Porphyromonas gingivalis gingipain is involved in the detachment and aggregation of Aggregatibacter actinomycetemcomitans biofilm

Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are major periodontal pathogens that cause several types of periodontal disease. Our previous study suggested that P. gingivalis gingipains secreted in the subgingival environment are related to the detachment of A.actinomycetemcomitans biofilms. However, it remains unclear whether arginine‐specific cysteine proteinase (Rgp) and lysine‐specific proteinase (Kgp) play different roles in the detachment of A. actinomycetemcomitans biofilm. The aim of this study was to investigate possible disruptive roles of Kgp and Rgp in the aggregation and attachment of A. actinomycetemcomitans. While P. gingivalis ATCC33277 culture supernatant has an ability to decrease autoaggregation and coaggregation of A. actinomycetemcomitans cells, neither the boiled culture supernatant of ATCC33277 nor the culture supernatant of KDP136 showed this ability. The addition of KYT‐1 and KYT‐36, specific inhibitors of Rgp and Kgp, respectively, showed no influence on the ability of P. gingivalis culture supernatant. The result of gelatin zymography suggested that other proteases processed by gingipains mediated the decrease of A. actinomycetemcomitans aggregations. We also examined the biofilm‐destructive effect of gingipains by assessing the detachment of A. actinomycetemcomitans from polystyrene surfaces. Scanning electron microscope analysis indicated that A. actinomycetemcomitans cells were detached by P. gingivalis Kgp. The quantity of A. actinomycetemcomitans in biofilm was decreased in co‐culture with P. gingivalis. However, this was not found after the addition of KYT‐36. These findings suggest that Kgp is a critical component for the detachment and decrease of A. actinomycetemcomitans biofilms.     

The Porphyromonas gingivalis hemagglutinins HagB and HagC are major mediators of adhesion and biofilm formation

Porphyromonas gingivalis is a bacterium associated with chronic periodontitis that possesses a family of genes encoding hemagglutinins required for heme acquisition. In this study we generated ΔhagB and ΔhagC mutants in strain W83 and demonstrate that both hagB and hagC are required for adherence to oral epithelial cells. Unexpectedly, a double ΔhagB/ΔhagC mutant had less severe adherence defects than either of the single mutants, but was found to exhibit increased expression of the gingipain‐encoding genes rgpA and kgp, suggesting that a ΔhagB/ΔhagC mutant is only viable in populations of cells that exhibit increased expression of genes involved in heme acquisition. Disruption of hagB in the fimbriated strain ATCC33277 demonstrated that HagB is also required for stable attachment of fimbriated bacteria to oral epithelial cells. Mutants of hagC were also found to form defective single and multi‐species biofilms that had reduced biomass relative to biofilms formed by the wild‐type strain. This study highlights the hitherto unappreciated importance of these genes in oral colonization and biofilm formation.     

In silico analysis of the competition between Streptococcus sanguinis and Streptococcus mutans in the dental biofilm

During dental caries, the dental biofilm modifies the composition of the hundreds of involved bacterial species. Changing environmental conditions influence competition. A pertinent model to exemplify the complex interplay of the microorganisms in the human dental biofilm is the competition between Streptococcus sanguinis and Streptococcus mutans. It has been reported that children and adults harbor greater numbers of S. sanguinis in the oral cavity, associated with caries‐free teeth. Conversely, S. mutans is predominant in individuals with a high number of carious lesions. Competition between both microorganisms stems from the production of H₂O₂ by S. sanguinis and mutacins, a type of bacteriocins, by S. mutans. There is limited evidence on how S. sanguinis survives its own H₂O₂ levels, or if it has other mechanisms that might aid in the competition against S. mutans, nonetheless. We performed a genomic and metabolic pathway comparison, coupled with a comprehensive literature review, to better understand the competition between these two species. Results indicated that S. sanguinis can outcompete S. mutans by the production of an enzyme capable of metabolizing H₂O₂. S. mutans, however, lacks the enzyme and is susceptible to the peroxide from S. sanguinis. In addition, S. sanguinis can generate energy through gluconeogenesis and seems to have evolved different communication mechanisms, indicating that novel proteins may be responsible for intra‐species communication.     

SMU.940 regulates dextran‐dependent aggregation and biofilm formation in Streptococcus mutans

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence‐stimulating peptide. Eight competence‐stimulating peptide‐dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran‐dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild‐type. GbpC is known to be involved in the dextran‐dependent aggregation of S. mutans. An SMU.940‐gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran‐dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran‐dependent aggregation and biofilm formation.     

Benzamidine derivatives inhibit the virulence of Porphyromonas gingivalis

We have previously shown that benzamidine‐type compounds can inhibit the activity of arginine‐specific cysteine proteinases (gingipains HRgpA and RgpB); well‐known virulence factors of Porphyromonas gingivalis. They also hinder in vitro growth of this important periodontopathogenic bacterium. Apparently growth arrest is not associated with their ability to inhibit these proteases, because pentamidine, which is a 20‐fold less efficient inhibitor of gingipain than 2,6‐bis‐(4‐amidinobenzyl)‐cyclohexanone (ACH), blocked P. gingivalis growth far more effectively. To identify targets for benzamidine‐derived compounds other than Arg‐gingipains, and to explain their bacteriostatic effects, P. gingivalis ATCC 33277 and P. gingivalis M5‐1‐2 (clinical isolate) cell extracts were subjected to affinity chromatography using a benzamidine–Sepharose column to identify proteins interacting with benzamidine. In addition to HRgpA and RgpB the analysis revealed heat‐shock protein GroEL as another ligand for benzamidine. To better understand the effect of benzamidine‐derived compounds on P. gingivalis, bacteria were exposed to benzamidine, pentamidine, ACH and heat, and the expression of gingipains and GroEL was determined. Exposure to heat and benzamidine‐derived compounds caused significant increases in GroEL, at both the mRNA and protein levels. Interestingly, despite the fact that gingipains were shown to be the main virulence factors in a fertilized egg model of infection, mortality rates were strongly reduced, not only by ACH, but also by pentamidine, a relatively weak gingipain inhibitor. This effect may depend not only on gingipain inhibition but also on interaction of benzamidine derivatives with GroEL. Therefore these compounds may find use in supportive periodontitis treatment.     

The GroEL protein of Porphyromonas gingivalis accelerates tumor growth by enhancing endothelial progenitor cell function and neovascularization

Porphyromonas gingivalis is a bacterial species that causes destruction of periodontal tissues. Additionally, previous evidence indicates that GroEL from P. gingivalis may possess biological activities involved in systemic inflammation, especially inflammation involved in the progression of periodontal diseases. The literature has established a relationship between periodontal disease and cancer. However, it is unclear whether P. gingivalis GroEL enhances tumor growth. Here, we investigated the effects of P. gingivalis GroEL on neovasculogenesis in C26 carcinoma cell‐carrying BALB/c mice and chick eggs in vivo as well as its effect on human endothelial progenitor cells (EPC) in vitro. We found that GroEL treatment accelerated tumor growth (tumor volume and weight) and increased the mortality rate in C26 cell‐carrying BALB/c mice. GroEL promoted neovasculogenesis in chicken embryonic allantois and increased the circulating EPC level in BALB/c mice. Furthermore, GroEL effectively stimulated EPC migration and tube formation and increased E‐selectin expression, which is mediated by eNOS production and p38 mitogen‐activated protein kinase activation. Additionally, GroEL may enhance resistance against paclitaxel‐induced cell cytotoxicity and senescence in EPC. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to the neovasculogenesis of tumor cells and resulting in accelerated tumor growth.     

EzrA,a cell shape regulator contributing to biofilm formation and competitiveness in Streptococcus mutans

Bacterial cell division is initiated by tubulin homologue FtsZ that assembles into a ring structure at mid‐cell to facilitate cytokinesis. EzrA has been identified to be implicated in FtsZ‐ring dynamics and cell wall biosynthesis during cell division of Bacillus subtilis and Staphylococcus aureus, the model rod and cocci. However, its role in pathogenic streptococci remains largely unknown. Here, the role of EzrA was investigated in Streptococcus mutans, the primary etiological agent of human dental caries, by constructing an ezrA in‐frame deletion mutant. Our data showed that the ezrA mutant was slow‐growing with a shortened length and extended width round cell shape compared to the wild type, indicating a delay in cell division with abnormalities of peptidoglycan biosynthesis. Additionally, FtsZ irregularly localized in dividing ezrA mutant cells forming angled division planes, potentially contributing to an aberrant cell shape. Furthermore, investigation using single‐species cariogenic biofilm model revealed that deletion of ezrA resulted in defective biofilm formation with less extracellular polysaccharides and altered three‐dimensional biofilm architecture. Unexpectedly, in a dual‐species ecological model, the ezrA mutant exhibited substantially lower tolerance for H₂O₂ and reduced competitiveness against one commensal species, Streptococcus sanguinis. Taken together, these results demonstrate that EzrA plays a key role in regulating cell division and maintaining a normal morphology in S. mutans and is required for its robust biofilm formation/interspecies competition. Therefore, EzrA protein represents a potential therapeutic target in the development of drugs controlling dental caries and other biofilm‐related diseases.     

Influence of surface properties of resin‐based composites on in vitro Streptococcus mutans biofilm development

The aim of this in vitro study was to evaluate the influence of physicochemical surface properties of resin‐based composites on Streptococcus mutans biofilm formation. Specimens were prepared from each of four resin‐based composites by polymerization against Mylar strips. Half of the number of specimens received no further surface treatment, whereas the other half were subjected to a polishing treatment. Surface roughness (SR) and topography were assessed using profilometry and atomic force microscopy. Surface free‐energy (SFE) was determined, and the chemical surface composition was analysed by X‐ray photoelectron spectroscopy (XPS). S. mutans biofilms were formed on the surface of the resin‐based composite specimens for either 48 or 96 h using an artificial mouth system (AMS). Polishing caused a significant decrease in SFE, and XPS analysis indicated an increase of surface silicon and a decrease of surface carbon. Only for Grandio was a significant increase in SR identified after polishing, which was probably related to the higher concentration of filler particles on its surface. Significantly less S. mutans biofilm formation was observed on polished resin‐based composites than on unpolished resin‐based composites. These results indicate that the proportions of resin matrix and filler particles on the surface of resin‐based composites strongly influence S. mutans biofilm formation in vitro, suggesting that minimization of resin matrix exposure might be useful to reduce biofilm formation on the surface of resin‐based composites.     

Towards defining the outer membrane proteome of Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram‐negative anaerobic pathogen found in subgingival plaque associated with progressive periodontitis. Proteins associated with the outer membrane (OM) of Gram‐negative pathogens are particularly important for understanding virulence and for developing vaccines. The aim of this study was to establish a reliable list of outer membrane associated proteins (Omps) for this organism. Starting with a list of 99 experimentally determined Omps, several bioinformatics tools were used to predict a further 52 proteins, leading to a predicted OM proteome of 151 proteins. The tools used included databases of protein families, prediction of OM β‐barrels and structural homology. The list includes 33 T9SS cargo proteins, 43 lipoproteins and 66 OM β‐barrel proteins with some overlap between categories. The proteins are discussed both in these structural categories as well as their various functions in OM biogenesis, nutrient acquisition, protein secretion, adhesion and efflux. Proteins that were previously shown to be part of large complexes are highlighted and cross reference is provided to a previous major study of protein localization in P. gingivalis. Finally, proteins were also scored according to their level of conservation within the Bacteroidales taxon. Low scores were shown to correlate with virulence factors and may be predictive of novel virulence factors.     

Spheres of influence: Porphyromonas gingivalis outer membrane vesicles

Outer membrane vesicles (OMVs) are asymmetrical single bilayer membranous nanostructures produced by Gram‐negative bacteria important for bacterial interaction with the environment. Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces OMVs that act as a virulence factor secretion system contributing to its pathogenicity. Despite their biological importance, the mechanisms of OMV biogenesis have not been fully elucidated. The ~14 times more curvature of the OMV membrane than cell outer membrane (OM) indicates that OMV biogenesis requires energy expenditure for significant curvature of the OMV membrane. In P. gingivalis, we propose that this may be achieved by upregulating the production of certain inner or outer leaflet lipids, which causes localized outward curvature of the OM. This results in selection of anionic lipopolysaccharide (A‐LPS) and associated C‐terminal domain (CTD) ‐family proteins on the outer surface due to their ability to accommodate the curvature. Deacylation of A‐LPS may further enable increased curvature leading to OMV formation. Porphyromonas gingivalis OMVs that are selectively enriched in CTD‐family proteins, largely the gingipains, can support bacterial coaggregation, promote biofilm development and act as an intercessor for the transport of non‐motile bacteria by motile bacteria. The P. gingivalis OMVs are also believed to contribute to host interaction and colonization, evasion of immune defense mechanisms, and destruction of periodontal tissues. They may be crucial for both micro‐ and macronutrient capture, especially heme and probably other assimilable compounds for its own benefit and that of the wider biofilm community.     

Influence of pH on inhibition of Streptococcus mutans by Streptococcus oligofermentans

Streptococcus oligofermentans is a novel strain of oral streptococcus that can specifically inhibit the growth of Streptococcus mutans. The aims of this study were to assess the growth of S. oligofermentans and the ability of S. oligofermentans to inhibit growth of Streptococcus mutans at different pH values. Growth inhibition was investigated in vitro using an interspecies competition assay. The 4‐aminoantipyine method was used to measure the initial production rate and the total yield of hydrogen peroxide in S. oligofermentans. S. oligofermentans grew best at pH 7.0 and showed the most pronounced inhibitory effect when it was inoculated earlier than S. mutans. In terms of the total yield and the initial production rate of hydrogen peroxide by S. oligofermentans, the effects of the different culture pH values were as follows: pH 7.0 > 6.5 > 6.0 > 7.5 > 5.5 = 8.0 (i.e. there was no significant difference between pH 5.5 and pH 8.0). Environmental pH and the sequence of inoculation significantly affected the ability of S. oligofermentans to inhibit the growth of S. mutans. The degree of inhibition may be attributed to the amount of hydrogen peroxide produced.