H. influenzae type b is an important pathogen in children. Approximately 10,000 infants acquire meningitis each year due to this organism with 500 deaths and neurologic sequelae in 50% of the survivors. Treatment with effective antimicrobial therapy is critical. The Beta-lactam antibiotics are a mainstay of therapy for H. influenzae disease. However, the mechanism of antibacterial action of these drugs on the cell wall of this pathogen is undetermined. In addition, the extreme variation in cellular morphology of H. influenzae is unique among Gram-negative bacteria pathogenic for man. Certain Beta-lactams induce pleomorphic changes in pathogenic Gram-negative bacteria which occur in vivo in type b H. influenzae. The isolation of naturally occurring strains of ampicillin-resistant non-B-lactamase producing H. influenzae with altered penicillin binding proteins (PBPs) is unique. These strains have provided the opportunity to study the PBPs in H. influenzae. This proposal seeks to define the structure and function of PBPs in H. influenzae. Specifically the PBPs will be characterized in terms of their number, relative amount, apparent molecular weights, and relative binding affinities to selected Beta-lactam antibiotics. The structural (morphology) and enzymatic (transpeptidase activity) roles of certain PBPs will be assessed by observation and assay after incubation with selected Beta-lactams. This data will define the killing targets for Beta-lactam antibiotics, allowing rapid evaluation of new Beta-lactam (or Beta-lactam-like) antimicrobials. The genetic basis of resistance in these novel strains with altered PBPs is unknown. This will be defined (chromosomal or plasmid mediated). The PBP genes will be mapped on the H. influenzae chromosome. Cloning of the PBP genes onto a plasmid vector will be performed to obtain amounts which will facilitate purification. Understanding the physiology and genetics of the cell wall of this important childhood pathogen will provide a basis for definition of its role in the bactericidal action of certain antibiotics, as well as in the serumcidal action of antibodies.
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