Lopatin Lab - research
current research
P.GINGIVALIS HSP90 RECOGNITION IN PERIODONTAL DISEASE
This proposal is submitted as part of the application for a Research Career Award for Domenica G. Sweier, D.D.S. A Research Career Award will enable the applicant to complete her graduate dentist scientist training ill the Oral Health Sciences Ph.D. Program at the University Of Michigan School Of Dentistry. The objective of this proposal is to investigate the role of the P. gingivalis Hsp90 homologue in the pathogenesis of the microorganism in periodontal disease. Heat shock proteins have been found to be important in many soft tissue infections and have been referred to as virulence factors in some models. For example, the overexpression of Saccharomyces cerevisiae Hsp90 enhances the virulence of this organism in mice (Hodgetts et al., 1996). In humans, anti-Hsp90 seroconversion is associated with a higher survival rate in systemic candidiasis. Moreover, in candidiasis, antibodies to a specific conserved epitope were shown to confer / protection (Matthews et al., 1995). Heat shock proteins have also been implicated in adherence mechanisms. A 66kDa Hsp of Salmonella typhimurium is believed to be responsible for binding to the intestinal mucosa and is considered a virulence factor in this microorganism (Ensgraber and Loos, 1992). I Elevated levels of serum antibodies to purified human Hsp90 in healthy adults were associated with periodontal health (manuscript in preparation). Healthy individuals had higher anti-Hsp90 serum antibodies than those individuals diagnosed with periodontal disease. Additionally, the P. gingivalis Hsp90 homologue has been found to cross-react with anti-human Hsp90 stress protein antibodies. We feel that further studies on the role of the Hsp90 homologue of P. gingivalis in host-bacteria interactions in periodontal disease will result in the development of new diagnostic and therapeutic modalities. The studies proposed in this application will address the following global hypothesis: Specific epitopes of the Porphyromonas gingivalis Hsp90 homologue are putative virulence detenninants that may be blocked by antibodies in sera of healthy subjects. In order to begin to address this hypothesis, we propose to: (1) clone and characterize the P. gingivalis hsp90 gene homologue, (2) express the P. gingivalis hsp90 gene homologue and harvest the protein, and (3) map the immunodominant epitopes recognized in health and disease.

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IMMUNODOMINANT STRESS PROTEINS OF P. GINGIVALIS
Studies performed in our laboratory implicate the Porphyromonas gingivalis HtpG stress protein, the prokaryotic homologue of Hsp90, in the etiology of periodontal disease. We have reported that elevated levels of anti-Hsp90 antibodies, concomitant with P. gingivalis colonization, are associated with periodontal health. Transcription of HtpG message was also found to be upregulated 7-10-fold in P. gingivalis obtained from diseased subgingival plaque. There is precedence for Hsp90 homologues contributing to pathogenicity of other microorganisms. Immunity to a single Hsp90 epitope of Candida albicans has been demonstrated to confer protection against systemic candidiasis. Studies performed by our laboratory have revealed that P. gingivalis HtpG has a significant degree of homology with human Hsp90, but remains clearly distinct from other HtpG proteins due to its unique C-terminal region. We have found that HtpG is localized to P. gingivalis membranes and extracellular vesicles, and that it cross-reacts with other prokaryotic and eukaryotic Hsp90 homologues. Our findings suggest that HtpG is readily accessible to participate in host cellular invasion processes, as well as to interfere with normal host cell functions once P. gingivalis enters the host cytoplasmic compartment. Transfection of KB cells with the P. gingivalis htpG gene stimulates IL-8 production by these cells.

This application proposes to extend our investigations into the role that molecular mimicry by HtpG plays in the pathogenicity of P. gingivalis. Previous studies of other pathogenic microorganisms which appear to use the Hsp90 homologue as a virulence factor have been purely descriptive. Our application is unique in that while will propose to evaluate the role of HtpG in adherence and invasion mechanisms, we also propose to elucidate novel pathogenic mechanism{s) by which microorganisms such as P. gingivalis utilize molecular mimicry to disrupt nonnal eukaryotic cell function{s). Since the most clearly defined eukaryotic Hsp90- mediated mechanisms involve signal transduction pathways, these will be the primary foci of our investigations. The hypothesis to be tested in this study is: 1) HtpGplays a role in adherence and invasion of host cells; and 2) once internalized, signal transduction mechanisms mediated by Hsp90ITRAP 1 within eukaryotic cells are disrupted by the HtpG of P. gingivalis through molecular mimicry. This leads to disruption ofnonnal inflammatory cytokine responses to microbial invasion by P. gingivalis and other oral microorganisms.

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