Molecular basis of Campylobacter antibiotic resistance and adaptation to the intestinal tract of chickens
Campylobacter is a major foodborne pathogen causing human enteric infections. Antibiotic treatments are needed for prolonged and severe cases of campylobacteriosis, but Campylobacter is increasingly resistant to antimicrobials, which has rendered antimicrobial therapy less effective. Multiple mechanisms contribute to antibiotic resistance in Campylobacter, but antibiotic efflux mediated by membrane transporters is considered a significant player in conferring intrinsic and acquired resistance to a broad spectrum of antimicrobials. The recently published genomic sequence revealed the presence of multiple genes encoding efflux transporters in C. jejuni, and work conducted in our laboratory identified that CmeABC, a RND (resistance nodulation and cell division) type efflux pump, serves as a major efflux pump in C. jejuni, contributing to antibiotic resistance, bile resistance, and in vivo colonization of the chicken intestinal tract. Work from our laboratory also found that that CmeABC is controlled by a transcriptional regulator named CmeR and this regulator appears to modulate the expression of multiple membrane transporters in C. jejuni. Despite these recent advances, it is unclear if CmeABC is present and functional in other Campylobacter spp; the functions and regulatory mechanisms of other efflux transporters are not known; and the role of the CmeR-regulated membrane transporters in Campylobacter pathophysiology remains to be determined. In this project, we conducted a series of studies to address these questions using both in vitro and in vivo approaches. In the first study, we successfully identified CmeABC in C. coli, C. upsaliensis, C. lari , and C. fetus and found that the genomic organizations and predicted structures CmeABC are conserved in the Campylobacter spp. Insertional mutagenesis of CmeABC showed that this efflux pump is functional in Campylobacter spp. and contributes to their intrinsic resistance to a range of structurally diverse antimicrobials and toxic compounds. In the second study, we found that Cj0035c (a putative major facilitator superfamily transporter) and Cj0561c (a periplasmic fusion protein) are directly regulated by CmeR. Specifically, CmeR binds to the promoter regions of Cj0035c and Cj0561c and represses their transcription. Bile salts, which are normally present in animal gut, induce the expression of both genes, suggesting that they may be upregulated during in vivo infection. Insertional mutagenesis and induction revealed that Cj0561c plays a moderate role in antimicrobial resistance in C. jejuni, but a role of Cj0035c in antimicrobial resistance was not detected. In addition, the Cj0561c mutant was outcompeted by the wild-type strain when co-inoculated into chickens, indicating that Cj0561c contributes to Campylobacter adaptation in the intestinal environment. In the third study, we investigated the regulation and function of DcuA and DcuB, which are homologs of C4-dicarboxylate transporters identified in other bacterial species. It was demonstrated that the expression of dcuA and dcuB in C. jejuni was suppressed by CmeR and both genes were inducible by low-oxygen conditions or fumurate. Insertional mutagenesis of the two genes indicated that dcuA and dcuB collectively contribute to C. jejuni colonization in chicken ceca. The coordinated regulation of the drug efflux transporters and C4-dicarboxylate transporters by CmeR suggests that the interplay between the two systems has an important physiological function in facilitating Campylobacter adaptation to various environments. Together, these findings have significantly improved our understanding of the efflux transporters in Campylobacter. Targeting the efflux transporters may not only control antibiotic resistance, but also potentially prevent Campylobacter colonization in the intestinal tract.