Efflux-mediated resistance to tetracyclines

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Use of tetracyclines to treat infectious diseases has been sharply curtailed because of resistance. Among two major mechanisms for tetracycline (Tc) resistance, active efflux is the most common, represented by more than 20 different Tc determinants. Since our discovery of active Tc efflux almost 25 years ago, this laboratory has focused on a prototype efflux protein, the class B Tet A (TetA(B)) protein specified by Tn10. The proposed project has three major aims directed at better understanding the molecular basis for Tet protein efflux activity. 1. Grow and analyze 2D and 3D crystals of the TetA(B) protein. This work will benefit from recent developments in membrane protein crystal structure work and improvements in the purification of Tet protein that have led to our ability to reproducibly obtain crystals. 2. Extend efforts aimed at identifying the substrate binding site(s): a. Prepare and use photoaffinity labeled Tcs to identify particular amino acids which comprise the Tc binding site. b. Use a FeTc complex and the Fenton reaction to cleave the protein near the binding site and so define the second such cleavage site. c. Perform site-directed mutagenesis of residues identified in "a" to see the effect on Tc resistance and Tc transport. 3. Continue the genetic analysis of the TetA(B) protein, focusing on the interdomain loop. By suppressor mutations, amino acid replacements and cross-linking studies, we shall study the physical neighborhood and functional role of the loop in terms of efflux and substrate specificity. Insights into the molecular structure and function of TetA protein, a prototype of an efflux protein of the Major Facilitator Superfamily, will help in understanding this common mechanism of drug resistance and suggests ways it could be overcome. This knowledge, in turn, could help lead to a renaissance for the tetracycline family of antibiotics which have a long history of being generally well-tolerated and safe. Such understanding will also enhance efforts to reverse efflux of other antibiotics and drugs used to treat infectious diseases, including bacteria, fungi and parasites, and cancer.