Efflux-mediated resistance to tetracyclines

Tufts University Boston, Boston MA

Investigators

Abstract

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