Rapid Chair-Side Test for Detection of Porphyromonas gingivalis.

Porphyromonas gingivalis is a keystone pathogen of chronic periodontitis, and its intraoral levels have been shown to predict disease progression (activity). An accurate and sensitive chair-side (point of care) test to determine disease activity is critical for early intervention and clinical management of disease. This study aimed to develop a rapid, chair-side, saliva-based detection of P. gingivalis. Monoclonal antibodies (mAbs) to the A1-adhesin domain of the P. gingivalis RgpA-Kgp proteinase-adhesin complex were screened by enzyme-linked immunosorbent assay and microbial flow cytometry, with 2 mAbs shown to recognize all laboratory and clinical strains tested, without significantly cross-reacting with other oral bacteria tested. With these mAbs, an immunochromatographic device was produced and shown in preclinical studies to detect, in inoculated saliva, all P. gingivalis laboratory strains and clinical isolates tested. The device was able to detect ≥1 × 105 P. gingivalis cells/mL. In a patient age- and sex-matched control clinical cohort, P. gingivalis levels in saliva-as measured by real-time polymerase chain reaction-positively correlated with P. gingivalis levels in subgingival plaque ( r = 0.819, P < 0.01) and clinical parameters of disease ( r = 0.633, P < 0.01). A positive device result strongly correlated with P. gingivalis levels >1 × 105 cells/mL in saliva ( r = 0.778, P < 0.001) and subgingival plaque ( r = 0.715, P < 0.001) with sensitivity, specificity, positive/negative predictive values, and accuracy levels of 95.0%, 93.3%, 90.5%, 96.6%, and 94.0%, respectively. The device result also positively correlated ( r = 0.695, P < 0.01) with disease severity as measured by probing depth. Detection of P. gingivalis in saliva was found to be rapid, taking 3 min from sample collection.

Candida virulence and ethanol-derived acetaldehyde production in oral cancer and non-cancer subjects.

OBJECTIVES: To compare biofilm-forming ability, hydrolytic enzymes and ethanol-derived acetaldehyde production of oral Candida isolated from the patients with oral cancer and matched non-oral cancer. MATERIAL AND METHODS: Fungal biofilms were grown in RPMI-1640 medium, and biofilm mass and biofilm activity were assessed using crystal violet staining and XTT salt reduction assays, respectively. Phospholipase, proteinase, and esterase production were measured using agar plate method, while fungal acetaldehyde production was assessed via gas chromatography. RESULTS: Candida isolated from patients with oral cancer demonstrated significantly higher biofilm mass (P = 0.031), biofilm metabolic activity (P < 0.001), phospholipase (P = 0.002), and proteinase (P = 0.0159) activity than isolates from patients with non-oral cancer. High ethanol-derived acetaldehyde-producing Candida were more prevalent in patients with oral cancer than non-oral cancer (P = 0.01). In univariate regression analysis, high biofilm mass (P = 0.03) and biofilm metabolic activity (P < 0.001), high phospholipase (P = 0.003), and acetaldehyde production ability (0.01) were significant risk factors for oral cancer; while in the multivariate regression analysis, high biofilm activity (0.01) and phospholipase (P = 0.01) were significantly positive influencing factors on oral cancer. CONCLUSION: These data suggest a significant positive association between the ability of Candida isolates to form biofilms, to produce hydrolytic enzymes, and to metabolize alcohol to acetaldehyde with their ability to promote oral cancer development.

The development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high-resolution melting curve analysis.

Accurate, rapid and economical fungal species identification has been a major aim in mycology. In this study, our goal was to examine the feasibility of a high-resolution melting curve analysis (HRMA) of internal transcribed regions ITS1 and ITS2 in ribosomal DNA (rDNA) for a rapid, simple and inexpensive differentiation of eight clinically relevant Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Candida guilliermondii, Candida dubliniensis and Candida lusitaniae). In addition, for the first time, we tested the applicability of HRMA to classify C. albicans strains into four previously described genotypes (A, B, C and D) using a primer set that spans the transposable intron region of 25S of rDNA. Type and unknown clinical oral isolates were used in this study and the melting curve analysis was compared with both amplicons' sequencing and agarose gel electrophoresis analysis. Real-time PCR and subsequent HRMA of the two described rDNA regions generated distinct melting curve profiles that were in accord with sequencing and gel electrophoresis analysis, highly reproducible, and characteristic of each of the eight Candida species and C. albicans genotypes. Moreover, results were obtained in 4 h and without the need for any post-amplification handling, so reducing time and cost. Owing to its simplicity and speed, this technique is a good fit for genotypic analysis of hundreds of clinical strains in large epidemiological settings.

Maculatin 1.1 disrupts Staphylococcus aureus lipid membranes via a pore mechanism.

Maculatin 1.1 (Mac1) showed potent activity against Staphylococcus aureus with an MIC of 7 µM. The mode of action of Mac1 was investigated by combining assays with S. aureus cells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs of S. aureus incubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans by S. aureus was observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores in S. aureus membranes.

Synergistic virulence of Porphyromonas gingivalis and Treponema denticola in a murine periodontitis model.

Chronic periodontitis is characterized by the destruction of the tissues supporting the teeth and has been associated with the presence of a subgingival polymicrobial biofilm containing Porphyromonas gingivalis and Treponema denticola. We have investigated the potential synergistic virulence of P. gingivalis and T. denticola using a murine experimental model of periodontitis. An inoculation regime of four intra-oral doses of 1 × 10(10) P. gingivalis cells induced significant periodontal bone loss compared with loss in sham-inoculated mice, whereas doses of 1 × 10(9) cells or lower did not induce bone loss. Inoculation with T. denticola with up to eight doses of 1 × 10(10) cells failed to induce bone loss in this model. However, four doses of a co-inoculum of a 1 : 1 ratio of P. gingivalis and T. denticola at 5 × 10(8) or 1 × 10(9) total bacterial cells induced the same level of bone loss as four doses of 1 × 10(10) P. gingivalis cells. Co-inoculation induced strong P. gingivalis-specific T-cell proliferative and interferon-γ-dominant cytokine responses, and induced a strong T. denticola-specific interferon-γ dominant cytokine response. Only at the higher co-inoculum dose of 1 × 10(10) total cells was a T. denticola-specific T-cell proliferative response observed. These data show that P. gingivalis and T. denticola act synergistically to stimulate the host immune response and to induce alveolar bone loss in a murine experimental periodontitis model.

The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.

Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing beta cells. While the role of CD4(+) T cells in the pathogenesis of T1D is accepted widely, the epitopes recognized by pathogenic human CD4(+) T cells remain poorly defined. None the less, responses to the N-terminal region of the insulin A-chain have been described. Human CD4(+) T cells from the pancreatic lymph nodes of subjects with T1D respond to the first 15 amino acids of the insulin A-chain. We identified a human leucocyte antigen-DR4-restricted epitope comprising the first 13 amino acids of the insulin A-chain (A1-13), dependent upon generation of a vicinal disulphide bond between adjacent cysteines (A6-A7). Here we describe the analysis of a CD4(+) T cell clone, isolated from a subject with T1D, which recognizes a new HLR-DR4-restricted epitope (KRGIVEQCCTSICS) that overlaps the insulin A1-13 epitope. This is a novel epitope, because the clone responds to proinsulin but not to insulin, T cell recognition requires the last two residues of the C-peptide (Lys, Arg) and recognition does not depend upon a vicinal disulphide bond between the A6 and A7 cysteines. The finding of a further CD4(+) T cell epitope in the N-terminal A-chain region of human insulin underscores the importance of this region as a target of CD4(+) T cell responses in human T1D.

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.

Role of RgpA, RgpB, and Kgp proteinases in virulence of Porphyromonas gingivalis W50 in a murine lesion model.

Extracellular Arg-x- and Lys-x-specific cysteine proteinases are considered important virulence factors and pathogenic markers for Porphyromonas gingivalis, a bacterium implicated as a major etiological agent of chronic periodontitis. Three genes. rgpA, rgpB, and kgp, encode an Arg-x-specific proteinase and adhesins (RgpA), an Arg-x-specific proteinase (RgpB), and a Lys-x-specific proteinase and adhesins (Kgp), respectively. The contribution to pathogenicity of each of the proteinase genes of P. gingivalis W50 was investigated in a murine lesion model using isogenic mutants lacking RgpA, RgpB, and Kgp. Whole-cell Arg-x-specific proteolytic activity of both the RgpA(-) and RgpB(-) isogenic mutants was significantly reduced (3- to 4-fold) relative to that of the wild-type W50. However, for the Kgp(-) isogenic mutant, whole-cell Arg-x activity was similar to that of the wild-type strain. Whole-cell Lys-x proteolytic activity of the RgpA(-) and RgpB(-) mutants was not significantly different from that of wild-type W50, whereas the Kgp(-) mutant was devoid of Lys-x whole-cell proteolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using proteinase-specific antibodies of cell sonicates of the wild-type and mutant strains showed that the proteinase catalytic domain of each of the mutants was not expressed. This analysis further showed that RgpB appeared as 72- and 80-kDa bands, and the catalytic domains of RgpA and Kgp appeared as processed 45-kDa and 48-kDa bands, respectively. In the murine lesion model, mice were challenged with three doses of each mutant and wild-type strain. At the lower dose (3.0 x 10(9) viable-cells), no lesions were recorded for each of the mutants, whereas wild-type W50 induced large ulcerative lesions. At a dose of 6.0 x 10(9) viable-cells, all the mice challenged with the wild-type strain died, whereas mice challenged with the RgpA(-) and RgpB(-) isogenic mutants did not die but developed lesions. Mice challenged with the Kgp(-) isogenic mutant at this dose did not develop lesions. At a 1.2 x 10(10) viable-cell dose, only 40% of mice challenged with the Kgp(-) mutant developed lesions, and these lesions were significantly smaller than lesions induced by the wild-type strain at the 3.0 x 10(9) viable-cell dose. All the mice challenged with the RgpA(-) mutant died at the 1.2 x 10(10) viable-cell dose, whereas only 20% died when challenged with the RgpB(-) mutant at this dose. Wild-type phenotype was restored to the RgpB(-) mutant by complementation with plasmid pNJR12::rgpB containing the rgpB gene. There was no difference between the pNJR12::rgpB-complemented RgpB(-) mutant and the wild-type W50 strain in whole-cell Arg-x activity, protein profile, or virulence in the murine lesion model. These results show that the three proteinases, RgpA, RgpB, and Kgp, all contributed to virulence of P. gingivalis W50 in the murine lesion model and that the order in which they contributed was Kgp >> RgpB > or = RgpA.

Kappacin, a novel antibacterial peptide from bovine milk.

Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk kappa-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans and Porphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S. mutans in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated kappa-casein (residues 106 to 169) [kappa-casein(106--169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)(149)kappa-casein-A(138--158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(P)(149)kappa-casein-A(138--158) and its nonphosphorylated counterpart kappa-casein-A(138--158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P)(149) kappa-casein-A(138--158) displayed growth-inhibitory activity against S. mutans (MIC, 59 microg/ml [26 microM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.

RgpA-Kgp peptide-based immunogens provide protection against Porphyromonas gingivalis challenge in a murine lesion model.

Porphyromonas gingivalis, a gram-negative bacterium, has been linked to the onset and progression of periodontitis, a chronic inflammatory disease of the supporting tissues of the teeth. A major virulence factor of P. gingivalis is an extracellular complex of Arg- and Lys-specific proteinases and adhesins designated the RgpA-Kgp complex (formerly the PrtR-PrtK complex). In this study we show that the RgpA-Kgp complex, when used as an immunogen with incomplete Freund adjuvant (IFA), protects against challenge with invasive and noninvasive strains of P. gingivalis in the murine lesion model. We identified a variety of peptide vaccine candidates from the RgpA and Kgp polyprotein sequences that involved the putative active site histidine of both proteinases and five repeat motifs in the adhesin domains of both polyproteins implicated in aggregation and binding to host substrates, designated adhesin-binding motif (ABM) peptides. These peptides were synthesized using standard, solid-phase protocols for 9-fluorenylmethoxy carbonyl chemistry with S-acetylmercaptoacetic acid (SAMA) as the N-terminal residue. The SAMA-peptides were then conjugated to diphtheria toxoid and used with IFA to immunize BALB/c mice. Both active-site peptides and three of the five ABM peptides gave protection (P < 0.005) against challenge with P. gingivalis in the murine lesion model. The three ABM peptide sequences that conferred protection exist within a 100-residue span in the RgpA44 and Kgp39 adhesins of the RgpA-Kgp complex. Protective anti-RgpA-Kgp complex mouse antisera recognized the RgpA27, Kgp39, and RgpA44 adhesins in an immunoblot. Epitope mapping of the RgpA27 adhesin using the protective anti-RgpA-Kgp antisera identified a major protective epitope that mapped immediately N terminal to one of the protective ABM peptides in the 100-residue span in RgpA44 and Kgp39. This identified protective epitope contains clusters of basic residues spatially surrounded by hydrophobic amino acids, a finding which is characteristic of a heparin binding motif.

Serum immunoglobulin G (IgG) and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis in adult periodontitis.

Serum immunoglobulin G (IgG), IgM, and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis were examined by enzyme-linked immunosorbent assay using adult periodontitis patients and age- and sex-matched controls. Twenty-five sera from subjects with adult periodontitis (diseased group) and 25 sera from healthy subjects (control group) were used for the study. Sera and subgingival plaque samples from 10 sites were collected from each patient at the time of clinical examination. The level of P. gingivalis in the plaque samples was determined using a DNA probe. Highly significant positive associations between the percentage of sites positive for P. gingivalis and measures of disease severity (mean pocket depth, mean attachment loss, and percentage of sites that bled on probing) were found. The diseased group had significantly higher specific IgG responses to the RgpA-Kgp complex than did the control group, and the responses were significantly associated with mean probing depths and percentage of sites positive for P. gingivalis. Analysis of the IgG subclass responses to the RgpA-Kgp complex revealed that the subclass distribution for both the diseased and control groups was IgG4 > IgG2 > IgG3 = IgG1. The IgG2 response to the complex was positively correlated with mean probing depth, whereas the IgG4 response was negatively correlated with this measure of disease severity. Immunoblot analysis of the RgpA-Kgp complex showed that sera from healthy subjects and those with low levels of disease, with high IgG4 and low IgG2 responses, reacted with the RgpA27, Kgp39, and RgpA44 adhesins; however, sera from diseased subjects with low IgG4 and high IgG2 responses reacted only with the RgpA44 and/or Kgp44 adhesins. Epitope mapping of the RgpA27 adhesin localized a major epitope recognized by IgG4 antibodies in sera from subjects with high IgG4 and low IgG2 responses to the RgpA-Kgp complex which was not recognized by sera from diseased subjects with low IgG4 and high IgG2 responses.

Histatin 5 is a substrate and not an inhibitor of the Arg- and Lys-specific proteinases of Porphyromonas gingivalis.

The salivary peptide histatin 5 has been reported to be an inhibitor of the Arg- and Lys-specific proteinases of Porphyromonas gingivalis, an oral pathogen associated with periodontitis. In this study a purified P. gingivalis proteinase preparation consisting of a complex of the Arg- and Lys-specific proteinases and adhesins was assayed using chromogenic substrates in the presence of histatin 5. Histatin 5 produced a concentration-dependent decrease in the initial rate of hydrolysis of the chromogenic substrates by both proteinases. However, pre-incubation of histatin 5 with the purified proteinase preparation or a P. gingivalis cell sonicate for 10 min prior to assay with the chromogenic substrates showed that under these conditions the salivary peptide did not decrease the initial rate of chromogen release. Mass spectrometric analysis revealed rapid degradation of histatin 5 at all four lysyl and all three arginyl residues by the P. gingivalis proteinases. This study demonstrates that histatin 5 is a substrate for the P. gingivalis extracellular Arg- and Lys-specific cysteine proteinases and not an inhibitor.

Purification and characterization of a putative fimbrial protein/receptor of Porphyromonas gingivalis.

Porphyromonas gingivalis has been implicated as a major aetiological agent in certain forms of periodontal disease, P. gingivalis is a Gram-negative, asaccharolytic bacterium that obtains energy from the fermentation of amino acids derived from the hydrolysis of host protein. Virulence factors of this bacterium include the capsule, fimbrial adhesins, cytotoxins and extracellular hydrolytic enzymes. A 43 kDa fimbrillin from P. gingivalis has been isolated and characterized. However, there is evidence that a second type of fimbria exists on the surface of P. gingivalis. A putative P. gingivalis fimbrial protein from a membrane preparation has been isolated and identified. This protein was shown to be reactive with sera from patients harbouring P. gingivalis. A 28 kDa protein fragment was purified by anion exchange, gel filtration and reversed-phase chromatography. N-terminal sequence analysis of the 28 kDa protein fragment revealed homology to the fimbrial precursor protein of Dichelobacter nodosus. A peptide corresponding to the N-terminal 26 amino acyl residues of the 28 kDa protein fragment was synthesized and used to raise antibodies to the protein. Western blot analysis after SDS-PAGE of a P. gingivalis membrane preparation using the antibodies raised to the synthetic peptide detected three proteins of 36, 41 and 67 kDa. When protease inhibitors were not included in the extraction procedure only the 36 and 41 kDa bands were detected. It would appear, therefore, that the intact protein has an M(r) of 67 kDa and that the 28, 36 and 41 kDa bands represent protein fragments produced by endogenous proteolytic activity. Based on sequence homology, the 67 kDa protein is possibly a sub-unit of a second P. gingivalis fimbrial type or a surface receptor.