PROJECT SUMMARYBacterial multidrug efflux transporters confer resistance to structurally diverse antimicrobials, which is one of themajor causes for clinical treatment failure. Campylobacter jejuni is a major enteric pathogen and has developedvarious mechanisms for antibiotic resistance. Recently, both the World Health Organization and the Centers forDisease Prevention and Control have designated Campylobacter as a ?serious antibiotic resistance threat?. InCampylobacter, the multidrug efflux pump CmeABC, a RND-type efflux system, plays a key role in the resistanceto various antimicrobials and in intestinal colonization by mediating bile resistance. In CmeABC, the threeproteins assemble to form a powerful tripartite machinery, allowing direct efflux of substrates across bothmembranes of the Gram-negative cellular envelope. CmeABC is essential for C. jejuni as it has an importantnatural function for bile resistance, which is required for Campylobacter colonization in animal intestine. Typically,CmeABC requires to function cooperatively with other resistance mechanisms (such as target mutations) toconfer clinically relevant antibiotic resistance. However, a ?super? resistance-enhancing variant of CmeABC (RE-CmeABC) has recently emerged in clinical isolates of C. jejuni. This variant pump has a distinct CmeB sequenceand is much more potent in conferring multidrug resistance. Additionally, we found that RE-CmeABC isincreasingly prevalent in clinical isolates and mediates exceedingly high-level resistance to fluoroquinolone, aclinically important antibiotic for treating campylobacteriosis. Our preliminary data further suggest the enhancedefflux function of RE-CmeABC is due to sequence variations in the RE-CmeB transporter. To begin to understandhow CmeABC extrudes antimicrobials, we have initiated work to decipher the structural basis of CmeABC-mediated efflux. Our preliminary crystallization data indicate that CmeB forms a homotrimer, where individualprotomers bind to and export substrates independently. Based on the solid preliminary data, we propose in thisapplication to pursue three specific aims to 1) identify the specific mutations responsible for the enhanced effluxfunction in RE-CmeABC, 2) define the structural basis of CmeB-mediated antibiotic efflux and how sequencepolymorphisms affect the structure-function relationship, and 3) determine the horizontal spread of RE-cmeABCand its impact on C. jejuni fitness in the absence and presence of antibiotic selection pressure. We will use ahigh throughput mapping strategy, a CRISPR-Cas9 based technique for efficient gene editing and replacement,in vitro and animal model systems, x-ray crystallography, and single-molecule FRET to achieve the goals of thethree specific aims. The team of investigators have strong and complementary expertise, and are uniquelypositioned to conduct the proposed work, which is expected to reveal novel mechanisms used by an RND-typetransporter for antibiotic extrusion and enhanced multidrug resistance. This gained knowledge should betransferrable to other bacterial efflux pumps, and the findings may facilitate the development of new strategiesto control the emergence and spread of multidrug resistant Campylobacter.