Our long-term goal is to elucidate the structures and fundamental mechanisms that give rise to drug transfer across the cell membrane by the AbgT-family of transporters. The primary targets of the proposed work are the Alcanivorax borkumensis YdaH and Neisseria gonorrhoeae MtrF transporters. Approximately 13,000 putative transporters of the family have been identified. However, no structural information has yet been available and even functional data are minimal for this family of membrane proteins. It has been hypothesized that the AbgT-family transporters contribute to the bacterial folate synthesis pathway by importing the catabolite p-aminobenzoyl- glutamate for producing this essential vitamin. To understand the structure and function of the AbgT family of transporters, we have recently cloned, expressed, purified and crystallized the full-length A. borkumensis YdaH and N. gonorrhoeae MtrF membrane proteins. We have also determined the X-ray structures of these two transporters at 2.9 and 3.9 resolutions, respectively. The structures reveal that these two transporters assemble as dimers with architectures distinct from all other families of transporters. Both YdaH and MtrF are bowl- shaped dimers with a solvent-filled basin extending from the cytoplasm halfway across the membrane bilayer. The monomers of YdaH and MtrF contain nine transmembrane helices and two hairpins. These structures directly suggest a plausible pathway for substrate transport. A combination of the crystal structure, genetic analysis and substrate accumulation assay indicates that both YdaH and MtrF behave as exporters, capable of removing the folate metabolite p-aminobenzoic acid from bacterial cells. Further experimental data based on drug susceptibility and radioactive transport assay suggest that both YdaH and MtrF participate as antibiotic efflux pumps, importantly mediating bacterial resistance to sulfonamide antimetabolite drugs. Our hypothesis is that many of these AbgT-family transporters act as exporters, thereby conferring bacterial resistance to sulfonamides.
The specific aim i s to define the structures and transport mechanisms of the AbgT family of transporters. We will use the structural, biochemical and computational approaches to determine the molecular mechanisms of the YdaH and MtrF transporters. Neisseria gonorrhoeae is an obligate human pathogen and the infectious agent for the sexually-transmitted disease gonorrhea. Although gonorrhea is one of the oldest described diseases, it remains a significant global problem with more than 100 million cases reported annually worldwide, with antibiotic resistance increasing at an alarming rate. Sulfonamides were used in the late 1930s and early 1940s to treat gonorrhea, but the rapid emergence of strains resistant to this class of drug resulted in the removal of sulfonamides from lists of recommended therapies. Therefore, elucidating the structures and mechanisms of the AbgT-family transporters will provide important new targets for the rational design of novel antibiotics to combat these bacterial infections.
Drug-resistant pathogens are on the rise. This proposal will determine the molecular mechanism for sulfonamide recognition and extrusion in transmembrane efflux transporters. Thus, the research will establish a body of knowledge about antimetabolite resistance in pathogenic bacteria, which will provide a platform for the design of novel antimetabolite-based antimicrobial therapeutic strategies.
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