The Campylobacter jejuni Response Regulator and Cyclic-Di-GMP Binding CbrR Is a Novel Regulator of Flagellar Motility.

A leading cause of bacterial gastroenteritis, Campylobacter jejuni is also associated with broad sequelae, including extragastrointestinal conditions such as reactive arthritis and Guillain-Barré Syndrome (GBS). CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), an enzyme that catalyzes the synthesis of c-di-GMP, a universal bacterial second messenger, from GTP. In C. jejuni DRH212, we constructed an unmarked deletion mutant, cbrR-, and complemented mutant, cbrR+. Motility assays indicated a hyper-motile phenotype associated with cbrR-, whereas motility was deficient in cbrR+. The overexpression of CbrR in cbrR+ was accompanied by a reduction in expression of FlaA, the major flagellin. Biofilm assays and scanning electron microscopy demonstrated similarities between DRH212 and cbrR-; however, cbrR+ was unable to form significant biofilms. Transmission electron microscopy showed similar cell morphology between the three strains; however, cbrR+ cells lacked flagella. Differential radial capillary action of ligand assays (DRaCALA) showed that CbrR binds GTP and c-di-GMP. Liquid chromatography tandem mass spectrometry detected low levels of c-di-GMP in C. jejuni and in E. coli expressing CbrR. CbrR is therefore a negative regulator of FlaA expression and motility, a critical virulence factor in C. jejuni pathogenesis.

Binding of Campylobacter jejuni FliW Adjacent to the CsrA RNA-Binding Pockets Modulates CsrA Regulatory Activity.

Campylobacter jejuni CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to E. coli CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), C. jejuni CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of C. jejuni CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.

RNA Binding by the Campylobacter jejuni Post-transcriptional Regulator CsrA.

Campylobacter jejuni is a Gram-negative rod-shaped bacterium that commensally inhabits the intestinal tracts of livestock and birds, and which also persists in surface waters. C. jejuni is a leading cause of foodborne gastroenteritis, and these infections are sometimes associated with the development of post-infection sequelae such as Guillain-Barré Syndrome. Flagella are considered a primary virulence factor in C. jejuni, as these organelles are required for pathogenicity-related phenotypes including motility, biofilm formation, host cell interactions, and host colonization. The post-transcriptional regulator CsrA regulates the expression of the major flagellin FlaA by binding to flaA mRNA and repressing its translation. Additionally, CsrA has previously been shown to regulate 120-150 proteins involved in diverse cellular processes. The amino acid sequence of C. jejuni CsrA is significantly different from that of Escherichia coli CsrA, and no previous research has defined the amino acids of C. jejuni CsrA that are critical for RNA binding. In this study, we used in vitro SELEX to identify the consensus RNA sequence mAwGGAs to which C. jejuni CsrA binds with high affinity. We performed saturating site-directed mutagenesis on C. jejuni CsrA and assessed the regulatory activity of these mutant proteins, using a reporter system encoding the 5' untranslated region (5' UTR) upstream of flaA linked translationally to the C. jejuni astA gene. These assays allowed us to identify 19 amino acids that were involved in RNA binding by CsrA, with many but not all of these amino acids clustered in predicted beta strands that are involved in RNA binding by E. coli CsrA. Decreased flaA mRNA binding by mutant CsrA proteins L2A and A36V was confirmed by electrophoretic mobility shift assays. The majority of the amino acids implicated in RNA binding were conserved among diverse Campylobacter species.