Antibiotics are arguably one of the most important discoveries impacting human health in the last century. Microbial resistance to antibiotics has steadily increased since they were first introduced. Tetracycline, once a widely prescribed broad specturum antimicrobial, has seen a sharp decline in effectiveness mainly due to the activity of resistance genes encoding multi-drug resistance pumps. TetL belongs to a family of tetracycline-specific transporters that account for the majority of resistance in most pathogenic bacteria responsible for diseases such as pneumonia, dysentery, cholera, and bacteremia. Detailed information about the molecular basis of interaction and mechanism of tetracycline binding and transport through TetL will be invaluable to the future development of antibiotics. The broad long term research objectives of this study are to use x-ray crystallagraphy to obtain the first complete picture of an efflux transporter-antibiotic complex and to characterize the mechanistic basis of this interaction.
The specific aims for the proposed studies are as follows:
Aim I. To understand the basis of efflux mediated tetracycline resistance, I propose to solve the structure of TetL, a tetracycline exporter from B.subtilis, alone and bound to an antibiotic. TetL has been crystallized and crystals currently diffract to 4.2 A. Improvement of these crystals to 3 A will allow for high resolution structural determination. Tetracycline analogs have been co-crystallized with TetL or soaked into TetL crystals, the first steps toward understanding the interaction of this multidrug resistance transporter with its substrate.
AIM II. To understand TetL substrate specificity and the functional basis of transport of tetracycline, I propose to examine the role of residues predicted to be involved in substrate recognition and transport and the significance of TetL dimeric oligomerization. Charged residues involved in tetracycline binding and transport predicted from alignments, homology modeling, and the crystal structure of TetL, will be probed with fluorescent binding experiments, transport assays in reconstituted systems, and in vivo activity experiments. Allosteric communication within the dimer will be examined using equilibrium binding experiments.
Drug-resistant pathogens are global threats to public health. This research seeks to demonstrate the molecular basis of a tetracycline interaction with a bacterial drug resistance pump. Understanding the structural basis of drug resistance will open the field to future development of new antibiotics.
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