VBNC, previously unrecognized in the life cycle of Porphyromonas gingivalis?

Bacteria are exposed to stresses during their growth and multiplication in their ecological systems to which they respond in multiple ways as expert survivalists. One such response mechanism is to convert to a viable but not culturable (VBNC) state. As the name indicates, bacteria in the VBNC state have lost their ability to grow on routine growth medium. A large number of bacteria including many pathogenic species have been reported to be able to enter a VBNC state. VBNC differs from culturable cells in various physiological properties which may result in changes in chemical resistance, adhesion, cellular morphology, metabolism, gene expression, membrane and cell wall composition and/or virulence. The ability of VBNC bacteria to return to the culturable state or resuscitate, when the stressor is removed poses a considerable threat to public health. There have been few publications that overtly describe the ability of oral pathogenic species to enter the VBNC state. However, the presence of VBNCs among oral pathogens such as Porphyromonas gingivalis in human chronic infections may be an important virulence factor and have severe implications for therapy. In this review, we intend to i) define and summarize the significance of the VBNC state in general and ii) discuss the VBNC state of oral bacteria with regard to P. gingivalis. Future studies focused on this phenomenon of intraoral VBNC would provide novel molecular insights on the virulence and persistence of oral pathogens during chronic infections and identify potential novel therapies.

Porphyromonas gingivalis W83 traffics via ICAM1 in microvascular endothelial cells and alters capillary organization in vivo.

Objective: Microvascular dysfunction is a feature of periodontal disease. P. gingivalis, one of the most common oral bacteria present in gingival tissue biofilms, has also been identified in the gingival capillaries of patients with chronic periodontitis. We sought to determine the effect of P. gingivalis W83 infection on microvascular endothelium in vivo and in vitro. Methods and Results: Interdental papillae of rats with P. gingivalis-induced alveolar bone loss had a more dilated and denser subepithelial capillary network than uninfected controls. P. gingivalis W83 was detected in the epithelial layers, the subepithelial connective tissue matrix, and subgingival capillaries. P. gingivalis invaded human dermal microvascular endothelial cells (HD-MVECS) and persisted up termination (24 h). Colocalization analysis at 2.5, 6, and 24 h post-inoculation showed that 79-88% of internalized bacteria were in ICAM-1 positive endosomes, and 10-39% were in Rab5, Rab7, or LAMP1 positive compartments, but never in autophagosomes. Antibody-based blockade of ICAM-1 significantly reduced W83 invasion in HD-MVECS. P. gingivalis infected HD-MVECS were unable to form vascular networks in Matrigel. Conclusions: P. gingivalis perturbs microvascular endothelial function and invasion of these cells via ICAM-1 may be important for microbial persistence within tissues.

Increased atherogenesis during Streptococcus mutans infection in ApoE-null mice.

Streptococcus mutans, a dental caries pathogen, also causes endocarditis and is detected in atheroscelerotic plaque. We investigated the potential for an invasive strain of S. mutans, OMZ175, to accelerate plaque growth in apolipoprotein E deficient (ApoE(null)) mice without and with balloon angioplasty (BA) injury, a model of restenosis. ApoE(null) mice were divided into 4 groups (N = 10), 2 with and 2 without BA. One each of the BA and non-BA groups was infected with S. mutans (Sm). S. mutans DNA, plaque area, inflammatory cell invasion, and Toll-like receptor (TLR) expression were measured at 6-20 weeks post-infection. S. mutans genomic DNA was detected in the aorta, liver, spleen, and heart. Plaque growth was significantly increased in infected mice with BA (Sm+BA) vs. those in the non-infected groups (p < 0.03). Plaque size was increased after infection without BA (Sm), but did not reach significance. Aortic specimens from both S. mutans and Sm+BA groups displayed increased numbers of macrophages, and TLR4 expression was increased in BA mice. In conclusion, S. mutans infection accelerated plaque growth, macrophage invasion, and TLR4 expression after angioplasty. S. mutans may also be associated with atherosclerotic plaque growth in non-injured arteries.

Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175.

INTRODUCTION: Dissemination of oral bacteria into the bloodstream has been associated with eating, oral hygiene, and dental procedures; including tooth extraction, endodontic treatment, and periodontal surgery. Recently, studies identified Streptococcus mutans, the primary etiological agent of dental caries, as the most prevalent bacterial species found in clinical samples from patients who underwent heart valve and atheromatous plaque surgery. METHODS: By using antibiotic protection assays, we tested the capacity of 14 strains of S. mutans to invade primary human coronary artery endothelial cells (HCAEC). RESULTS: Serotype e strain B14 and serotype f strain OMZ175 of S. mutans were able to efficiently invade HCAEC. Among the tested strains, serotype f S. mutans OMZ175 was the most invasive, whereas strains of serotype c S. mutans, the most prevalent serotype in dental plaque, were not invasive. Based on its high invasion rate, we further investigated the invasive properties of serotype f OMZ175. Using transmission electron microscopy and antibiotic protection assays we demonstrate that S. mutans OMZ175 is capable of attaching to the HCAEC surface, entering the cells and surviving in HCAEC for at least 29 h. DISCUSSION: Our findings highlight a potential role for S. mutans in the pathogenesis of certain cardiovascular diseases.

Analysis of a band 7/MEC-2 family gene of Porphyromonas gingivalis.

In vivo-induced antigen technology has previously been used to identify 115 genes induced in Porphyromonas gingivalis W83 during human infection. The aim of this study was to determine if one of these genes, PG1334, was important for the virulence of P. gingivalis. Analysis of plaque samples from persons with periodontitis revealed that PG1334 was expressed in 88.0% of diseased sites, compared with 42.1% of healthy sites, even though P. gingivalis was detected in equal numbers from both sites. A mutant of PG1334 was found to adhere to and to invade better than the parent strain, but did not persist as well in human coronary artery endothelial cells. Additionally, the mutant did not persist as well in a mouse abscess model. This gene appears to be important for the virulence of P. gingivalis, both in vivo and in vitro.

Invasive differences among Porphyromonas gingivalis strains from healthy and diseased periodontal sites.

BACKGROUND AND OBJECTIVE: The purpose of this study was to determine any difference between Porphyromonas gingivalis isolates from periodontally healthy sites as compared to those from diseased sites with respect to the ability to invade host cells. MATERIAL AND METHODS: Subgingival plaque samples were obtained from periodontally healthy and diseased sites using paper points. P. gingivalis colonies were isolated and tested, using an antibiotic protection assay, for their ability to invade KB cells. P. gingivalis 381 and Escherichia coli MC1061 were used as controls. RESULTS: Mean values of 16.79 +/- 0.86 x 10(3) colony-forming units/mL and 26.14 +/- 2.11 x 10(3) colony-forming units/mL were observed in invasion assays for isolates from periodontally healthy and diseased sites, respectively. P. gingivalis present in diseased sites had significantly greater invasive abilities than strains isolated from healthy sites. No statistical difference was noted between male or female subjects concerning the degree of invasion; isolates from diseased sites from both genders had significantly greater invasion abilities than those from healthy sites. A significant correlation was found between the increased invasive capabilities of P. gingivalis isolates vs. an increased probing depth. CONCLUSION: The increased invasion noted with P. gingivalis isolates from diseased sites vs. healthy sites, and the increased invasive capabilities with increasing probing depth, indicate that P. gingivalis isolates have a varying ability to invade host cells in the periodontal pocket.

Invasion of vascular cells in vitro by Porphyromonas endodontalis.

AIM: The objective of this study was to determine whether laboratory strains and clinical isolates of microorganisms associated with root canal infections can invade primary cultures of cardiovascular cells. METHODOLOGY: Quantitative levels of bacterial invasion of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) were measured using a standard antibiotic protection assay. Transmission electron microscopy was used to confirm and visualize internalization within the vascular cells. RESULTS: Of the laboratory and clinical strains tested, only P. endodontalis ATCC 35406 was invasive in an antibiotic protection assay using HCAEC and CASMC. Invasion of P. endodontalis ATCC 35406 was confirmed by transmission electron microscopy. DISCUSSION: Certain microorganisms associated with endodontic infections are invasive. If bacterial invasion of the vasculature contributes to the pathogenesis of cardiovascular disease, then microorganisms in the pulp chamber represent potential pathogens.

Porphyromonas gingivalis traffics to autophagosomes in human coronary artery endothelial cells.

Porphyromonas gingivalis is a periodontal pathogen that also localizes to atherosclerotic plaques. Our previous studies demonstrated that P. gingivalis is capable of invading endothelial cells and that intracellular bacteria are contained in vacuoles that resemble autophagosomes. In this study, we have examined the trafficking of P. gingivalis 381 to the autophagic pathway. P. gingivalis 381 internalized by human coronary artery endothelial (HCAE) cells is located within vacuoles morphologically identical to autophagosomes. The progression of P. gingivalis 381 through intracellular vacuoles was analyzed by immunofluorescence microscopy. Vacuoles containing P. gingivalis colocalize with Rab5 and HsGsa7p early after internalization. At later times, P. gingivalis colocalizes with BiP and then progresses to a vacuole that contains BiP and lysosomal glycoprotein 120. Late endosomal markers and the lysosomal cathepsin L do not colocalize with P. gingivalis 381. The intracellular survival of P. gingivalis 381 decreases over 8 h in HCAE cells pretreated with the autophagy inhibitors 3-methyladenine and wortmannin. In addition, the vacuole containing P. gingivalis 381 lacks BiP but contains cathepsin L in the presence of wortmannin. These results suggest that P. gingivalis 381 evades the endocytic pathway to lysosomes and instead traffics to the autophagosome.

The effect of monoclonal antibody and route of immunization on the humoral immune response against Porphyromonas gingivalis.

Immunomodulation mediated by exogenous antibodies has been proposed as a vaccine strategy to improve immune protection against pathogenic microorganisms and suggested to contribute to protection following passive immunization. To test whether a monoclonal antibody directed against an adhesion epitope of the periodontal pathogen Porphyromonas gingivalis could influence the humoral immune response following mucosal immunization, BALB/c mice were immunized orally or intranasally with P. gingivalis alone or P. gingivalis coated with monoclonal antibody 61BG1.3. Differences in antigenic specificity of anti- P. gingivalis serum immunoglobulin G (IgG) were demonstrated between groups of mice that received monoclonal antibody-coated P. gingivalis versus those that received P. gingivalis alone by either route of immunization. Binding of monoclonal antibody 61BG1.3 to P. gingivalis prior to immunization did not influence the serum IgG subclass distribution. However, minor differences in subclass distribution were observed between the various routes of mucosal immunization. These results support the hypothesis that specific monoclonal antibody bound to a bacterial vaccine can alter the quality of the humoral immune response to that organism.

Identification of lysine decarboxylase as a mammalian cell growth inhibitor in Eikenella corrodens: possible role in periodontal disease.

The pathogenesis of inflammatory periodontal disease was studied by examining the mechanism of HeLa and HL60 cell growth inhibition by cell-free saline-soluble extracts of Eikenella corrodens and bacterial plaque. Previous studies identified a protein (p80) as causing growth inhibition by E. corrodens extracts. After purification by two-dimensional SDS-PAGE, p80 was digested with protease lysC. Amino acid sequences were obtained and backtranslated for use as PCR primers. A 5840 nucleotide sequence containing a lysine decarboxylase gene was obtained from a Sau3 A1 genomic library of E. corrodens DNA. Lysine decarboxylase activity was present at physiologic pH in the E. corrodens extracts containing p80, and also in bacterial plaque. Both extracts caused growth inhibition by depleting lysine from cell culture media through conversion to cadaverine. Adding lysine, or immune goat IgG to a peptide derived from the active site sequence of E. corrodens lysine decarboxylase, retarded lysine depletion and growth inhibition. epsilon-Amino caproic acid specifically enhanced lysine decarboxylase activity at the low lysine concentration in HL60 cell culture media, and also increased the growth inhibition. Thus, lysine decarboxylases such as p80 inhibit growth by removing lysine from mammalian cell culture media. A new role for lysine decarboxylase activity in the microbial aetiology of periodontal disease is discussed.

IVIAT: a novel method to identify microbial genes expressed specifically during human infections.

In vivo induced antigen technology (IVIAT) is a novel technology that can quickly and easily identify in vivo induced genes in human infections, without the use of animal models. This technology is expected to facilitate the discovery of new targets for vaccines, antimicrobials and diagnostic strategies in a wide range of microbial pathogens.

Invasion of endothelial and epithelial cells by strains of Porphyromonas gingivalis.

Porphyromonas gingivalis is a periodontal pathogen that may also be involved in the pathogenesis of coronary heart disease. This microorganism has the ability to invade several cell lines. In this study, 26 different strains of P. gingivalis were tested for invasion of human umbilical vein endothelial cells and KB cells, a human oral epidermoid cell line. Abilities to invade both cell lines by an individual strain were similar, and their invasion efficiencies could be assembled into four groups: high, moderate, low and non-invasive. Of the 26 strains, only P. gingivalis AJW4 was non-invasive. Since the fimbriae are implicated as having a key role in invasion by this species, the presence of fimbriae on strain AJW4 was investigated. Using polymerase chain reaction (PCR), strain AJW4 was found to contain the fimA gene. Sequence analysis revealed it to be type IV according to the typing scheme developed by Amano et al. Further, fimA is transcribed in this strain as demonstrated by reverse transcription PCR and is expressed on the cell surface as visualized by negative staining and electron microscopy. The adherence+invasion of strain AJW4 was 38.7% of the most invasive strain (strain 381). However, the CFU ml(-1) of strain AJW4 recovered from within cells was 2.9% of strain 381. Even though strains AJW4 and W50 have the same type IV fimbriae, strain AJW4 is 8.9-fold more adhesive yet is internalized 170-fold less. These data indicate that the invasion efficiency of P. gingivalis is variable among the different strains, and that the expression of FimA is not sufficient for invasion.

Long-term immunological memory induced by recombinant oral Salmonella vaccine vectors.

We have previously shown that Salmonella enterica serovar Typhimurium expressing the hagB hemagglutinin gene from Porphyromonas gingivalis can induce primary and recall immune responses in serum and secretions in mice; however, the longevity of memory induced by oral Salmonella carriers has not been adequately demonstrated. In this study, we examined the capacity of mice to mount a recall response 52 weeks after primary immunization. Recall responses were seen in serum immunoglobulin G (IgG) and IgA following boosting at week 52, and in most cases, they were equal to or greater than the primary responses. Significant mucosal IgA recall responses in saliva and vaginal wash were also detected following boosting at week 52. In addition, there was a considerable residual response in secretions at week 51, prior to boosting. These results indicate that oral Salmonella vectors can induce long-term memory to recombinant HagB and are particularly effective at inducing long-lasting mucosal responses as well as at inducing the capacity for mucosal recall responses.

Expression and immunogenicity of hemagglutinin A from Porphyromonas gingivalis in an avirulent Salmonella enterica serovar typhimurium vaccine strain.

Porphyromonas gingivalis is a major etiologic agent of periodontitis, a chronic inflammatory disease that ultimately results in the loss of the supporting tissues of the teeth. Previous work has demonstrated the usefulness of avirulent Salmonella enterica serovar Typhimurium strains as antigen delivery systems for protective antigens of pathogens that colonize or cross mucosal surfaces. In this study, we constructed and characterized a recombinant S. enterica serovar Typhimurium avirulent vaccine strain which expresses hemagglutinin A and carries no antibiotic resistance markers. HagA, a major virulence-associated surface protein, is a potentially useful immunogen that contains an antigenic epitope which, in humans, elicits an immune response that is protective against subsequent colonization by P. gingivalis. The hagA gene, including its promoter, was cloned into a balanced-lethal Salmonella vector and transferred to the vaccine strain. Heterologous expression of HagA was demonstrated in both Escherichia coli JM109 and S. enterica serovar Typhimurium vaccine strain chi4072. The HagA epitope was present in its native configuration as determined by immunochemistry and immunoelectron microscopy. Purified recombinant HagA was recognized by sera from mice immunized with the S. enterica serovar Typhimurium vaccine strain. The HagA-specific antigen of the vaccine was also found to be recognized by serum from a periodontal patient. This vaccine strain, which expresses the functional hemagglutinin protein, induces a humoral immune response against HagA and may be useful for developing a protective vaccine against periodontal diseases associated with P. gingivalis.

Invasion of human coronary artery cells by periodontal pathogens.

There is an emerging paradigm shift from coronary heart disease having a purely hereditary and nutritional causation to possibly having an infectious etiology. Recent epidemiological studies have shown a correlation between periodontal disease and coronary heart disease. However, to date, there is minimal information as to the possible disease mechanisms of this association. It is our hypothesis that invasion of the coronary artery cells by oral bacteria may start and/or exacerbate the inflammatory response in atherosclerosis. Since a few periodontal pathogens have been reported to invade oral epithelial tissues, we tested the ability of three putative periodontal pathogens-Eikenella corrodens, Porphyromonas gingivalis, and Prevotella intermedia-to invade human coronary artery endothelial cells and coronary artery smooth muscle cells. In this study we demonstrate by an antibiotic protection assay and electron microscopy that specific species and strains invade coronary artery cells at a significant level. Actin polymerization and eukaryotic protein synthesis in metabolically active cells were required since the corresponding inhibitors nearly abrogated invasion. Many intracellular P. gingivalis organisms were seen to be present in multimembranous vacuoles resembling autophagosomes by morphological analysis. This is the first report of oral microorganisms invading human primary cell cultures of the vasculature.

Porphyromonas gingivalis virulence factors and invasion of cells of the cardiovascular system.

Our laboratory is interested in the genes and gene products involved in the interactions between Porphyromonas gingivalis (Pg) and the host. These interactions may occur in either the periodontal tissues or other non-oral host tissues such as those of the cardiovascular system. We have previously reported the cloning of several genes encoding hemagglutinins, surface proteins that interact with the host tissues, and are investigating their roles in the disease process. Primary among these is HagA, a very large protein with multiple functional groups that have significant sequence homology to protease genes of this species. Preliminary evidence indicates that an avirulent Salmonella typhimurium strain containing hagA is virulent in mice. These data indicate that HagA may be a key virulence factor of Pg. Additionally, we are investigating the invasion of primary human coronary artery endothelial cells (HCAEC) by Pg because of the recent epidemiological studies indicating a correlation between periodontal disease (PD) and coronary heart disease (CHD). We found that some, but not all, strains of Pg are able to invade these cells. Scanning electron microsopy of the infected HCAEC demonstrated that the invading organisms initially attached to the host cell surface as aggregates and by a "pedestal"-like structure. By transmission electronmicroscopy it could be seen that internalized bacteria were present within multimembranous compartments localized with rough endoplasmic reticulum. In addition, invasion of the HCAEC by Pg resulted in an increase in the degradation of long-lived cellular proteins. These data indicate that Pg are present within autophagosomes and may use components of the autophagic pathway as a means to survive intracellularly. However, Pg presence within autophagosomes in KB cells could not be observed or detected. It is therefore likely that Pg uses different invasive mechanisms for different host cells. This and the role of HagA in invasion is currently being investigated further.

Invasion of human oral epithelial cells by Prevotella intermedia.

Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of Prevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4 degreesC) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of P. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion.

The number of direct repeats in hagA is variable among Porphyromonas gingivalis strains.

The coding sequence for the surface protein hemagglutinin A (HagA) of Porphyromonas gingivalis 381 has previously been shown to contain four direct 1.35-kb repeats, designated repHA. This study was performed to determine if the number of repHA units in hagA is consistently 4 or if allelic polymorphism exists among strains and/or upon multiple passage of P. gingivalis. To this end, primers which were homologous to the regions directly 5' and 3' of the repeat domain in hagA were synthesized. PCR conditions which allowed amplification of the 8.4-kb repeat region between the primers in P. gingivalis 381 were established. Genomic DNA templates from 13 other P. gingivalis strains and 9 fresh clinical isolates from patients were analyzed under the same conditions as used above. Analysis of these PCR products demonstrated that the strains tested had different numbers (two to four) of repHA units in the respective hagA genes. The PCR products of 8.4, 7.0, and 5.7 kb represent four, three, and two repeats, respectively. One strain from each group (381, four repeats; W83, three repeats; and AJW4, two repeats) was also tested to determine if the number of repeats remained invariant upon passaging onto solid medium. No variability in the number of repeats in hagA within a strain was detected after 18 passages. P. gingivalis 381 was chosen for further testing in a mouse abscess model to determine if conditions of in vivo growth would select for deletions or duplications of the repeated sequences. Five days after infection, no change in the number of repeats was detected in cells recovered from either nonimmunized or preimmunized mice. This data indicates an interstrain variability of the number of repeat units and hence a size variability of the HagA protein of P. gingivalis, but unlike some surface antigens of other pathogenic species, the number of repeats remains relatively stable given the conditions of growth tested here.

The porphyromonas gingivalis prtP/kgp homologue exists as two open reading frames in strain 381.

P. gingivalis is considered to be a major pathogen of adult periodontitis. Among its cadre of putative virulence factors are hemagglutinins (adhesins) and proteases. We here report the cloning, sequencing and characterization of two genes, designated kgp(381) and hagD. Kgp(381), an open reading frame (ORF) of 1095 bp encoding a 40.1 kda protein, has high homology to the proteolytic domain of cysteine protease/hemagglutinin genes. HagD, an ORF of 4077 bp encoding a 147.1 kda protein, contains one HArep sequence which establishes it as an additional member of the HArep multigene family. Although similar in sequence to kgp and prtP which were identified from strains HG66 and W12, respectively, the kgp(381)-hagD genes have several characteristics which distinguish them from kgp and prtP. Foremost among these is a single base difference which produces a termination codon and an immediate frame shift resulting in two ORFs in strain 381 as compared to one ORF in strains HG66 and W12. In addition, a 172 amino acid sequence near the C-terminal end of hagD has very low identity (20.5-27.8%) to the corresponding region of kgp and prtP. These demonstrate that the homologue of kgp and prtP in strain 381 occurs as two separate genes which may genetically separate the adhesive and enzymatic domains of Kgp and PrtP proteins. Reverse polymerase chain reaction (PCR) analysis indicates that hagD expression is regulated by hemin concentration.