H. influenzae is an important pathogen. Approximately 10,000 infants acquire meningitis each year due to the type b organism with 500 deaths and neurologic sequelae in 50% of the survivors. In addition, montypable H. influenzae have been increasingly implicated in neonatal sepsis and is a respiratory pathogen in both children and adults. Unfortunately, the recently licensed PRP vaccine is not effective in prevention of disease due to the nontypable strains and is only effective in prevention of type b disease when given after 24 months of age; 80% of invasive disease occurs before this age. Thus treatment with effective antimicrobial therapy remains critical. The beta-lactam antibiotics are a mainstay of therapy for H. influenzae disease. However, the mode of action of beta-lactams on the cell wall of this pathogen is undetermined at the biochemical level. The proposed targets of the beta-lactams, the penicillin binding proteins (PBPs), have only recently been described for H. influenzae. Compared to other rod shaped Gram-negative bacteria, the PBPs of H. influenzae are unique in terms of number, molecular weight, and binding affinities for certain beta- lactams. The major """"""""killing target"""""""" of H. influenzae has not been defined. Furthermore, the other killing targets, those PBPs essential for cell shape and cell division, have not been determined. The isolation of naturally occurring strains of ampicillin- resistant-non-beta-lactamase producing H. influenzae with altered PBPs have provided a means to define these targets. The techniques of molecular biology will be used to define the genes involved with acquisition of beta-lactam resistance; these genes will be cloned and mapped on the H. influenzae chromosome. The essential PBPs in H. influenzae (the major killing targets for beta-lactam antibodies) will be determined; these data will define those involved in cell shape and cell division and those with transpeptidase activity. In addition, the genes of selected PBPs from a wild-type susceptible isolate wil be cloned and the gene products studied in order to confirm their precise structural and functional roles. The cell wall structure of this pathogen will be examined with thin layer chromatography and high pressure liquid chromatography allowing confirmation of the enzymatic assays by analysis of their products (monomer and dimer fractions) and determination of the effects of beta-lactams on cell wall structure.
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