly prevalent in C. jejuni (67.9%) and C. coli (84%). Campylobacter autotransporter proteins were also shown to be present in truncated and full length forms. Interestingly, full length CapC was shown to be primarily associated with the ST-45, ST-283 and ST-573 clonal complexes in C. jejuni and the ST-828 clonal complex in C. coli. Furthermore, this study detailed the identification of a novel autotransporter in Campylobacter species, tentatively designated as CapD. This autotransporter was found to be genetically distinct from CapC and is the most prevalent autotransporter identified in Campylobacter species. The studies presented in this thesis indicate that CapC is a strain-specific virulence determinant in Campylobacter species that is associated with select lineages of C. jejuni and C. coli. CapC contributes to the integral infection process of adhesion however further studies are required to fully elucidate the exact nature of this interaction. Additionally, it can be concluded that possession of Campylobacter autotransporter proteins is dependent on genetic background