EFFLUX-MEDIATED RESISTANCE TO TETRACYCLINES

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The tetracyclines are antibiotics commonly used to treat a wide range of infectious disease agents. However, their efficacy has been severely curtailed by the emergence of resistance in many different organisms. This proposal seeks funding for the molecular analysis of tetracycline resistance mediated by an active efflux, a common mechanism in resistant bacteria. The studies will be devoted primarily to the Class B (Tn10-like) encoded Tet protein as a prototype efflux carrier for more than seven related, but different, classes of tetracycline resistance determinant. This transport protein has two complementing genetic domains and appears to act as a multimer. The active site for binding of tetracycline to Tet will be sought using site-directed mutagenesis of conserved, charged amino acid residues predicted to be within the membrane, using selection of mutant Tet proteins leading to atypical resistance to tetracycline analogs, and using location of tetracycline bound covalently by UVinduced photoaffinity labeling. The role and interactions of the two complementary alpha and beta domains of Tet (comprising the proximal and distal halves of the polypeptide respectively) will be studied using genetic complementation and dominance assays of mutant and wild type Tet proteins. Active revertants of inactive interclass hybrid proteins (which contain an alpha domain from one Tet protein and a beta domain from another) will be characterized; such revertants presumably have remedied an incorrect alpha - beta domain interaction. Biochemical studies on the predicted multimerization of Tet monomers will be done using cross-linking agents and size chromatography of detergent extracts. Whether tetracycline transport is accompanied by proton antiport and the role of magnesium in this process will be investigated. The functional and physical topography of Tet within the membrane will be further characterized using polyclonal antibodies to Tet and Tet peptides. Finally, efforts will be directed at purifying active Tet and reconstituting it into liposomes. Efflux as a mechanism for drug resistance is becoming increasingly more recognized. The proposed studies are important for understanding not only resistance to the widely used antibiotic tetracycline, but also for understanding other efflux systems and membrane transport in general.