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.