Functional Characterization of a Lipoprotein-Encoding Operon in Campylobacter jejuni

Abstract

BackgroundBacterial lipoproteins have important functions in bacterial pathogenesis and physiology. In Campylobacter jejuni, a major foodborne pathogen causing gastroenteritis in humans, the majority of lipoproteins have not been functionally characterized. Previously, we showed by DNA microarray that CmeR, a transcriptional regulator repressing the expression of the multidrug efflux pump CmeABC, modulates the expression of a three-gene operon (cj0089, cj0090, and cj0091) encoding a cluster of lipoproteins in C. jejuni.Methodology/Principal FindingsIn this work, we characterized the function and regulation of the cj0089-cj0090-cj0091 operon. In contrast to the repression of cmeABC, CmeR activates the expression of the lipoprotein genes and the regulation is confirmed by immunoblotting using anti-Cj0089 and anti-Cj0091 antibodies. Gel mobility shift assay showed that CmeR directly binds to the promoter of the lipoprotein operon, but the binding is much weaker compared with the promoter of cmeABC. Analysis of different cellular fractions indicated that Cj0089 was associated with the inner membrane, while Cj0091 was located on the outer membrane. Inactivation of cj0091, but not cj0089, significantly reduced the adherence of C. jejuni to INT 407 cells in vitro, indicating that Cj0091 has a function in adherence. When inoculated into chickens, the Cj0091 mutant also showed a defect in early colonization of the intestinal tract, suggesting that Cj0091 contributes to Campylobacter colonization in vivo. It was also shown that Cj0091 was produced and immunogenic in chickens that were naturally infected by C. jejuni.Conclusion/SignificanceThese results indicate that the lipoprotein operon is subject to direct regulation by CmeR and that Cj0091 functions as an adhesion mechanism in C. jejuni and contributes to Campylobacter colonization of the intestinal tract in animal hosts.

Effects of Lipooligosaccharide Inner Core Truncation on Bile Resistance and Chick Colonization by Campylobacter jejuni

“…The construction of complemented strains of LOS-truncated mutants was based on a previous study [40]. Each target gene encoding the biosynthetic enzyme and promoter region of cmeABC was amplified independently and used as a template for overlapping extension PCR.…”

Key Role of Capsular Polysaccharide in the Induction of Systemic Infection and Abortion by Hypervirulent Campylobacter jejuni

“…The entire coding region of the kpsS gene was amplified from strain IA3902 by PCR using primers ST101_F and ST101_R (Table 3). The PCR product was digested with SacI and SacII and cloned into the plasmid construct pRY112-pABC (54,55) to generate pRY112-kpsS, in which the kpsS gene was fused to the constitutively expressed promoter of cmeABC. The constructed plasmid was confirmed by PCR.…”

Molecular mechanisms and biological role ofCampylobacter jejuniattachment to host cells

“…Reduced adhesion of 81-176/ peb1A mutant to HeLa cell-line [43] No difference in adhesion of F38011/peb1A mutant to LMH cell-line [17] No difference in adhesion in strain 81-176/peb1A mutant to T84 cell-line [24] that Cj0091 mediates binding of C. jejuni to INT 407 cells and is necessary for colonisation of the gastrointestinal tract of chickens [26]. Since cj0091 mutation affected colonisation at the early stages of the infectious process, these data suggest that Cj0091 is required only for initial adherence.…”