Investigating the Functions of the Campylobacter jejuni Type VI Secretion System and Associated Effectors in Bacterial Competition and Host cell Interaction

Campylobacter jejuni is the leading global cause of foodborne bacterial gastroenteritis. An increasing number of C. jejuni isolates possess a Type VI Secretion system (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite this emerging prevalence, the role of the T6SS in C. jejuni strains is still not fully understood. To address this, T6SS-positive C. jejuni 488 strain (human clinical isolate) was used to further characterise the C. jejuni T6SS. Preliminary transcriptional and translational assays demonstrated the C. jejuni 488 T6SS is expressed and functionally active under in vitro conditions. However, qRT-PCR analysis revealed essential T6SS structural genes (tssB, tssC and tssD) are differentially expressed in response to physiological concentrations of the bile salts sodium taurocholate and sodium deoxycholate in a time- and dose- dependent manner.

The role(s) of T6SS are inextricably linked to the activities of effectors secreted through the apparatus. Bioinformatic analysis of the C. jejuni 488 genome identified putative effector and immunity protein-encoding genes downstream of the main T6SS locus. Mutants were constructed for the genes encoding predicted Tox-REase-7 domain-containing proteins (CJ488_0980 and CJ488_0982) and zinc-dependent metalloprotease/peptidase (CJ488_1003) to enable phenotypic characterisation.

The T6SS predominately functions to mediate interbacterial competition. During co-culture assays, the C. jejuni 488 strain exhibited previous unexplored T6SS-mediated competitive behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains. Further investigation revealed that this antagonism was contact-dependent and contingent upon the functionality of CJ488_0980 and CJ488_0982. Examination of anti-eukaryotic activities revealed the T6SS contributes to the virulence of the C. jejuni 488 strain in the Galleria mellonella infection model. However, this phenotype was not linked to the effectors investigated in this study. Nevertheless, infection of human intestinal epithelial cells (IEC) demonstrated the T6SS enhances the interactive and invasive capacity of the C. jejuni 488 strain, with CJ488_1003 suggested to modulate host cell interactions. This study presents novel findings advancing knowledge of the role of the T6SS as an emerging C. jejuni virulence determinant and notably the first exploratory characterisation of C. jejuni T6SS cargo effectors.