Neisseria gonorrhoeae is the causative agent of gonorrhea, an STD that continues to be a public health problem. Antibiotic resistance in the gonococci, as well as in many other infectious microorganisms, is an increasing problem worldwide. Although penicillin used to be the antibiotic of choice in treating a gonococcal infection, increased resistance to this antibiotic has necessitated its replacement by 3rd generation cephalosporins or fluorinated quinolones for the treatment of infected individuals. Gonococcal resistance to tetracycline has also dramatically risen. Because of the rapid acquisition of resistance to previously efficacious antibiotics, resistance to currently used antibiotics can not be far behind. The resistance of the gonococci to penicillin occurs either through the plasmid-mediated production of penicillinase or chromosomally-mediated alterations in both membrane permeability and penicillin-binding proteins (PBPs). In chromosomally-mediated resistant N. gonorrhoeae (CMRNG), alterations of primary structures of two essential PBPs (PBPs 1 and 2) lead to a decrease in the affinities of these PBPs for penicillin, thereby rendering the organism resistant to that antibiotic. Susceptible gonococci can be transformed in the laboratory to higher levels of resistance using donor DNA from a highly resistant strain. Interestingly, however, it has proven very difficult to transform gonococci from intermediate-level penicillin resistance to a level of resistance equivalent to the donor strain. The highest level of penicillin resistance is correlated with the presence of an altered PBP1, which indicates that the PBP1 gene is involved in the genetic transformation. The investigator has recently cloned the gene encoding gonococcal PBP1, which will allow him to elucidate the mechanisms involved in the acquisition of high level penicillin resistance in N. gonorrhoeae. A five year research plan has been organized around four specific aims. The investigator proposes to clone the gene encoding PBP 1 from penicillin-resistant strains, identify the mutations in PBP1 that lead to the decrease in affinity for penicillin, and investigate the origin of these mutations. The biochemical and functional activities of PBP1 also will be determined. Using the cloned gene, the genetic events underlying the acquisition of high-level penicillin resistance in CMRNG will be investigated. Finally, the investigator will continue efforts to purify and crystallize a soluble form of PBP2 from both susceptible and resistant gonococci, with the long term goal to define the structural changes in PBP2 from resistant strains that result in a lower affinity for penicillin. The three-dimensional structure of a lethal target of beta-lactam antibiotic also may lead to the rational design of new antibiotics based on molecular modeling of the beta- lactam binding site.
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