Identification of a Czc-like operon of the periodontal pathobiont Porphyromonas gingivalis involved in metal ion efflux.

OBJECTIVE: The study aimed to investigate the role of the PGN2012 gene of the periodontitis contributing pathobiont Porphyromonas gingivalis. PGN2012 is a homolgue of TolC and is a gene our group previously showed was overexpressed in hyperinvasive cells. METHODS: The study used a combination of bioinformatics, knockout mutagenesis, growth experiments, biofilm assays and human cell invation assays to investigate PGN2012 function. RESULTS: Bioinformatics identified that PGN2012 is part of one of four TolC containing gene loci in P. gingivalis that we predicted may encode a metal resistance RND family tripartite pump, similar to those present in other Gram-negative bacteria, but which are not well understood in anaerobic bacteria. A ΔPGN2012 deletion displayed slightly reduced growth in liquid culture but did not effect biofilm formation or human cell invasion. When metal ions were included in the medium the mutant displayed significantly increased sensitivity to the divalent metal ions Zn2+ (500 µM), Co2+ (2 mM), and Cd2+(0.1 mM) but not Cu2+. CONCLUSIONS: We propose to rename the PGN2012-2014 genes czcCBA, which we suggest plays a role in intracellular stress resistance where zinc is often employed by host cells in antibacterial defence with implications for chronic infection in humans.

Porphyromonas gingivalis Outer Membrane Vesicles Increase Vascular Permeability.

Periodontitis is increasingly associated with increased risk of cardiovascular and other systemic diseases. The Gram-negative anaerobe, Porphyromonas gingivalis, is a key periodontal pathogen, and several lines of evidence link the presence of this bacterium in the circulation with vascular disease. The outer membrane vesicles (OMVs) produced by P. gingivalis have been shown to play a role in periodontitis, although, to date, little is known about their interaction with the vasculature; therefore, this study assessed the effects of P. gingivalis OMVs on the endothelium. OMVs were isolated from wild-type strain W83 and the gingipain-deficient strain ΔK/R-ab. Immunoblotting along with cryo-EM showed gingipain expression in W83 but not ΔK/R-ab-derived OMVs, where gingipains were localized to the cell wall surface. Confluent endothelial cell monolayers infected with either W83 or W83-derived OMV displayed significantly increased dextran permeability over those infected with ΔK/R-ab or its OMV. Moreover, W83-derived OMVs induced significantly more vascular disease in a zebrafish larvae systemic infection model over 72 h compared to those injected with gingipain-deficient OMVs or controls. In line with these data, human microvascular endothelial cells (HMEC-1) displayed an OMV-associated, gingipain-dependent decrease in cell surface levels of the intercellular adhesion molecule PECAM-1 (CD31) when examined by flow cytometry. These data show, for the first time, that OMVs from P. gingivalis mediate increased vascular permeability, leading to a diseased phenotype both in vitro and in vivo. Moreover, these data strongly implicate gingipains present on the OMV surface in mediating these vascular events, most likely via a mechanism that involves proteolytic cleavage of endothelial cell-cell adhesins such as PECAM-1. These data provide important evidence for the role of bacterial-derived OMVs in mediating systemic disease.

Identification of Novel Bacteriophages with Therapeutic Potential That Target Enterococcus faecalis.

The Gram-positive opportunistic pathogen Enterococcus faecalis is frequently responsible for nosocomial infections in humans and represents one of the most common bacteria isolated from recalcitrant endodontic (root canal) infections. E. faecalis is intrinsically resistant to several antibiotics routinely used in clinical settings (such as cephalosporins and aminoglycosides) and can acquire resistance to vancomycin (vancomycin-resistant enterococci). The resistance of E. faecalis to several classes of antibiotics and its capacity to form biofilms cause serious therapeutic problems. Here, we report the isolation of several bacteriophages that target E. faecalis strains isolated from the oral cavity of patients suffering root canal infections. All phages isolated were Siphoviridae with similar tail lengths (200 to 250 nm) and icosahedral heads. The genome sequences of three isolated phages were highly conserved with the exception of predicted tail protein genes that diverge in sequence, potentially reflecting the host range. The properties of the phage with the broadest host range (SHEF2) were further characterized. We show that this phage requires interaction with components of the major and variant region enterococcal polysaccharide antigen to engage in lytic infection. Finally, we explored the therapeutic potential of this phage and show that it can eradicate E. faecalis biofilms formed in vitro on a standard polystyrene surface but also on a cross-sectional tooth slice model of endodontic infection. We also show that SHEF2 cleared a lethal infection of zebrafish when applied in the circulation. We therefore propose that the phage described here could be used to treat a broad range of antibiotic-resistant E. faecalis infections.

Characterisation and pure culture of putative health-associated oral bacterium BU063 (Tannerella sp. HOT-286) reveals presence of a potentially novel glycosylated S-layer.

Tannerella HOT-286 (phylotype BU063) is a recently identified novel filamentous Gram-negative anaerobic oral bacterium cultured for the first time recently in co-culture with Propionibacterium acnes. In contrast to the related periodontal disease-associated pathobiont Tannerella forsythia, it is considered a putative health-associated bacterium. In this paper, we identified that this organism could be grown in pure culture if N-acetyl muramic acid (NAM) was provided in the media, although surprisingly the genetic basis of this phenomenon is not likely to be due to a lack of NAM synthesis genes. During further microbiological investigations, we showed for the first time that T. HOT-286 possesses a prominent extracellular S-layer with a novel morphology putatively made up of two proteins modified with an unknown glycan. These data further our knowledge of this poorly understood organism and genus that is an important part of the oral and human microbiome.

Lactose-crosslinked fish gelatin-based porous scaffolds embedded with tetrahydrocurcumin for cartilage regeneration.

Tetrahydrocurcumin (THC) is one of the major colourless metabolites of curcumin and shows even greater pharmacological and physiological benefits. The aim of this work was the manufacturing of porous scaffolds as a carrier of THC under physiological conditions. Fish-derived gelatin scaffolds were prepared by freeze-drying by two solutions concentrations (2.5% and 4% w/v), cross-linked via addition of lactose and heat-treated at 105 °C. This cross-linking reaction resulted in more water resistant scaffolds with a water uptake capacity higher than 800%. Along with the cross-linking reaction, the gelatin concentration affected the scaffold morphology, as observed by scanning electron microscopy images, by obtaining a reduced porosity but larger pores sizes when the initial gelatin concentration was increased. These morphological changes led to a scaffold's strength enhancement from 0.92 ±â€¯0.22 MPa to 2.04 ±â€¯0.18 MPa when gelatin concentration was increased. THC release slowed down when gelatin concentration increased from 2.5 to 4% w/v, showing a controlled profile within 96 h. Preliminary in vitro test with chondrocytes on scaffolds with 4% w/v gelatin offered higher metabolic activities and cell survival up to 14 days of incubation. Finally the addition of THC did not influence significantly the cytocompatibility and potential antibacterial properties were demonstrated successfully against Staphylococcus aureus.

Inhibition of sialidase activity and cellular invasion by the bacterial vaginosis pathogen Gardnerella vaginalis.

Bacterial vaginosis is a genital tract infection, thought to be caused by transformation of a lactobacillus-rich flora to a dysbiotic microbiota enriched in mixed anaerobes. The most prominent of these is Gardnerella vaginalis (GV), an anaerobic pathogen that produces sialidase enzyme to cleave terminal sialic acid residues from human glycans. Notably, high sialidase activity is associated with preterm birth and low birthweight. We explored the potential of the sialidase inhibitor Zanamavir against GV whole cell sialidase activity using methyl-umbelliferyl neuraminic acid (MU-NANA) cleavage assays, with Zanamavir causing a 30% reduction in whole cell GV sialidase activity (p < 0.05). Furthermore, cellular invasion assays using HeLa cervical epithelial cells, infected with GV, demonstrated that Zanamivir elicited a 50% reduction in cell association and invasion (p < 0.05). Our data thus highlight that pharmacological sialidase inhibitors are able to modify BV-associated sialidase activity and influence host-pathogen interactions and may represent novel therapeutic adjuncts.

Preparation and Antibacterial Properties of Silver-Doped Nanoscale Hydroxyapatite Pastes for Bone Repair and Augmentation.

The treatment of deep bone infections remains a significant challenge in orthopaedic and dental surgery. The relatively recent commercial manufacture of nanoscale hydroxyapatite has provided surgeons with an injectable biomaterial that promotes bone tissue regeneration, and with further modification it may be possible to incorporate antimicrobial properties into these devices. Silver-doped nanoscale hydroxyapatite pastes (0, 2, 5 and 10 mol.% silver) were prepared using a rapid mixing method. When the process was modified to prepare a 10 mol.% silver-doped material, silver phosphate was detected in addition to nanoscale hydroxyapatite. Thermal decomposition occurred more readily with greater silver content following calcination at 1000 °C for 2 h. Silver-doped nanoscale hydroxyapatite pastes showed antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa in a dose dependent manner using both agar diffusion assays and suspension cultures. It was concluded that the enhanced antibacterial activity of the silver-doped pastes was due to the action of diffusible silver ions. Based on these results, silver-doped nanoscale hydroxyapatite pastes represent a highly promising new biomaterial system for the prevention and treatment of deep infections in bone tissue.

Characterization of an α-l-fucosidase from the periodontal pathogen Tannerella forsythia.

The periodontal pathogen Tannerella forsythia expresses several glycosidases which are linked to specific growth requirements and are involved in the invasion of host tissues. α-l-Fucosyl residues are exposed on various host glycoconjugates and, thus, the α-l-fucosidases predicted in the T. forsythia ATCC 43037 genome could potentially serve roles in host-pathogen interactions. We describe the molecular cloning and characterization of the putative fucosidase TfFuc1 (encoded by the bfo_2737 = Tffuc1 gene), previously reported to be present in an outer membrane preparation. In terms of sequence, this 51-kDa protein is a member of the glycosyl hydrolase family GH29. Using an artificial substrate, p-nitrophenyl-α-fucose (KM 670 µM), the enzyme was determined to have a pH optimum of 9.0 and to be competitively inhibited by fucose and deoxyfuconojirimycin. TfFuc1 was shown here to be a unique α(1,2)-fucosidase that also possesses α(1,6) specificity on small unbranched substrates. It is active on mucin after sialidase-catalyzed removal of terminal sialic acid residues and also removes fucose from blood group H. Following knock-out of the Tffuc1 gene and analyzing biofilm formation and cell invasion/adhesion of the mutant in comparison to the wild-type, it is most likely that the enzyme does not act extracellularly. Biochemically interesting as the first fucosidase in T. forsythia to be characterized, the biological role of TfFuc1 may well be in the metabolism of short oligosaccharides in the periplasm, thereby indirectly contributing to the virulence of this organism. TfFuc1 is the first glycosyl hydrolase in the GH29 family reported to be a specific α(1,2)-fucosidase.

Gingipain-dependent degradation of mammalian target of rapamycin pathway proteins by the periodontal pathogen Porphyromonas gingivalis during invasion.

Porphyromonas gingivalis and Tannerella forsythia are gram-negative pathogens strongly associated with periodontitis. Their abilities to interact, invade and persist within host cells are considered crucial to their pathogenicity, but the mechanisms by which they subvert host defences are not well understood. In this study, we set out to investigate whether P. gingivalis and T. forsythia directly target key signalling molecules that may modulate the host cell phenotype to favour invasion and persistence. Our data identify, for the first time, that P. gingivalis, but not T. forsythia, reduces levels of intracellular mammalian target of rapamycin (mTOR) in oral epithelial cells following invasion over a 4-h time course, via the action of gingipains. The ability of cytochalasin D to abrogate P. gingivalis-mediated mTOR degradation suggests that this effect is dependent upon cellular invasion. We also show that levels of several other proteins in the mTOR signalling pathway are modulated by gingipains, either directly or as a consequence of mTOR degradation including p-4E-BP1. Taken together, our data suggest that P. gingivalis manipulates the mTOR pathway, providing evidence for a potentially novel mechanism by which P. gingivalis mediates its effects on host cell responses to infection.

A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression.

Tannerella forsythia is a pathogen implicated in periodontitis, an inflammatory disease of the tooth-supporting tissues often leading to tooth loss. This key periodontal pathogen is decorated with a unique glycan core O-glycosidically linked to the bacterium's proteinaceous surface (S)-layer lattice and other glycoproteins. Herein, we show that the terminal motif of this glycan core acts to modulate dendritic cell effector functions to suppress T-helper (Th)17 responses. In contrast to the wild-type bacterial strain, infection with a mutant strain lacking the complete S-layer glycan core induced robust Th17 and reduced periodontal bone loss in mice. Our findings demonstrate that surface glycosylation of this pathogen may act to ensure its persistence in the host likely through suppression of Th17 responses. In addition, our data suggest that the bacterium then induces the Toll-like receptor 2-Th2 inflammatory axis that has previously been shown to cause bone destruction. Our study provides a biological basis for pathogenesis and opens opportunities in exploiting bacterial glycans as therapeutic targets against periodontitis and a range of other infectious diseases.

Identification of bistable populations of Porphyromonas gingivalis that differ in epithelial cell invasion.

Bistable populations of bacteria give rise to two or more subtypes that exhibit different phenotypes. We have explored whether the periodontal pathogen Porphyromonas gingivalis exhibits bistable invasive phenotypes. Using a modified cell invasion assay, we show for the first time that there are two distinct subtypes within a population of P. gingivalis strains NCTC 11834 and W50 that display differences in their ability to invade oral epithelial cells. The highly invasive subtype invades cells at 10-30-fold higher levels than the poorly invasive subtype and remains highly invasive for approximately 12-16 generations. Analysis of the gingipain activity of these subtypes revealed that the highly invasive type had reduced cell-associated arginine-specific protease activity. The role of Arg-gingipain activity in invasion was verified by enhancement of invasion by rgpAB mutations and by inclusion of an Arg-gingipain inhibitor in invasion assays using wild-type bacteria. In addition, a population of ΔrgpAB bacteria did not contain a hyperinvasive subtype. Screening of the protease activity of wild-type populations of both strains identified high and low protease subtypes which also showed a corresponding reduction or enhancement, respectively, of invasive capabilities. Microarray analysis of these bistable populations revealed a putative signature set of genes that includes oxidative stress resistance and iron transport genes, and which might be critical to invasion of or survival within epithelial cells.

Molecular analysis of the pmo (particulate methane monooxygenase) operons from two type II methanotrophs.

The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the alpha-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, consensus sequences for sigma(70) promoters were identified, suggesting that these pmo genes are recognized by sigma(70) and negatively regulated under low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies of pmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copy of pmo genes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the gamma-Proteobacteria, Methylococcus capsulatus Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices. Hybridization experiments showed that there are two copies of pmoC in both M. trichosporium OB3b and Methylocystis sp. strain M, and not three copies as found in M. capsulatus Bath.