application abstract) Neisseria gonorrhoeae is an obligate human pathogen and has the capacity to infect and cause disease at numerous sites. However, this capacity requires that gonococci resist antibacterial substances that naturally bathe mucosal surfaces or become available due to inflammation. The emphasis of this project is to understand the mechanism(s) used by gonococci to resist antibiotic-like substances that bathe certain mucosal sites. The mtr (multiple transferable resistance)and far (fatty acid resistance) loci contain operons that encode efflux pump proteins that export structurally diverse antibacterial hydrophobic agents (HAs), including bile salts, fatty acids, lysosomal proteins and antibiotics. The MtrCDE and FarAB proteins belong to families of bacterial proteins that form efflux pumps that remove structurally diverse antimicrobial agents from either the periplasm or cytoplasm. Expression of these efflux pump operons are subject to both negative and positive transcriptional control systems. For instance, the MtrR protein down-regulates expression of the mtrCDE operon through its capacity to bind to the mtrCDE promoter and this results in enhanced susceptibility of gonococci to certain HAs. Conversely, expression of the farAB operon, which encodes an efflux pump that exports long-chained fatty acids with potent antigonococcal activity, seems to be dependent on MtrR. Through the use of modern techniques in microbial genetics, molecular biology and biochemistry, we will determine the mechanisms by which MtrR exerts transcriptional control over these efflux pump operons and other gonococcal genes (Specific Aims I and 3). Expression of the mtrCDE operon can also be induced during exposure of gonococci to sub-lethal levels of HAs. This induction process requires a transcriptional activator, MtrA, that belongs to the AraC/XylS family of DNA-binding proteins. The mechanisms by which MtrA exerts its control over gonococcal gene expression will be determined (Specific Aim 2). Dr. Shafer'sins group has recently identified a novel protein (MtrF) that seems to act as a component of the mtrCDE-encoded efflux pump. MtrF counterparts exist in several other bacteria but their function has yet to be determined. Given its apparent wide-spread distribution, they will determine its role in efflux. pump activity (Specific Aim 4). The results from these studies will advance our knowledge regarding how gonococci and other pathogens resist antimicrobial agents at mucosal surfaces, antibiotics used in therapy of bacterial diseases, and topical microbicides that have been proposed for use to prevent sexually transmitted diseases.
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