The PI is interested in studying fluoroquinolone resistance in Staphylococcus spp. for three reasons. First, staphylococci are important pathogens and are growing more and more resistant to fluoroquinolones and other antibiotics. Understanding the mechanism of quinolone resistance, which appears to be different from that seen in the gram-negative bacteria, is thus an important goal. A second reason is that topoisomerase IV seems to be the main fluoroquinolone target in the gram positive bacteria rather than DNA gyrase, and this makes it possible to investigate the function and regulation of topoisomerases in ways not possible in the gram-negative bacteria. Finally, the PI is interested in the mechanism and regulation of multidrug efflux transporters that provide yet another mechanism of fluoroquinolone resistance in addition to alterations in topoisomerase itself. More specifically, the PI proposes the following: 1. To determine how mutations in the two topoisomerase IV subunits, ParC and ParD, make the topoisomerase less susceptible to fluoroquinolone action. To this end, the PI will purify and characterize mutant topoisomerases to determine if their binding of the antibiotic is reduced or affect the catalytic activity of the enzyme. Results of this analysis should indicate not only how the resistance mechanism works but whether quinolones act by reducing enzyme activity or more indirectly by stabilizing enzyme-DNA complexes. 2. To determine the location of topoisomerase IV relative to the DNA replication complex. Topoisomerase IV is thought to act by decatenation of newly replicated daughter chromosomes to allow segregation. Fluoroquinolones can be used to trap enzyme-DNA complexes and thus determine the sites where topoisomerase acts. Sites preferentially used by topo IV will be identified. The PI will also determine how rapidly the antibiotic stops new DNA synthesis, an indication of whether the topo IV acts near or far from the replication complex. The PI has isolated a new type of resistance-producing mutation that he hopes may shed light on the involvement of topo IV in DNA replication. These mutants will be characterized in detail. 3. To characterize the regulation and function of NorA, a multidrug efflux pump that mediates fluoroquinolone resistance. Gene fusions will be used to follow expression and the effect of various known global regulators will be determined. The promoter of norA will be located. NorA function will be assessed by purifying NorA protein and incorporating it in liposomes. 4. To characterize factors affecting the evolution of fluoroquinolone resistance. It has been noted that methicillin-resistant S. aureus strains are far more likely to become resistant to fluoroquinolones than methicillin-sensitive strains. The PI has evidence suggesting that this might be due to linkage of the methicillin resistance gene, mecA, to gyrA genes that have already started on the mutation pathway to detectable resistance. The PI will also determine whether the induction of fibronectin-binding proteins by quinolones in MSSA strains is responsible for the tendency of the MSSA that become quinolone resistant to spread clonally, in contrast to MRSA.
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