The Type IX Secretion System and Its Role in Bacterial Function and Pathogenesis.

Porphyromonas, Tannerella, and Prevotella species found in severe periodontitis use the Type IX Secretion System (T9SS) to load their outer membrane surface with an array of virulence factors. These virulence factors are then released on outer membrane vesicles (OMVs), which penetrate the host to dysregulate the immune response to establish a positive feedback loop of chronic, inflammatory destruction of the tooth's supporting tissues. In this review, we present the latest information on the molecular architecture of the T9SS and provide mechanistic insight into its role in secretion and attachment of cargo proteins to produce a virulence coat on cells and OMVs. The recent molecular structures of the T9SS motor comprising PorL and PorM as well as the secretion pore Sov, together with advances in the overall interactome, have provided insight into the possible mechanisms of secretion. We propose the presence of PorL/M motors arranged in a circle at the inner membrane with bent periplasmic rotors interacting with the PorN protein. At the outer membrane, we envisage a slide carousel model where the PorN protein is driven around a circular track composed of PorK. Cargo proteins are transported by PorN to PorW and the Sov translocon just as slides are rotated to the projection window. Secreted proteins are proposed to then be shuttled along highways consisting of the PorV shuttle protein to an array of attachment complexes distributed around the cell. The cell surface attachment of cargo is a hallmark of the T9SS, and in Porphyromonas gingivalis and Tannerella forsythia, this attachment is achieved via covalent bonding to a linking sugar synthesized by the Wbp/Vim pathway. The cell-surface attached cargo are enriched on OMVs, which are then released from the cell.

Gingival crevicular fluid proteomes in health, gingivitis and chronic periodontitis.

OBJECTIVE: The aim of this study was to compare the proteome composition of gingival crevicular fluid obtained from healthy periodontium, gingivitis and chronic periodontitis affected sites. BACKGROUND: Owing to its site-specific nature, gingival crevicular fluid is ideal for studying biological processes that occur during periodontal health and disease progression. However, few studies have been conducted into the gingival crevicular fluid proteome due to the small volumes obtained. METHODS: Fifteen males were chosen for each of three different groups, healthy periodontium, gingivitis and chronic periodontitis. They were categorized based on clinical measurements including probing depth, bleeding on probing, plaque index, radiographic bone level, modified gingival index and smoking status. Gingival crevicular fluid was collected from each patient, pooled into healthy, gingivitis and chronic periodontitis groups and their proteome analyzed by gel electrophoresis and liquid chromatography electrospray ionization ion trap tandem mass spectrometry. RESULTS: One hundred and twenty-one proteins in total were identified, and two-thirds of these were identified in all three conditions. Forty-two proteins were considered to have changed in abundance. Of note, cystatin B and cystatin S decreased in abundance from health to gingivitis and further in chronic periodontitis. Complement proteins demonstrated an increase from health to gingivitis followed by a decrease in chronic periodontitis. Immunoglobulins, keratin proteins, fibronectin, lactotransferrin precursor, 14-3-3 protein zeta/delta, neutrophil defensin 3 and alpha-actinin exhibited fluctuations in levels. CONCLUSION: The gingival crevicular fluid proteome in each clinical condition was different and its analysis may assist us in understanding periodontal pathogenesis.

Mass spectrometric analysis of gingival crevicular fluid biomarkers can predict periodontal disease progression.

BACKGROUND AND OBJECTIVE: Gingival crevicular fluid has been suggested as a possible source of biomarkers for periodontal disease progression. This paper describes a technique for the analysis of gingival crevicular fluid from individual sites using mass spectrometry. It explores the novel use of mass spectrometry to examine the relationship between the relative amounts of proteins and peptides in gingival crevicular fluid and their relationship with clinical indices and periodontal attachment loss in periodontal maintenance patients. The aim of this paper was to assess whether the mass spectrometric analysis of gingival crevicular fluid may allow for the site-specific prediction of periodontal disease progression. MATERIAL AND METHODS: Forty-one periodontal maintenance subjects were followed over 12 mo, with clinical measurements taken at baseline and every 3 mo thereafter. Gingival crevicular fluid was collected from subjects at each visit and was analysed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Samples were classified based upon pocket depth, modified gingival index (MGI), plaque index and attachment loss, and were analysed within these groups. A genetic algorithm was used to create a model based on pattern analysis to predict sites undergoing attachment loss. RESULTS: Three hundred and eighty-five gingival crevicular fluid samples were analysed. Twenty-five sites under observation in 14 patients exhibited attachment loss of > 2 mm over the 12-mo period. The clinical indices pocket depth, MGI, plaque levels and bleeding on probing served as poor discriminators of gingival crevicular fluid mass spectra. Models generated from the gingival crevicular fluid mass spectra could predict attachment loss at a site with a high specificity (97% recognition capability and 67% cross-validation). CONCLUSIONS: Gingival crevicular fluid mass spectra could be used to predict sites with attachment loss. The use of algorithm-generated models based on gingival crevicular fluid mass spectra may provide utility in the diagnosis of periodontal disease.

Production of a high gel strength whey protein concentrate from cheese whey.

In order to develop a process for the production of a whey protein concentrate (WPC) with high gel strength and water-holding capacity from cheese whey, we analyzed 10 commercially available WPC with different functional properties. Protein composition and modification were analyzed using electrophoresis, HPLC, and mass spectrometry. The analyses of the WPC revealed that the factors closely associated with gel strength and water-holding capacity were solubility and composition of the protein and the ionic environment. To maintain whey protein solubility, it is necessary to minimize heat exposure of the whey during pretreatment and processing. The presence of the caseinomacropeptide (CMP) in the WPC was found to be detrimental to gel strength and water-holding capacity. All of the commercial WPC that produced high-strength gels exhibited ionic compositions that were consistent with acidic processing to remove divalent cations with subsequent neutralization with sodium hydroxide. We have shown that ultrafiltration/diafiltration of cheese whey, adjusted to pH 2.5, through a membrane with a nominal molecular weight cut-off of 30,000 at 15 degrees C substantially reduced the level of CMP, lactose, and minerals in the whey with retention of the whey proteins. The resulting WPC formed from this process was suitable for the inclusion of sodium polyphosphate to produce superior functional properties in terms of gelation and water-holding capacity.

Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire.

The gingipains are cell surface Arg- and Lys-specific proteinases of the bacterium Porphyromons gingivalis, which has been associated with periodontitis, a disease that results in the destruction of the teeth-s supporting tissues. The proteinases are encoded by three genes designated rgpA, rgpB and kgp. Arg-specific proteolytic activity is encoded by rgpA/B and the Lys-specific activity by kgp. RgpA and Kgp are polyproteins comprising proteinases with C-terminal adhesin domains that are proteolytically processed. After processing, the domains remain non-covalently associated as complexes on the cell surface. RgpB is also a cell surface proteinase but does not associate with adhesin domains. Using gene knockout P. gingivalis mutants, the proteolytic processing of the gingipain domains has been shown to involve the gingipains themselves as well as C-terminal processing by a carboxypeptidase. A motif in the C-terminal domain of each protein/polyprotein has been identified that is suggested to be involved in attachment to LPS on the cell surface. RgpB lacks a C-terminal adhesin binding motif found in the catalytic domains of RgpA and Kgp. This adhesin binding motif is proposed to be responsible for the non-covalent association of the RgpA and Kgp catalytic domains into the cell surface complexes with the processed adhesin domains. The RgpA-Kgp proteinase-adhesin complexes, through the adhesin domains A1 and A3, have been implicated in colonization of P. gingivalis by binding to other bacteria in subgingival plaque and also binding to crevicular epithelial cells. The RgpA-Kgp complexes also bind to fibrinogen, laminin, collagen type V, fibronectin and hemoglobin. Amino acid sequences likely to be involved in binding to these host proteins have been identified in adhesin domains A1 and A3. It is proposed that these adhesins target the proteolytic activity to host cell surface matrix proteins and receptors. The continual cycle of binding and degradation of the surface proteins/receptors on epithelial, fibroblast and endothelial cells by the RgpA-Kgp complexes in the gingival tissue leading to cell death would contribute to inflammation, tissue destruction and vascular disruption (bleeding). P. gingivalis has an obligate growth requirement for iron and protoporphyrin IX, which it preferentially utilizes in the form of hemoglobin. Kgp proteolytic activity is essential for rapid hydrolysis of hemoglobin and it is suggested therefore that a major role of the RgpA-Kgp complexes is in vascular disruption and the binding and rapid degradation of hemoglobin for heme assimilation by P. gingivalis. The RgpA-Kgp complexes also have a major role in the evasion and dysregulation of the host-s immune response. It is proposed that host pro-inflammatory cytokines and cellular receptors close to the infection site may be rapidly and efficiently degraded by the gingipains while the proteinases at lower concentrations distally could result in the promotion of an inflammatory response through activation of proteinase-activated receptors and cytokine release. The culmination of this dysregulation would be tissue destruction and bone resorption. In animal models of disease the RgpA-Kgp complex when used as a vaccine to produce a high titre antibody response protects against challenge with P. gingivalis. Using recombinant domains of RgpA and Kgp as vaccines, it has been demonstrated that the A1 and A3 domains confer protection.

Identification of a novel heterodimeric outer membrane protein of Porphyromonas gingivalis by two-dimensional gel electrophoresis and peptide mass fingerprinting.

Porphyromonas gingivalis is a Gram-negative, anaerobic bacterium associated with chronic periodontitis. A 2D electrophoretic analysis of the outer membrane of P. gingivalis W50 revealed a dominant train of spots at 40-41 kDa. The proteins in the train of spots were digested in-gel with trypsin and identified by MS. The train of spots represented two proteins, designated Omp40 and Omp41 that share 47% sequence identity. Preparation of outer membranes in the absence of protease inhibitors resulted in partial cleavage of Omp40 and Omp41 to produce an N-terminal and C-terminal fragment of both proteins. The N-terminal fragments displayed the same isoelectric heterogeneity as the intact proteins. Almost 100% of the amino-acid sequence of these N-terminal fragments in each 2D gel spot was verified suggesting lack of post-translational modification. Re-subjecting a single N-terminal domain spot to 2D electrophoresis resulted in the complete series of spots being reproduced, suggesting that the heterogeneity was related to conformational equilibria. Under reduced conditions and without heating, Omp40 and Omp41 migrated as 34- to 35-kDa proteins in SDS/PAGE whereas under nonreduced conditions the proteins migrated as 70-kDa proteins, suggesting the formation of dimers through intersubunit disulfide bonds. The proteins each contain two cysteine residues in the conserved sequence RPVSCPECPE. Tryptic peptides generated from the nonreduced forms of the proteins confirmed the presence of heterodimers stabilized through intersubunit disulfide bond formation. With the exception of heterodimer formation, the two proteins share several similarities with OmpA-like porins of other Gram-negative bacteria including consensus sequence, abundance, modification by heat, overall length and positioning of domains.