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Porphyromonas gingivalis is a keystone oral pathogen in the progression of periodontal disease and has been implicated in the etiology of many other systemic human diseases. P. gingivalis produces primary virulence factors termed the gingipains, a group of cysteine proteases. There is a high demand for high purity gingipains for downstream in vitro studies where P.g. is implicated, both oral and systemic. Gingipain production candidate mutant P.g. strains were generated to isolate each gingipain for large scale purification and the purified gingipains characterized. Two mutants were determined not to be viable for large scale purification of gingipains, each for different reasons, which may give some insight into novel mechanisms of adaption in P. gingivalis.

Porphyromonas gingivalis (Pg), one of the ‘red-complex’ perio-pathogens known to play a critical role in the development of periodontitis, has been used in various animal models to mimic human bacteria-induced periodontitis. In order to achieve a more realistic animal model of human Pg infection, the present study investigated whether repeated small-volume topical applications of Pg directly into the gingival pocket can induce local infection, including periodontitis and systemic vascular inflammation in wild-type mice. Freshly cultured Pg was topically applied directly into the gingival pocket of the second molars for 5 weeks (3 times/week). After the final application, the mice were left in cages for 4 or 8 weeks and sacrificed. The status of Pg colony formation in the pocket, gingival inflammation, alveolar bone loss, the expression levels of pro-inflammatory cytokines in the serum and aorta, the presence of anti-Pg lipopolysaccharide (LPS) and gingipain (Kpg and RgpB) antibodies in the serum, as well as the accumulation of Pg LPS and gingipain aggregates in the gingiva and arterial wall were evaluated. The topical application of Pg into the gingival pocket induced the following local and systemic pathohistological changes in mice when examined at 4 or 8 weeks after the final topical Pg application: Pg colonization in the majority of gingival pockets; increased gingival pocket depths; gingival inflammation indicated by the increased expression of TNF-α, IL-6 and IL-1β; significant loss of alveolar bone at the sites of topical Pg application; and increased levels of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-17, IL-13, KC and IFN-γ in the serum in comparison to those from mice receiving PBS. In addition, the Pg application/colonization model induced anti-Pg LPS and gingipain antibodies in serum, as well as the accumulation of Pg LPS and gingipain aggregates in the gingivae and arterial walls. To the best of our knowledge, this mouse model represents the first example of creating a more sustained local infection in the gingival tissues of wild-type mice and may prove to be useful for the investigation of the more natural and complete pathogenesis of the bacteria in the development of local oral and systemic diseases, such as atherosclerosis. It may also be useful for the determination of a treatment/prevention/efficacy model associated with Pg-induced colonization periodontitis in mice.

Background: Porphyromonas gingivalis, a major pathogen associated with chronic periodontitis, secretes variety of proteins, majority of which begins with glutamine. Several of these proteins were found with pyroglutamate (pGlu) at N-terminus suggesting the presence of this posttranslational modification pathway in P.gingivalis. The observation that N-terminal glutamine is over-represented as the first amino acid after signal peptide cleavage, and subsequent confirmation of pGlu formation on the nascent protein via mass spectrometry, led us to conclude that an enzyme must be present as the executor of this reaction. Hypothesis: PG2157 is a glutaminyl cyclase and is responsible for the cyclization of N-terminal glutamine residues. Methods: A homology search was used to identify a gene (PG2157) encoding a protein homologous to human glutaminyl cyclase (QC) in the P. gingivalis genome. The gene was cloned, expressed in E. coli and recombinant PgQC purified. The protein was crystalized, and structure determined by molecular replacement. The rPgQC activity was characterized with respect to pH, ionic strength, optimum substrate specificity, and sensitivity to inhibition by an array of non-specific and specific inhibitors. Finally, subcellular localization of PgQC in P. gingivalis was determined. Results: PgQC specificity is restricted for N-terminal glutamine. The enzyme converts this residue to pGlu with kcat/Km at 1.34 s-1. The reaction was fastest at low ionic strength and at pH around 8.0. The activity was inhibited by o-phenanthroline (≥100µM) and EDTA (≥100mM EDTA). Cu2+ and Zn2+ at ≥100nM exerted ≥90% inhibition. The activity was also significantly affected by cysteamine, imidazole, and reduced glutathione. In bacterial cells PgQC was found associated with the inner membrane as a lipoprotein facing the periplasm. The crystalline structure of PgQC showed strong similarity to human QC on the atomic level. Nevertheless, an inhibitor specific for human QC had a limited effect on the PgQC activity. Conclusions: PgQC is an enzyme resembling mammalian QC and it is responsible for pyroglutamination of proteins secreted by the T9SS of P. gingivalis. This activity is likely essential for bacterium viability since all attempts to produce a viable PgQC knockout failed. Taking into account that also T. forsythia and P. intermedia possess similar enzymes and the frequency of the Q value of Bacteriodetes it is likely that similar post-translatonal modification plays a pivotal role in protein secretion by these periodontal pathogens. Therefore, inhibition of bacterial QC may represent a novel approach to treat periodontal diseases.

Periodontitis is a chronic inflammatory disease that is characterized by severe tissue destruction of the gingiva and other tooth supporting structures; if left untreated, tooth loss and disintegration of the alveolar bone occurs. This chronic inflammatory state has been linked to other systemic diseases such as cardiovascular disease, diabetes, rheumatoid arthritis, and Alzheimer’s disease. Porphyromonas gingivalis is the major pathogenic microbe in periodontitis. The main virulence factors of P. gingivalis are the Arg-aa and Lys-aa gingipains, which are proteolytic enzymes implicated in a plethora of activities that allow P. gingivalis to subvert the human immune system in the oral cavity and cause host tissue destruction. Here, we report that Arg-aa gingipains, RgpB and HRgpA stimulate fibroblasts to express fibronectin, a glycoprotein associated with tissue injury and repair, through the transcription of its gene promoter in a dose and time dependent fashion. Interestingly, when using a protease activated receptor 2 antagonists, gingipain induced fibronectin promoter activity was attenuated. Furthermore, gingipains stimulate fibroblasts to produce a pro-inflammatory matrix triggering production of IL-6 and IL-8 by monocytes that correlated with fibronectin-EDA expression. Taken together, our data suggests Arg-gingipains act on host fibroblasts to produce fibronectin which, stimulates immune cell activation. Taken together, these data suggest that gingipains promote and environment that disrupts host homeostasis.