FLAGELLAR CHAPERONES AND CONTROL OF CAMPYLOBACTER JEJUNI MOTILITY

Abstract

Campylobacter jejuni is a leading cause of bacterial gastroenteritis, with an annual incidence of 140 million cases globally and 1.3 million cases in the United States. Approximately 1/1000 of C. jejuni infections lead to the onset of Guillain-Barré Syndrome, the world’s leading cause of acute paralysis. While the pathogenic mechanisms of C. jejuni are incompletely understood, it is known that flagellar motility is a primary virulence factor. Flagella are involved in host cell adherence and invasion, biofilm formation, and chick colonization. Flagellar assembly is dependent on the coordinated regulation of flagellar subunit synthesis via transcriptional and post-transcriptional regulators, as well as flagellar chaperones which maintain flagellar subunits in an unfolded state. In C. jejuni, the post-transcriptional regulator CsrA regulates flagellar biogenesis in C. jejuni by binding to the mRNA for flaA, repressing FlaA synthesis. CsrA regulatory activity on flagellar and non-flagellar targets is predicted to be modulated by protein-protein interactions with the flagellar chaperone FliW. Both FliW and a second flagellar chaperone FliS are predicted to participate in flagellar assembly by binding to FlaA. Therefore, we investigated the roles of FliW and FliS in CsrA regulation and flagellar biogenesis by constructing and characterizing a fliS mutant of C. jejuni strain 81-176, and by testing for the presence of protein-protein interaction(s) among CsrA, flagellin, and flagellar chaperones. We created an in-frame deletion mutant of fliS, and determined that the deletion of fliS resulted in a loss of motility and reduced the capacity of C. jejuni to autoagglutinate and form biofilm. We also used a bacterial two-hybrid system to study possible binding among the flagellarrelated proteins FlaA, FliS, FliW, and CsrA. Additionally, we performed deletion analysis of fliW in pT25, with the goal of identifying the region of FliW that mediates binding to CsrA. CsrA bound to full-length FliW, but no other protein-protein interactions were evident using the two-hybrid system. Surprisingly, CsrA did not interact with fragments of FliW, suggesting that the CsrA-binding site of FliW may be complex. These results show that flagellar biogenesis is accomplished by interactions of flagellar chaperones that link motility with the regulation of Campylobacter pathogenesis-related properties.