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.
Highlights
IntroductionSurveillance Network (FoodNet) in the United States in 2016–2019 [1], Campylobacter jejuni causes 450 million annual cases of bacterial gastroenteritis worldwide [2] and approximately 40,000 deaths each year in children aged five and younger [3]
wild type (WT), cbrR−, and cbrR+ complemented mutant were cultured on Mueller–Hinton (MH) agar plates or in biphasic cultures constituted of a layer of MH agar overlaid with 25 mL MH broth in a 75 cm2 tissue culture flask, supplemented with streptomycin
DRH212, whereas flagellin expression in cbrR+ was demonstrably lower than both WT and cbrR− lysates (Figure 4D), consistent with the electron microscopy findings
Summary
Surveillance Network (FoodNet) in the United States in 2016–2019 [1], Campylobacter jejuni causes 450 million annual cases of bacterial gastroenteritis worldwide [2] and approximately 40,000 deaths each year in children aged five and younger [3]. Infection does not lead to lifelong immunity against the bacteria; individuals are left at risk of subsequent Campylobacter infection [11]. Gastrointestinal symptoms are not the only threat associated with Campylobacter, as several peripheral neuropathies including Guillain–Barré syndrome (GBS) have been linked to the bacteria [4,12,13]. C. jejuni is able to vary its gene expression in response to diverse in vivo and environmental cues; it does not contain nearly the number of regulatory proteins that many other bacteria do.
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