The overall goal of this project is to study the flux (both in and out) of small molecules across the surface layers of bacteria. As is well-known, multiple resistance in pathogenic microorganisms is becoming a serious threat to human health. Recent studies in our own and other laboratories suggest that in many cases such resistance is the synergistic effect of two factors, the cell surface layer acting as permeability barrier for the influx of drugs (outer membrane of Gram-negative bacteria or mycolate-containing cell wall of mycobacteria) and active, multidrug efflux pumps. In fact, for more advanced antimicrobial agents that resist the enzymatic inactivation of bacteria, such as fluoroquinolones, this mechanism that prevents the access of drugs to the target has become a primary mechanism of resistance. In this study, we plan to continue our characterization of both of these resistance mechanisms. Thus we will characterize the """"""""slow"""""""" porins of organism such as Pseudomonas aeruginosa, the proteins that contribute to the generalized intrinsic resistance of these bacteria by slowing down the influx of antimicrobial agents across their outer membrane. We will study the mechanism in which the cell wall of mycobacteria drastically slows down the entry of most agents by producing an organized layer of mycolic acid and other lipids. At the other end, we will investigate the mechanism of multidrug efflux pumps, especially those pumps that show an incredibly wide range of specificity, such as AcrAB of Escherichia coli that extrudes almost any lipophilic or amphiphilic compounds, including dyes, disinfectants, detergents, solvents, and practically all antibiotic (except aminoglycosides). These pumps appear to become expressed more strongly when the bacteria are under stress, and the pathway of regulation will be defined. Finally, as a prototype of ABC transporter, which includes P-glycoprotein and CFTR, the maltose transporter complex of a hyperthermophile Pyrococcus furiosus will be purified, because proteins from hyperthermophiles tend to produce well-diffracting crystals, and there is great need to obtain crystallographic data on this important class of transporters.
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