Synergistic biofilm formation by Treponema denticola and Porphyromonas gingivalis

Biofilm formation is an important step in the etiology of periodontal diseases. In this study, in vitro biofilm formation by Treponema denticola and Porphyromonas gingivalis 381 displayed synergistic effects. Confocal microscopy demonstrated that P. gingivalis attaches to the substratum first as a primary colonizer followed by coaggregation with T. denticola to form a mixed biofilm. The T. denticola flagella mutant as well as the cytoplasmic filament mutant were shown to be essential for biofilm formation as well as coaggregation with P. gingivalis. The major fimbriae and Arg-gingipain B of P. gingivalis also play important roles in biofilm formation with T. denticola.     

1,2,3-Triazole-based inhibitors of Porphyromonas gingivalis adherence to oral streptococci and biofilm formation

The development and use of small-molecule inhibitors of the adherence of Porphyromonas gingivalis to oral streptococci represents a potential therapy for the treatment of periodontal disease as these organisms work in tandem to colonize the oral cavity. Earlier work from these laboratories demonstrated that a small synthetic peptide was an effective inhibitor of the interaction between P. gingivalis and Streptococcus gordonii and that a small-molecule peptidomimetic would provide a more stable, less expensive and more effective inhibitor. An array of 2-(azidomethyl)- and 2-(azidophenyl)-4,5-diaryloxazoles having a full range of hydrophobic groups were prepared and reacted with substituted arylacetylenes to afford the corresponding ‘click’ products. The title compounds were evaluated for their ability to inhibit P. gingivalis’ adherence to oral streptococci and several were found to be inhibitory in the range of (IC₅₀) 5.3–67 μM.     

Glycosylation of the OMP85 homolog of Porphyromonas gingivalis and its involvement in biofilm formation

OMP85 is a highly conserved outer membrane protein in all Gram-negative bacteria. We studied an uncharacterized OMP85 homolog of Porphyromonas gingivalis, a primary periodontal pathogen forming subgingival plaque biofilms. Using an outer-loop peptide antibody specific for the OMP85 of P. gingivalis, loop-3 Ab, we found a difference in the mobility of OMP85 on SDS-PAGE gel between the P. gingivalis wild-type and the isogenic galE mutant, a deglycosylated strain, suggesting that OMP85 naturally exists in a glycosylated form. This was also supported by a shift in OMP85 PAGE mobility after chemical deglycosylation treatment. Further, loop-3 Ab cross-reacted with the galE mutant stronger than the wild-type strain; and could inhibit biofilm formation in the galE mutant more than in the wild-type strain. In conclusion, this is the first report providing the evidence of OMP85 glycosylation and the involvement of OMP85 in biofilm formation.     

Time course of gene expression during Porphyromonas gingivalis strain ATCC 33277 biofilm formation

Chronological gene expression patterns of biofilm-forming cells are important to understand bioactivity and pathogenicity of biofilms. For Porphyromonas gingivalis ATCC 33277 biofilm formation, the number of genes differentially regulated by more than 1.5-fold was highest during the growth stage (312/2,090 genes), and some pathogen-associated genes were time-dependently controlled.     

A universal stress protein of Porphyromonas gingivalis is involved in stress responses and biofilm formation

Porphyromonas gingivalis is recognized as one of the major periodontal pathogens in subgingival plaque, which is implicated in the progression of chronic periodontal disease. We analyzed the role of upsA in P. gingivalis 381 and its uspA-deficient mutant CW301 under various stress conditions. In general, the uspA mutant was less tolerant to a variety of environmental stresses relative to the parental strain. In addition, gene expression of uspA is upregulated during biofilm formation. Biofilm formation of the uspA mutant was also less than that of strain 381. In conclusion, the uspA gene affecting the stress responses of P. gingivalis is required for optimal biofilm formation.     

LuxS-based signaling in Streptococcus gordonii: autoinducer 2 controls carbohydrate metabolism and biofilm formation with Porphyromonas gingivalis

Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm. S. gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi. An isogenic mutant of S. gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence in V. harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis. Complementation of the luxS mutation in S. gordonii restored normal biofilm formation with the luxS-deficient P. gingivalis. Differential display PCR demonstrated that the inactivation of S. gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-beta-D-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase. However, S. gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S. gordonii and P. gingivalis, was unaffected by inactivation of luxS. The results suggest that S. gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. gordonii.     

Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures

Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated with epithelial cells in a 24-well plate for 4 h. Epithelial cell detachment was assessed using imaging. The activity of arginine-gingipain (Rgp) and gene expression profiles of P. gingivalis cultures were examined using a gingipain assay and quantitative PCR, respectively. P. gingivalis biofilms induced significantly higher cell detachment and displayed higher Rgp activity compared to the planktonic cultures. The genes involved in gingipain post-translational modification, but not rgp genes, were significantly up-regulated in P. gingivalis biofilms. The results underline the importance of including biofilms in the study of bacterial and host cell interactions.     

Role of the Clp system in stress tolerance, biofilm formation, and intracellular invasion in Porphyromonas gingivalis

Clp proteases and chaperones are ubiquitous among prokaryotes and eukaryotes, and in many pathogenic bacteria the Clp stress response system is also involved in regulation of virulence properties. In this study, the roles of ClpB, ClpC, and ClpXP in stress resistance, homotypic and heterotypic biofilm formation, and intracellular invasion in the oral opportunistic pathogen Porphyromonas gingivalis were investigated. Absence of ClpC and ClpXP, but not ClpB, resulted in diminished tolerance to high temperatures. Response to oxidative stress was not affected by the loss of any of the Clp proteins. The clpC and clpXP mutants demonstrated elevated monospecies biofilm formation, and the absence of ClpXP also enhanced heterotypic P. gingivalis-Streptococcus gordonii biofilm formation. All clp mutants adhered to gingival epithelial cells to the same level as the wild type; however, ClpC and ClpXP were found to be necessary for entry into host epithelial cells. ClpB did not play a role in entry but was required for intracellular replication and survival. ClpXP negatively regulated the surface exposure of the minor fimbrial (Mfa) protein subunit of P. gingivalis, which stimulates biofilm formation but interferes with epithelial cell entry. Collectively, these results show that the Clp protease complex and chaperones control several processes that are important for the colonization and survival of P. gingivalis in the oral cavity.     

The introduction of colonic-Bacteriodes shuttle plasmids into Porphyromonas gingivalis: Identification of a putative P. gingivalis insertion-sequence element

Two Escherichia coli-Bacteroides plasmid-shuttle vectors pNJR5 and pNJR12 were introduced for the first time into Porphyromonas gingivalis W83 by conjugal transfer from E. coli. The transfer frequencies were comparable to those obtained when using colonic Bacteroides as recipients. Both plasmids were maintained in P. gingivalis W83 and could be isolated and introduced back into E. coli. Plasmid DNA extracted from one P. gingivalis W83 pNJR12 transconjugant had an additional 1.5 kb of inserted DNA. Southern-blot analysis of P. gingivalis W83 chromosomal DNA using this inserted DNA as a probe revealed the presence of multiple copies of this sequence on the chromosome. We propose that this DNA represents a P. gingivalis insertion sequence (IS) element and should be referred to as IS1126. This is the first IS element to be isolated from a Gram-negative oral anaerobic bacterium.     

Autophagy: a highway for Porphyromonas gingivalis in endothelial cells

P. gingivalis, an important periodontal pathogen associated with adult periodontitis and a likely contributing factor to atherosclerosis and cardiovascular disease, traffics in endothelial cells via the autophagic pathway. Initially, P. gingivalis rapidly adheres to the host cell surface followed by internalization via lipid rafts and incorporation of the bacterium into early phagosomes. P. gingivalis activates cellular autophagy to provide a replicative niche while suppressing apoptosis. The replicating vacuole contains host proteins delivered by autophagy that are used by this asaccharolytic pathogen to survive and replicate within the host cell. When autophagy is suppressed by 3-methyladenine or wortmannin, internalized P. gingivalis transits to the phagolysosome where it is destroyed and degraded. Therefore, the survival of P. gingivalis depends upon the activation of autophagy and survival of the endothelial host cell, but the mechanism by which P. gingivalis accomplishes this remains unclear.     

Distinct roles of long/short fimbriae and gingipains in homotypic biofilm development by Porphyromonas gingivalis

Background  

Porphyromonas gingivalis, a periodontal pathogen, expresses a number of virulence factors, including long (FimA) and short (Mfa) fimbriae as well as gingipains comprised of arginine-specific (Rgp) and lysine-specific (Kgp) cysteine proteinases. The aim of this study was to examine the roles of these components in homotypic biofilm development by P. gingivalis, as well as in accumulation of exopolysaccharide in biofilms.     

Porphyromonas gingivalis in oral squamous cell carcinoma: a review

Oral cancer contributes significantly to the global cancer burden. Oral bacteria play an important role in the spread of oral cancer, according to mounting evidence. The most proven instance is the carcinogenic implications of Porphyromonas gingivalis, a key pathogen in chronic periodontitis. It is imperative to understand the pathogenesis of P. gingivalis in OSCC. This review aims to gather and assess scientific shreds of evidence on the involvement of P. gingivalis in the molecular mechanism of oral squamous cell carcinoma.     

Thrombospondin-1 Production Is Enhanced by Porphyromonas gingivalis Lipopolysaccharide in THP-1 Cells

Periodontitis is a chronic inflammatory disease caused by gram-negative anaerobic bacteria. Monocytes and macrophages stimulated by periodontopathic bacteria induce inflammatory mediators that cause tooth-supporting structure destruction and alveolar bone resorption. In this study, using a DNA microarray, we identified the enhanced gene expression of thrombospondin-1 (TSP-1) in human monocytic cells stimulated by Porphyromonas gingivalis lipopolysaccharide (LPS). TSP-1 is a multifunctional extracellular matrix protein that is upregulated during the inflammatory process. Recent studies have suggested that TSP-1 is associated with rheumatoid arthritis, diabetes mellitus, and osteoclastogenesis. TSP-1 is secreted from neutrophils, monocytes, and macrophages, which mediate immune responses at inflammatory regions. However, TSP-1 expression in periodontitis and the mechanisms underlying TSP-1 expression in human monocytic cells remain unknown. Here using real-time RT-PCR, we demonstrated that TSP-1 mRNA expression level was significantly upregulated in inflamed periodontitis gingival tissues and in P. gingivalis LPS-stimulated human monocytic cell line THP-1 cells. TSP-1 was expressed via Toll-like receptor (TLR) 2 and TLR4 pathways. In P. gingivalis LPS stimulation, TSP-1 expression was dependent upon TLR2 through the activation of NF-κB signaling. Furthermore, IL-17F synergistically enhanced P. gingivalis LPS-induced TSP-1 production. These results suggest that modulation of TSP-1 expression by P. gingivalis plays an important role in the progression and chronicity of periodontitis. It may also contribute a new target molecule for periodontal therapy.     

Rapid viability loss on exposure to air in a superoxide dismutase-deficient mutant of Porphyromonas gingivalis.

Porphyromonas gingivalis, an obligate anaerobe, exhibits a relatively high degree of aerotolerance and possesses superoxide dismutase (SOD) which is induced by exposure to air. To clarify roles for SOD in this organism, the gene encoding SOD (sod) on the P. gingivalis chromosome was disrupted in a gene-directed way by use of a suicide plasmid containing a mutated sod. A sod mutant thus obtained showed no SOD activity in crude extracts and exhibited a rapid viability loss immediately after exposure to air, whereas the wild-type parent showed no decrease in viability for at least 5 h under aerobic conditions. These results clearly indicate that SOD is essential for aerotolerance in P. gingivalis.