<italic>Porphyromonas gingivalis</italic> Hsp90 recognition in periodontal disease.

We reported that elevated levels of serum antibody to Hsp90 stress protein in individuals colonized with Porphyromonas gingivalis were associated with periodontal health. We also identified a P. gingivalis Hsp90 homologue (HtpG) that cross-reacts with human Hsp90 and appears to be localized to membrane fractions. P. gingivalis has been implicated in the etiology of Chronic Periodontal Disease which is a treatment challenge for dentistry. Additionally, associations between microbial virulence and stress protein expression have been identified in important human infections. This thesis describes the studies addressing the following hypothesis: Specific epitopes of the Porphyromonas gingivalis Hsp90 homologue are putative virulence determinants that may be blocked by antibodies in sera of healthy subjects. We have (i) cloned and characterized the P. gingivalis htpG gene and protein, (ii) created a P. gingivalis htpG disruption mutant and looked at growth, adherence and invasion into human cells, (iii) generated polyclonal rabbit anti-P. gingivalis recombinant HtpG (Pg rHtpG) antiserum, and (iv) mapped the immunogenic regions of the P. gingivalis HtpG dominant in recognition in human health and periodontal disease. The P. gingivalis ATCC 33277 htpG sequence has Genbank accession number AF176245. The translated HtpG is 684 amino acids and contains a unique C-terminal insert of 65 amino acids not found in other HtpGs/Hsp90s sequenced to date. The P. gingivalis htpG disruption mutant does not appear to affect growth at optimal conditions, and there is no difference in adherence and invasion compared to the wild type onto/into human epithelial cells. I identified immunoreactive regions of the P. gingivalis HtpG using rabbit anti-Pg rHtpG and human serum with a fusion protein library consisting of Pg HtpG peptides linearly representative of the Pg HtpG. Healthy human serum showed a trend of higher anti-Pg HtpG peptide titer compared to Chronic Periodontal Disease. Statistically significant differences exist between healthy serum and Aggressive Periodontal Disease when measuring anti-Pg HtpG and anti-Pg HtpG peptides. My work, in conjunction with ongoing studies in our laboratory, suggests a modulatory mechanism mediated by P. gingivalis HtpG which suppresses the proinflammatory cytokine response and is attenuated by the presence of epitope-specific anti-Hsp90 antibodies.Ph.D.DentistryHealth and Environmental SciencesImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124193/2/3122053.pd

Immunoglobulin G (IgG) Class, but Not IgA or IgM, Antibodies to Peptides of the Porphyromonas gingivalis Chaperone HtpG Predict Health in Subjects with Periodontitis by a Fluorescence Enzyme-Linked Immunosorbent Assay▿

Chaperones are molecules found in all cells and are critical in stabilization of synthesized proteins, in repair/removal of defective proteins, and as immunodominant antigens in innate and adaptive immunity. Subjects with gingivitis colonized by the oral pathogen Porphyromonas gingivalis previously demonstrated levels of anti-human chaperone Hsp90 that were highest in individuals with the best oral health. We hypothesized that similar antibodies to pathogen chaperones might be protective in periodontitis. This study examined the relationship between antibodies to P. gingivalis HtpG and clinical statuses of healthy and periodontitis-susceptible subjects. We measured the humoral responses (immunoglobulin G [IgG], IgA, and IgM) to peptides of a unique insert (P18) found in Bacteroidaceae HtpG by using a high-throughput, quantitative fluorescence enzyme-linked immunosorbent assay. Indeed, higher levels of IgG class anti-P. gingivalis HtpG P18 peptide (P < 0.05) and P18α, consisting of the N-terminal 16 amino acids of P18 (P < 0.05), were associated with better oral health; these results were opposite of those found with anti-P. gingivalis whole-cell antibodies and levels of the bacterium in the subgingival biofilm. When we examined the same sera for IgA and IgM class antibodies, we found no significant relationship to subject clinical status. The relationship between anti-P18 levels and clinical populations and individual subjects was found to be improved when we normalized the anti-P18α values to those for anti-P18γ (the central 16 amino acids of P18). That same ratio correlated with the improvement in tissue attachment gain after treatment (P < 0.05). We suggest that anti-P. gingivalis HtpG P18α antibodies are protective in periodontal disease and may have prognostic value for guidance of individual patient treatment

Serum antibodies to Porphyromonas gingivalis chaperone HtpG predict health in periodontitis susceptible patients.

Chaperones are ubiquitous conserved proteins critical in stabilization of new proteins, repair/removal of defective proteins and immunodominant antigens in innate and adaptive immunity. Periodontal disease is a chronic inflammatory infection associated with infection by Porphyromonas gingivalis that culminates in the destruction of the supporting structures of the teeth. We previously reported studies of serum antibodies reactive with the human chaperone Hsp90 in gingivitis, a reversible form of gingival disease confined to the oral soft tissues. In those studies, antibodies were at their highest levels in subjects with the best oral health. We hypothesized that antibodies to the HSP90 homologue of P. gingivalis (HtpG) might be associated with protection/resistance against destructive periodontitis.ELISA assays using cloned HtpG and peptide antigens confirmed gingivitis subjects colonized with P. gingivalis had higher serum levels of anti-HtpG and, concomitantly, lower levels of attachment loss. Additionally, serum antibody levels to P. gingivalis HtpG protein were higher in healthy subjects compared to patients with either chronic or aggressive periodontitis. We found a negative association between tooth attachment loss and anti-P. gingivalis HtpG (p = 0.043) but not anti-Fusobacterium nucleatum (an oral opportunistic commensal) HtpG levels. Furthermore, response to periodontal therapy was more successful in subjects having higher levels of anti-P. gingivalis HtpG before treatment (p = 0.018). There was no similar relationship to anti-F. nucleatum HtpG levels. Similar results were obtained when these experiments were repeated with a synthetic peptide of a region of P. gingivalis HtpG.OUR RESULTS SUGGEST: 1) anti-P. gingivalis HtpG antibodies are protective and therefore predict health periodontitis-susceptable patients; 2) may augment the host defence to periodontitis and 3) a unique peptide of P. gingivalis HtpG offers significant potential as an effective diagnostic target and vaccine candidate. These results are compatible with a novel immune control mechanism unrelated to direct binding of bacteria