description) At the current time, we have a poor understanding of how NTHi cause otitis media in children. We hypothesize: (1) that there are as yet unidentified genes in NTHi which are important in the ability of this organism to cause otitis media; and (2) that there is a set of genes which are known but whose role in pathogenesis is uncertain or unsuspected. We propose using three complementary molecular strategies to identify these novel virulence-associated genes or to assign a role to those as yet undefined determinants: subtractive hybridization; differential fluorescence induction using promoter probe constructs; and signature tag mutagenesis. Genes unique to NTHi, genes differentially expressed in NTHi and/or genes obligatorily required at one or more stages of the infectious process will be identified by sequence determination. Some of the genes we identify in NTHi will have homology to genes present in the published strain H. influenzae Rd genome. Sequences with no homology to Rd genes but with homology to known virulence determinants of other organisms, or unique genes which have no homology to previously identified genes, will be cloned and sequenced. Isogenic mutants, deficient in newly identified putative virulence-associated determinants, and promoter probe constructs will be used in a battery of in vitro and in vivo biological assays to dissect the role of these determinants in disease. We will utilize a number of established systems for assessing NTHi adherence, colonization and bacterial cell uptake to initially analyze mutants for absence of these virulence- associated phenotypes. We will subsequently employ several chinchilla models of both frank disease induction as well as one of disease progression to more fully characterize the role of identified determinants in pathogenesis. Chinchilla models of active and passive protection will also ultimately be used to assess the relative efficacy of antibodies directed against selected newly identified antigens of interest in prevention of otitis media. The long term goals of this proposal are to gain an increased understanding of the pathogenic mechanisms operative in otitis media induced by NTHi. The identification of new potential vaccine candidates as well as novel targets for antimicrobial therapeutics could contribute significantly to the goal of developing more effective and accepted methods to both manage otitis media and ultimately prevent it.
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