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

Abstract

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.

Extracellular c-di-GMP Plays a Role in Biofilm Formation and Dispersion of Campylobacter jejuni

“…The expression of genes involved in chemotactic motility and quorum sensing in C.jejuni 11168-O ( cheA , tlp6 , tlp8 and luxS ) were examined in the presence and absence of ex-c-di-GMP by RT-PCR. CbrR gene as the only gene in the C. jejuni genome encoding GGDEF-like motif, reported to be potentially involved in c-di-GMP synthesis [ 48 ] was also included. The expression of, tlp6 , LuxS and CheA was upregulated in presence of 200 µM ex-c-di-GMP by 1.9 to 3-fold ( Figure 7 ); however, no significant effect on the expression of tlp8 was observed.…”

“…The ability of bacteria to auto-aggregate was assessed as described by [ 21 , 48 ]. Briefly, C. jejuni cells were harvested from overnight MHA plates, washed with PBS and resuspended in PBS to OD 600 0.8.…”

“…Those studies suggested that using c-di-GMP in combination with other antimicrobial agents, may be a promising novel approach to treating or inhibiting biofilms [ 47 ]. Recently, the c-di-GMP signalling system has been reported in C. jejuni DRH212 [ 48 ]; however, the presence and the role of c-di-GMP in other C. jejuni strains, particularly C. jejuni NCTC 11168, are still unknown.…”

Two-component regulatory systems in Helicobacter pylori and Campylobacter jejuni: Attractive targets for novel antibacterial drugs

“…Sodium deoxycholate resistance, flagellum biosynthesis, biofilm formation, chemotaxis (Raphael et al, 2005;Parrish et al, 2007;Cox et al, 2022) CheY (cj1118c) Chemotaxis (Lertsethtakarn et al, 2011;Zautner et al, 2012) CheA (cj0284c) Chemotaxis (Lertsethtakarn et al, 2011;Zautner et al, 2012) essential for H. pylori motility (Spohn and Scarlato, 1999;McDaniel et al, 2001;Brahmachary et al, 2004). The internal signal sensed by FlgS that triggers its autophosphorylation and subsequent activation of FlgR remains unknown (Alvarado et al, 2019).…”

“…A second C. jejuni orphan RR, the protein CbrR, is an atypical RR that lacks a DNA-binding domain, but contains a C-terminal GGDEF motif commonly observed in diguanylate cyclase enzymes ( Raphael et al., 2005 ; Cox et al., 2022 ). Deletion mutants of CbrR exhibited increased sensitivity to sodium deoxycholate, enhanced motility and reduced ability to colonize chickens ( Raphael et al., 2005 ; Cox et al., 2022 ), whereas CbrR overexpression resulted in loss of flagella and a significant reduction in biofilm formation ( Cox et al., 2022 ). Yeast two-hybrid screenings have showed that CbrR interacts with CheA ( Parrish et al., 2007 ), which seems to indicate a putative role in chemotaxis.…”