Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Campylobacter jejuni is a zoonotic, foodborne pathogen causing gastroenteritis in humans. The multidrug efflux pump CmeABC plays a key role in antimicrobial resistance by extruding structurally diverse compounds and is essential for intestinal colonization by mediating bile resistance. Expression of cmeABC is under the control of CmeR, a TetR family transcriptional regulator, and CosR, an oxidative stress response regulator. However, the molecular basis and functional consequences of differential CmeABC expression as well as the interactive role of CosR and CmeR in modulating cmeABC expression are still unknown. To address these questions, we performed two sets of studies. In the first study, we evaluated differential expression of cmeABC in naturally occurring C. jejuni isolates and identified the mutations associated with overexpression of cmeABC. It was found that 67% of examined C. jejuni isolates exhibited a CmeABC-overexpressed phenotype as determined by immunoblotting and real-time RT-PCR. This phenotype was further linked to mutations in the cmeABC promoter sequence that decreased the binding of CmeR to the promoter DNA or a reduced cmeR expression. Consequently, both types of mutation increased expression of cmeABC. Additionally, the CmeABC-overexpressed phenotype was associated with increased emergence of ciprofloxacin-resistant mutants in cultures treated with a fluoroquinolone antibiotic.
In the second study, we demonstrated that CmeR and CosR simultaneously bound to two separate sites in the cmeABC promoter, providing dual repression of cmeABC expression. The two regulators interact with the cmeABC promoter independently, but maximal repression by CmeR and CosR requires a 17 bp spacer between the binding sites as shortening the spacer interferes with CmeR binding of the promoter in the presence of CosR. Additionally, we demonstrated that CosR utilizes the single cysteine residue (C218) to sense oxidative stress as oxidation of C218 inhibited CosR binding to the promoter, providing a mechanistic explanation for oxidative-stress-induced, CosR-mediated overexpression of cmeABC.
Together, these results reveal sophisticated mechanisms that modulate expression of cmeABC and identify a new signal (oxidative stress) that interacts with this efflux system. Considering the important role of CmeABC in Campylobacter pathobiology, the diverse mechanisms influencing cmeABC expression may facilitate Campylobacter adaptation to diverse environmental conditions.
Tara Leigh Grinnage-Pulley
Grinnage-Pulley, Tara Leigh, "Regulatory and mutational mechanisms for differential expression of the CmeABC multidrug efflux pump in Campylobacter jejuni" (2013). Graduate Theses and Dissertations. 13495.