Identification of Campylobacter jejuni secreted proteins

Sophia A. Pacheco

Campylobacter jejuni is a leading cause of bacterial gastrointestinal illness worldwide. Several studies indicate the importance of the bacterium's flagellum as an essential component for maximal and efficient invasion into host cells. To further understand the role of the flagellum in C. jejuni pathogenesis, studies were undertaken to characterize the flagellum as an apparatus that serves dual purposes for motility and secretion of virulence proteins. Specifically, we hypothesize that C. jejuni secretes virulence proteins termed, Campylobacter secreted proteins (CSP), in a Type III Secretion System (T3SS) dependent manner and that these proteins play a role in C. jejuni-mediated disease. Previous work indicated that C. jejuni secretes 8-16 proteins in vitro via the flagellum. Since, the flagellum shares evolutionary homology to the T3SS injectisome, we surmised that the CSP must harbor characteristics that allow for recognition and secretion from the flagellum in a T3SS dependent manner. To test our hypothesis, we utilized the Yersinia enterocolitica flagellar T3SS to determine if homologous flagellar machinery would recognize and secrete the CSP, Campylobacter invasion antigen B (CiaB). Secretion of CiaB from the Y. enterocolitica flagellar apparatus provided proof of concept for the generation of a genetic screen to identify the remaining Cia proteins. 321 amino termini of previously uncharacterized C. jejuni open reading frames (orfs) were tested for secretion by fusing putative secretion signals with the Y. enterocolitica YplA reporter. Recombinants were then tested for secretion under Y. enterocolitica flagellar induction conditions. One putative CSP that drove YplA secretion was encoded by the C. jejuni Cj1242 orf. A Cj1242 mutant was generated and subsequently characterized for the ability to invade host cells. A secretion profile from the Cj1242 mutant, designated ciaC, revealed an altered C. jejuni secretion profile whereby one band that corresponded to the predicated mass of CiaC was missing. The ciaC mutant also exhibited an invasion deficient phenotype, as judged by the gentamicin protection assay. In conclusion, our C. jejuni genome-wide screen resulted in the identification of at least one novel C. jejuni virulence determinant. Additional studies are underway to determine how CiaC contributes to C. jejuni-mediated disease.

Identification of Campylobacter jejuni secreted proteins

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