Technology for Analysis of Porphyromonas gingivalis

University Of Florida, Gainesville FL

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Abstract

DESCRIPTION (provided by applicant): Porphyromonas gingivalis is considered an important etiologic agent of adult periodontal diseases and is also implicated in some systemic diseases such as cardiovascular disease and low birth weight. The goal of this application is to apply a fundamentally new approach to identify P. gingivalis genes that are expressed during in vivo but not in vitro growth. Such genes are likely to be important to the ability of P. gingivalis to cause disease. The approach, called In Vivo Induced Antigen Technology (IVIAT), is superior to other related technologies in a number of ways, but most importantly because it does not rely on animal models to mimic the growth of the pathogen in humans. IVIAT uses antibodies present in pooled sera from infected patients as probes to identify the pathogen genes of interest. This will be accomplished in three Specific Aims. In the first Specific Aim, pooled sera from periodontitis patients will be exhaustively adsorbed with in vitro grown whole P. gingivalis cells and cell extracts. The resulting serum will be used to probe a genomic expression library of P. gingivalis in Escherichia. coli using colony-blotting methods. In Specific Aim 2, the cloned DNA inserts in reactive clones will be sequenced and analyzed to determine the cloned open reading frames (ORFs) likely to be responsible for expression of the in vivo induced (IVI) antigens. The pertinent ORFs will be subcloned into an appropriate expression vector and at least 1 mg of the expressed protein will be purified to homogeneity. In Specific Aim 3, a direct proof that P. gingivalis produces IVI antigens during an actual infectious process will be performed by probing P. gingivalis cells recovered from plaque samples of infected patients with labeled antibodies specifically directed against the IVI antigens. The results of these studies are expected to improve understanding of the pathogenic mechanisms employed by P. gingivalis by identifying virulence-associated genes that would not be found by conventional methods. These genes and their products are excellent candidates for therapeutic and diagnostic targets and for vaccine design.