Dietary Nitrite Drives Disease Outcomes in Oral Polymicrobial Infections.

Streptococcus mutans resides in the oral polymicrobial biofilm and is a major contributor to the development of dental caries. Interestingly, high salivary nitrite concentrations have been associated with a decreased prevalence of dental caries. Moreover, the combination of hydrogen peroxide-producing oral commensal streptococci and nitrite has been shown to mediate the generation of reactive nitrogen species, which have antimicrobial activity. The goal of this study was to examine whether nitrite affects S. mutans virulence during polymicrobial infections with the commensal Streptococcus parasanguinis. Here, we report that the combination of S. parasanguinis and nitrite inhibited S. mutans growth and biofilm formation in vitro. Glucan production, which is critical for S. mutans biofilm formation, was also inhibited in 2-species biofilms with S. parasanguinis containing nitrite as compared with biofilms that contained no nitrite. In the in vivo caries model, enamel and dentin carious lesions were significantly reduced in rats that were colonized with S. parasanguinis prior to infection with S. mutans and received nitrite in the drinking water, as compared with animals that had a single S. mutans infection or were co-colonized with both bacteria and received no nitrite. Last, we report that S. mutans LiaS, a sensor kinase of the LiaFSR 3-component system, mediates resistance to nitrosative stress. In summary, our data demonstrate that commensal streptococci and nitrite provide protection against S. mutans pathogenesis. Modulating nitrite concentrations in the oral cavity could be a useful strategy to combat the prevalence of dental caries.

92K-GL (MMP-9) and 72K-GL (MMP-2) are produced in vivo by human oral squamous cell carcinomas and can enhance FIB-CL (MMP-1) activity in vitro.

Previous studies have shown a correlation between the production of certain matrix metalloproteinases (MMPs), especially the gelatinases, by malignant tumors and the progression of these cancers as they invade and metastasize through the extracellular matrix and basement membranes. However, very few of these studies examined this relationship in human oral cancer in vivo, and none addressed the issue of how combinations of the MMPs may further enhance tumor progression. To determine which MMPs are produced in vivo by human oral cancers, we used specific anti-human-MMP antibodies and immunocytochemistry (ICC) methods to examine oral cancer tissue specimens from 20 surgery patients. The ICC data indicated that 72-kDa (72K-GL) and 92-kDa gelatinases (92K-GL) were produced in vivo by discreet clusters of tumor cells and by stromal fibroblasts, vascular endothelial cells (72K-GL), and PMNs (92K-GL). Some stromal fibroblasts near the tumors also appeared to produce fibroblast-type collagenase (FIB-CL), a finding confirmed by Western blot analysis of media conditioned by oral tumor explant cultures. ICC results indicated that 5 of the 20 tumors coincidentally produced all three MMPs. To examine how the two gelatinases and FIB-CL may interact in vitro to degrade fibrillar type I collagen, a major structural component of the extracellular matrix, we used a modified FIB-CL activity assay. Combinations of the gelatinases and FIB-CL were incubated with a 3H-collagen substrate, with the results compared with the combination of stromelysin-1 (SL-1, a superactivator of FIB-CL) and FIB-CL. 92K-GL caused a nine-fold increase in collagenase activity, equivalent to SL-1, while 72K-GL produced a four-fold increase. These results indicate that human oral cancers produce 92K-GL, 72K-GL, and FIB-CL in vivo and that the gelatinases and FIB-CL cooperate to enhance collagen degradation greatly in vitro.

Induction of matrix metalloproteinases and a collagen-degrading phenotype in fibroblasts and epithelial cells by secreted Porphyromonas gingivalis proteinase.

Periodontitis is characterized by advancement of a narrow band of epithelium (1-10 cells wide) through the collagenous periodontal ligament in response to bacterial accumulation and infection. A modulating role by epithelial cells in the progression of periodontitis was hypothesized due to the close proximity of the advancing epithelium to both the etiological bacteria and to the collagen fibers of the ligament. We demonstrate that rat mucosal epithelial cells and human fibroblasts are similarly stimulated to degrade a collagen type I cellular substrate by thiol-dependent activity released by the major periodontal pathogen Porphyromonas gingivalis. A purified, extracellular bacterial thiol-proteinase from P. gingivalis ATCC 33277 stimulated mucosal epithelial cells to upregulate expression of collagenase and stromelysin, and to degrade a collagen type I fibril matrix. Stimulation of the epithelial cells with this purified proteinase was associated with morphological changes in the cells and with accumulation of secreted latent procollagenase throughout the culture medium. Release of active collagenase was minimal and collagen degradation by the epithelial cells was discreet and localized subcellularly suggesting the possibility that activation of secreted procollagenase was cell-associated. We conclude that a collagen-degrading phenotype can be stimulated in relatively quiescent mucosal epithelial cells and fibroblasts by the presence of bacterial proteinase. These experiments suggest roles for the P. gingivalis thiol-proteinase and the epithelial cell in the pathogenesis of periodontal disease and demonstrate the potential for dysregulation of extracellular matrix remodeling events during healing of other bacterially infected wounds.

Activation and novel processing of matrix metalloproteinases by a thiol-proteinase from the oral anaerobe Porphyromonas gingivalis.

A critical outcome of periodontal disease is degradation of the collagenous periodontal ligament that connects teeth to bone in the dental arch. Periodontal diseases occur in response to bacterial colonization of the teeth, but their molecular pathogenesis is still speculative. One family of enzymes, known as the matrix metalloproteinases (MMPs), has been implicated in the degradation of the periodontal ligament. MMPs, which are also suspected to play a role in many other physiologic and pathologic remodeling processes, can be secreted by epithelial cells surrounding the teeth and are found in relative abundance in tissues and fluids near periodontally diseased sites. Since most MMPs are secreted as inactive zymogens which may be activated by limited proteolysis, it has been suggested that proteinases expressed by the infecting periodontal pathogens might activate latent host MMPs to initiate or accelerate degradation of the collegenous periodontal ligament. The aim of this work was to examine interactions between purified host MMPs and bacterial proteinase. In this article, we demonstrate that a proteinase isolated from the periodontopathogen Porphyromonas gingivalis can activate MMP-1, MMP-3, and MMP-9 and can catalyze the superactivation of MMP-1 by MMP-3. Activation of these MMPs is demonstrated to result from initial hydrolysis within their propeptide. Also, for MMP-1 and MMP-9, the P. gingivalis proteinase cleaves the MMP propeptide following a lysine residue at a previously unreported site which, for both MMPs, is one residue NH2-terminal to the known autocatalytic cleavage site. These data describe a mode of virulence for the periodontopathogen Porphyromonas gingivalis that involves activation of host-degradative enzymes.

Hemagglutinin activity and heterogeneity of related Porphyromonas gingivalis proteinases.

Thiol-dependent proteinases that are expressed and released by Porphyromonas gingivalis are considered virulence factors in periodontitis because of their potential to effect matrix degradation and inflammation. A number of P. gingivalis thiol-proteinases have been described, however, with similar biochemical characteristics. In this report we demonstrate that an isolate P. gingivalis proteinase consists of noncovalently associated peptides and that slight variations in the association pattern of these peptides could result in different proteinases with different affinities and activities. We also describe the co-purification of thiol-proteinase activity with hemagglutinin activity and demonstrate that each type of activity has similar inhibition profiles. With the use of monoclonal antibodies against the P. gingivalis proteinase we follow proteinase released into the culture medium over the course of 10 days and, by Western blot analysis, demonstrate that many of the proteinases with varying molecular weight are related. The identification of a single, immunoreactive, 140 kDa proteinase detected early in the culture and in association with the P. gingivalis cells suggests that multiple proteinase may originate from a single 140 kDa proteinase.

Functional domains of human TIMP-1 (tissue inhibitor of metalloproteinases).

To define domains of tissue inhibitor of metalloproteinases (TIMP-1) that are important to its ability to inhibit fibroblast-type collagenase (FIB-CL), two different approaches were used: (i) competition with synthetic peptides modeled after the human TIMP-1 sequence and (ii) localization of epitopes of blocking antibodies. TIMP-1 consists of six loops, held in place by six disulfide bonds arranged in three knotlike structures. Several long peptides (n = 20-34), together covering three-fourths of the human TIMP-1 sequence, were able to block inhibition of human FIB-CL by TIMP-1. While most of these peptides were modeled after sequences in the NH2-terminal domain of the molecule (loops 1, 2, and 3), they also included two-thirds of the residues of the COOH-terminal domain including loops 4 and 5 and the COOH-terminal tail but not loop 6. Refinement by competition with shorter peptides (7-10 residues) showed that the region surrounding the second "disulfide knot" (Cys13-Cys124, Cys127-Cys174) plays a major role in the inhibition of FIB-CL. This region consists of two strands, residues 10-25 and 121-129, connected through Cys13-Cys124. Peptides from this region also directly inhibited FIB-CL in the absence of TIMP-1. Additional competing peptides included T2-11 of the NH2-terminal domain and T34-42, a highly conserved region in the middle of loop 1. Among a series of monoclonal and polyclonal antibodies (mAbs and pAbs) to TIMP-1, we identified two, one mAb and one pAb, that neutralized the activity of TIMP-1 against FIB-CL. Both recognized epitopes in loop 3. The epitope for the mAb was located in the sequence that marks the transition between loops 3 and 4, GCEEC127, a region also identified as important by peptide competition experiments. By contrast, the epitope for a nonblocking mAb was located in a short 9-residue segment of loop 4, and a nonblocking pAb recognized epitopes in loop 1, loop 6, and the COOH-terminal tail. Our findings suggest that the FIB-CL-TIMP-1 complex possesses multiple contact sites that involve several different subdomains of the inhibitor.

Characterization and serial propagation of mouse prostate epithelial cells in serum-free medium.

Epithelial cell cultures derived from the ventral prostate of normal adult mice have been propagated in serum-free medium. The cultures were initiated and maintained in Ham's F-12 nutrient mixture supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), cholera toxin (10 ng/ml), bovine pituitary extract (25 micrograms protein/ml) and antibiotics. The cells exhibited microvilli on cell surfaces, interdigitations and junctional complexes including desmosomes between cells, and cytokeratins in cytoplasm which are characteristic of epithelial cells. In addition, the cells exhibited the tissue-specific markers, prostatic acid phosphatase and prostate-specific antigen.