Porphyromonas gingivalis, a Gram-negative anaerobe, is a major etiologic agent of severe adult periodontitis. P. gingivalis possesses a number of virulence factors including the ability to invade the epithelial cells of the gingiva. In primary cultures of human gingival epithelial cells (GECs) the internal bacteria rapidly locate in the cytoplasm, predominantly in the perinuclear area, where they can replicate and reach a high density. The molecules of P. gingivalis that direct these events have yet to be determined. GECs are being used as a model system to study host-pathogen interactions involved in human periodontal disease. We propose to take a comprehensive, proteomics based approach to the study of P. gingivalis invasion and virulence by examining changes in global protein expression during invasion for the pathogen. For a select group of proteins, timecourse measurements of protein expression by mass spectrometry will be compared to mRNA levels measured using semi-quantitative real time reverse transcriptase polymerase chain reaction (RTPCR) and Northern blot analyses. Expected changes in P. gingivalis hydrophobic membrane bound proteins will be assayed as well, using non-aqueous reversed-phase HPLC technology developed at the University of Washington and (or) the MudPIT approach (Multidimensional Protein Identification Technology) of the Yates laboratory, both of which expand the range of proteins that can be analyzed beyond what can be done with the current state-of-the-art in 2D gel electrophoresis in terms of isoelectric point and hydrophobicity. The transcription analysis will be complemented by our ability to map observed proteins to the P. gingivalis genome in a semi-automated fashion directly using mass spectral fragmentation data. The data sets from these experiments will be used to gain insights into the precise molecular determinants of P. gingivalis invasiveness, that will aid in the characterization of potential targets for therapeutic agents that could serve to inhibit the transformation of P. gingivalis from a harmless commensal colonizer into a highly invasive pathogen.
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