Complete genome sequence of the oral pathogenic Bacterium porphyromonas gingivalis strain W83

The complete 2,343,479-bp genome sequence of the gram-negative, pathogenic oral bacterium Porphyromonas gingivalis strain W83, a major contributor to periodontal disease, was determined. Whole-genome comparative analysis with other available complete genome sequences confirms the close relationship between the Cytophaga-Flavobacteria-Bacteroides (CFB) phylum and the green-sulfur bacteria. Within the CFB phyla, the genomes most similar to that of P. gingivalis are those of Bacteroides thetaiotaomicron and B. fragilis. Outside of the CFB phyla the most similar genome to P. gingivalis is that of Chlorobium tepidum, supporting the previous phylogenetic studies that indicated that the Chlorobia and CFB phyla are related, albeit distantly. Genome analysis of strain W83 reveals a range of pathways and virulence determinants that relate to the novel biology of this oral pathogen. Among these determinants are at least six putative hemagglutinin-like genes and 36 previously unidentified peptidases. Genome analysis also reveals that P. gingivalis can metabolize a range of amino acids and generate a number of metabolic end products that are toxic to the human host or human gingival tissue and contribute to the development of periodontal disease.     

Porphyromonas gingivalis influences actin degradation within epithelial cells during invasion and apoptosis

Porphyromonas gingivalis, a Gram-negative oral pathogen, has been shown to induce apoptosis in human gingival epithelial cells, yet the underlining cellular mechanisms controlling this process are poorly understood. We have previously shown that the P. gingivalis proteases arginine and lysine gingipains, are necessary and sufficient to induce host cell apoptosis. In the present study, we demonstrate that 'P. gingivalis-induced apoptosis' is mediated through degradation of actin leading to cytoskeleton collapse. Stimulation of human gingival epithelial cells with P. gingivalis strains 33277 and W50 at moi:100 induced β-actin cleavage as early as 1 h and human serum inhibited this effect. By using gingipain-deficient mutants of P. gingivalis and purified gingipains, we demonstrate that lysine gingipain is involved in actin hydrolysis in a dose and time-dependent manner. Use of Jasplakinolide and cytochalasin D revealed that P. gingivalis internalization is necessary for actin cleavage. Further, we also show that lysine gingipain from P. gingivalis can cleave active caspase 3. Taken together, we have identified actin as a substrate for lysine gingipain and demonstrated a novel mechanism involved in microbial host cell invasion and apoptosis.     

P. gingivalis accelerates gingival epithelial cell progression through the cell cycle

P. gingivalis, an opportunistic pathogen in periodontal disease, can reside within the epithelial cells that line the gingival crevice. A proteomic analysis revealed that infection of gingival epithelial cells with P. gingivalis induces broadly based changes in the level and phosphorylation status of proteins that exert multi-level control on the eukaryotic cell cycle. Pathways that were impacted by P. gingivalis included those involving cyclins, p53 and PI3K. The predicted infection-dependent phenotype was confirmed by cytofluorimetry that showed an enhanced proliferation rate of gingival epithelial cells infected with P. gingivalis associated with accelerated progression through the S-phase. Elevated cell proliferation was dependent on the presence of the long fimbriae of P. gingivalis. The ability of P. gingivalis, a common inhabitant of the subgingival crevice, to accelerate cell cycling could have biological consequences for barrier and signaling functions, and for physiological status, of the gingival epithelium.     

Porphyromonas gingivalis SerB-mediated dephosphorylation of host cell cofilin modulates invasion efficiency

Porphyromonas gingivalis, a host-adapted opportunistic pathogen, produces a serine phosphatase, SerB, known to affect virulence, invasion and persistence within the host cell. SerB induces actin filament rearrangement in epithelial cells, but the mechanistic basis of this is not fully understood. Here we investigated the effects of SerB on the actin depolymerizing host protein cofilin. P. gingivalis infection resulted in the dephosphorylation of cofilin in gingival epithelial cells. In contrast, a SerB-deficient mutant of P. gingivalis was unable to cause cofilin dephosphorylation. The involvement of cofilin in P. gingivalis invasion was determined by quantitative image analysis of epithelial cells in which cofilin had been knocked down or knocked in with various cofilin constructs. siRNA-silencing of cofilin led to a significant decrease in numbers of intracellular P. gingivalis marked by an absence of actin colocalization. Transfection with wild-type cofilin or constitutively active cofilin both increased numbers of intracellular bacteria, while constitutively inactive cofilin abrogated invasion. Expression of LIM kinase resulted in reduced P. gingivalis invasion, an effect that was reversed by expression of constitutively active cofilin. These results show that P. gingivalis SerB activity induces dephosphorylation of cofilin, and that active cofilin is required for optimal invasion into gingival epithelial cells.     

Clathrin-dependent entry of a gingipain adhesin peptide and Porphyromonas gingivalis into host cells

Porphyromonas gingivalis, a Gram-negative oral anaerobe, is associated with periodontitis, a disease that in some form affects up to 80% of the adult population in the USA. The organism interacts with gingival epithelium and surrounding tissue, and in this study we analysed interactions initiated by P. gingivalis and by a peptide derived from the adhesin domain of arg-gingipain A, a member of a family of surface cysteine proteinases. Recombinant peptide A44 blocked adherence of bacteria to host cell monolayers, and bound to components of the cell membrane fraction. In pull-down assays A44 associated with proteins involved in a clathrin-dependent endocytosis pathway. Inhibitor studies confirmed a role for clathrin, and confocal microscopy demonstrated that both A44-coated beads and intact bacteria colocalized with GFP-clathrin in host cells. Finally, we used siRNA to determine whether clathrin or caveolin-1 was involved in association of peptide and intact bacteria with host cells. Again, the results of these assays indicated that association of both A44 and P. gingivalis depended on the presence of clathrin, and support a working model in which A44 initiates a clathrin-dependent pathway that potentially leads to internalization of peptide or bacteria by host epithelial cells.     

Porphyromonas gingivalis proteinases as virulence determinants in progression of periodontal diseases

Porphyromonas gingivalis, one of the major causative agents of periodontal diseases, produces large amounts of arginine- and lysine-specific cysteine proteinases in cell-associated and secretory forms, which are now referred to as Arg-gingipain (Rgp) and Lys-gingipain (Kgp), respectively. A number of studies have revealed that these proteinases are closely associated with the periodontopathogenesis of this bacterium: destruction of periodontal connective tissues, disruption of host defense mechanisms, and development and maintenance of inflammation in periodontal pockets. With respect to the physiology of the bacterium, Rgp and Kgp are indispensable for it to obtain nutrients from the environment, since it cannot utilize saccharides as carbon/energy sources for growth and totally depends on peptides and amino acids that are provided from environmental proteins by Rgp and Kgp. Furthermore, proteolytic activities of Rgp and Kgp contribute to processing/maturation of various cell-surface proteins of P. gingivalis, such as fimA fimbrilin (a subunit of major fimbriae), 75-kDa protein (a subunit of minor fimbriae), hemagglutinins, and the hemoglobin receptor protein, which are important for the bacterium to colonize and proliferate in the gingival crevice and to invade the periodontium. These findings strongly indicate critical roles of Rgp and Kgp in the virulence of P. gingivalis.     

The Mga virulence regulon: infection where the grass is greener

Co-ordinate regulation of virulence gene expression in response to different host environments is central to the success of the group A streptococcus (GAS, Streptococcus pyogenes) as an important human pathogen. Mga represents a ubiquitous stand-alone virulence regulator that controls genes (Mga regulon) whose products are necessary for adherence, internalization and host immune evasion. Mga highly activates a core set of virulence genes, including its own gene, by directly binding to their promoters. Yet, Mga also influences expression of over 10% of the GAS genome, primarily genes and operons involved in metabolism and sugar utilization. Expression of the Mga regulon is influenced by conditions that signify favourable growth conditions, presumably allowing GAS to take advantage of promising new niches in the host. The ability of Mga to respond to growth signals clearly involves regulation of mga expression via global regulatory networks such as RALPs, Rgg/RopB and the catabolite control protein CcpA. However, the presence of predicted PTS regulatory domains (PRDs) within Mga suggests an intriguing model whereby phosphorylation of Mga by the PTS phosphorelay might link growth and sugar utilization with virulence in GAS. As Mga homologues have been found in several important Gram-positive pathogens, the Mga regulon could provide a valuable paradigm for increasing our understanding of global virulence networks in bacteria.     

Calcium oscillations in gingival epithelial cells infected with Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis modulates epithelial cell signal transduction pathways including Ca2+ signaling, and internalizes within the host cell cytoplasm. Since nuclear and cytoplasmic [Ca2+] increases can induce different host cell responses, P. gingivalis-related [Ca2+] changes in these compartments were measured by digital fluorescent imaging microscopy. Non-deconvolved and deconvolved fura-2 images showed that P. gingivalis exposure caused human gingival epithelial cells cultured in physiologic [Ca2+] levels to undergo sustained oscillations of [Ca2+] in nuclear and cytoplasmic spaces. However, P. gingivalis invasion was not tightly correlated with intracellular [Ca2+] oscillations, since invasion could significantly precede, or even occur in the absence of, oscillations. [Ca2+] oscillations required a Ca2+ influx, which was completely inhibited by La3+ or 2-APB (2-aminoethoxydiphenyl borate), indicating Ca2+ entry was via a Ca(2+)-permeable channel. Ca2+ entry was likely not via a store-operated channel, since Ca2+ release from intracellular stores was not observed during cellular uptake of P. gingivalis. Hence, uptake of P. gingivalis in gingival epithelial cells induces oscillations in nuclear and cytoplasmic spaces by activating a Ca2+ influx through Ca2+ channels.     

Colocalization of Porphyromonas gingivalis with CD4+ T cells in periodontal disease

Porphyromonas gingivalis, an anaerobic, asaccharolytic gram-negative bacterium, is a causative agent in chronic periodontitis. It has many virulence factors that facilitate infection of the gingiva, but little is known about the local immune cells that respond to this bacterium. The aims of this study were to quantify P. gingivalis in gingival biopsies from patients with periodontitis using laser capture microdissection (LCM) plus qRT-PCR and to determine the phenotype of immune cells associated with the bacteria using immunofluorescence. The presence of P. gingivalis was confirmed in periodontitis gingival tissue from 10 patients, and differences in bacterial distribution in the epithelium and connective tissue with or without inflammatory infiltrates were observed. Immune cells found in the biopsy tissues, including CD20+ mature B cells and CD138+ plasma cells, were associated with the Th2-type immune response. Most P. gingivalis was in direct contact with CD4+ T cells. This study revealed for the first time the colocalization of P. gingivalis with immune cells. Use of LCM combined with qRT-PCR enabled quantitative analysis of bacteria in a selected area of a biopsy sample without any tissue degradation. Observation of the immune cells associated with these bacteria was also performed by immunofluorescence.     

Human lactoferrin binds and removes the hemoglobin receptor protein of the periodontopathogen Porphyromonas gingivalis

Porphyromonas gingivalis possesses a hemoglobin receptor (HbR) protein on the cell surface as one of the major components of the hemoglobin utilization system in this periodontopathogenic bacterium. HbR is intragenically encoded by the genes of an arginine-specific cysteine proteinase (rgpA), lysine-specific cysteine proteinase (kgp), and a hemagglutinin (hagA). Here, we have demonstrated that human lactoferrin as well as hemoglobin have the abilities to bind purified HbR and the cell surface of P. gingivalis through HbR. The interaction of lactoferrin with HbR led to the release of HbR from the cell surface of P. gingivalis. This lactoferrin-mediated HbR release was inhibited by the cysteine proteinase inhibitors effective to the cysteine proteinases of P. gingivalis. P. gingivalis could not utilize lactoferrin for its growth as an iron source and, in contrast, lactoferrin inhibited the growth of the bacterium in a rich medium containing hemoglobin as the sole iron source. Lactoferricin B, a 25-amino acid-long peptide located at the N-lobe of bovine lactoferrin, caused the same effects on P. gingivalis cells as human lactoferrin, indicating that the effects of lactoferrin might be attributable to the lactoferricin region. These results suggest that lactoferrin has a bacteriostatic action on P. gingivalis by binding HbR, removing it from the cell surface, and consequently disrupting the iron uptake system from hemoglobin.