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.

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.

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.

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.

Sequence analysis of the cloned streptococcal cell surface antigen I/II.

The gene spa P (formerly designated as spa P1) encoding the Mr 185,000 surface antigen (I/II) of Streptococcus mutans, serotype c (NG5), has been sequenced. The gene (4683 bp) encodes a protein of 1561 amino acid residues including putative signal peptide (residues 1-38) and transmembrane (residues 1537-1556) sequences. The N-terminal region (60-550) has alanine-rich repeats and is predicted to be alpha-helical. However, the C-terminal region (800-1540) is proline-rich and favours an extended structure. Except for a short central variable region the sequences appear to be highly conserved for S. mutans serotype c. N-Terminal sequencing of separated antigen I and antigen II polypeptides suggests that the former represents the N-terminal and the latter the C-terminal portions of the intact antigen.

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.

Evidence for independent molecular identity and functional interaction of the haemagglutinin and cysteine proteinase (gingivain) of Porphyromonas gingivalis.

The sequence of events involved in haemagglutination and lysis of erythrocytes by washed cells, vesicles and the culture supernate of Porphyromonas gingivalis strain W83 was monitored by 51Cr release and transmission electronmicroscopy. All preparations, except capsular material and lipopolysaccharide, caused haemagglutination and, by a slow process of attachment and specific attack on the surface structures of the red blood cells, produced minute pores and eventual leakage of cellular contents. N-acetylglucosamine, N-acetylgalactosamine and several other sugars such as glucose and sucrose had no effect on haemagglutination. Antiserum raised against a cloned haemagglutinin of P. gingivalis strain 381 inhibited the activity of strain W83 cells, vesicles and supernate. The antiserum-neutralised supernate lost 70-80% of its hydrolytic activity towards alpha-N-benzoyl-L-arginine-4-nitroanilide but the residual activity behaved in a manner similar to the native supernate in that it was completely inhibited by the addition of 2,2'-dipyridyl disulphide and was fully restored upon addition of a low-Mr mercaptan. Binding of the antiserum to the haemagglutinin epitope of P. gingivalis still permitted titration of the active centre cysteinyl thiol group of the proteinase. Purified gingivain caused lysis of erythrocytes and was not neutralised by antiserum to the haemagglutinin. These results suggest that, although the haemagglutinin and gingivain are probably separate molecules, they are closely associated on the outer membrane of P. gingivalis and may be functionally related.

Cloning and inactivation of the gene responsible for a major surface antigen on Streptococcus mutans.

To understand more fully the biological function(s) and investigate the reported cross-reactivity with heart tissue of antigen P1 (I/II) of Streptococcus mutans (serotype c), this molecular biological study of the responsible gene, spaP, was undertaken. A 5.2 kb Hin dIII fragment of strain NG5 was cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. Recombinant SM2949 expressed a P1 fusion protein under the control of the streptococcal promoter. Southern analysis revealed hybridization of pSM2949 with DNA from Strep. mutans (serotypes c, e, f), Strep. cricetus (a) and Strep. sobrinus (d), but not Strep. sobrinus (g), Strep. rattus (b) or Strep. downei (h). Recombinant (r) antigen was detected in E. coli periplasm, indicating the presence of a signal sequence. This product (of Mr 155K) showed partial identity to the native streptococcal P1 antigen by Ouchterlony double-diffusion analysis. The N-terminal 28 amino acid residues of rP1 were determined by Edman degradation analysis and an end-labelled oligonucleotide probe corresponding to residues 8-13 was used to determine the 5'-3' orientation of spaP by Southern hybridization with restriction enzyme digests of pSM2949. Rabbit antisera made against native and rP1 did not cross-react with human heart tissue. Isogenic mutants of strain NG8 were isolated after transformation with insertionally inactivated spaP. Each mutant was non-reactive with anti-P1 antisera. Selected mutants were shown to have a defective spaP gene incorporated into their chromosomal DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Restriction fragment length polymorphism analysis of two hemagglutinin loci, serotyping and agglutinating activity of Porphyromonas gingivalis isolates.

Restriction fragment length polymorphisms (RFLPs) of two hemagglutinin loci were analyzed in 36 Porphyromonas gingivalis isolates from human and monkey origins using portions of hagA and hagB as probes. The P. gingivalis strains were differentiated into 9 RFLP groups based on the heterogeneity of the hagA locus and 10 different groups based on hybridization with hagB. Homology to hagA was detected in all human derived and all but three monkey derived strains. All P. gingivalis isolates exhibited DNA homologous to hagB. Multiple alleles of the hemagglutinin genes were detected for most P. gingivalis strains. No DNA homologous to either hemagglutinin gene could be detected in 6 other bacterial species tested. Serotyping and hemagglutination titers of each P. gingivalis isolate were obtained in an attempt to establish a correlation between these pheno-typic parameters and RFLP group. Although no correlations were found with these parameters, a correlation between RFLP group and invasiveness in the mouse abscess model was noted.

Cloning and sequencing of two type 4 (N-methylphenylalanine) pilin genes from Eikenella corrodens.

Eikenella corrodens is a Gram-negative microaerophilic rod which is gaining recognition as an important human pathogen. We have previously reported the cloning and expression in Escherichia coli of a 3.6 kb Eik. corrodens genomic DNA fragment which encodes a 31.5 kDa haemagglutinin. Maxicell analysis revealed that this fragment also encodes two proteins of approximately 14 kDa. Nucleotide sequencing of the 2.2 kb fragment upstream of the haemagglutinin gene revealed two open reading frames with strong homology to genes encoding pilin subunit proteins of the type 4 or N-methylphenylalanine class. The two pilin genes, ecpA and ecpB, are complete and are expressed in E. coli. Southern analysis of ten additional Eik. corrodens strains revealed that all possess fragments homologous to ecpA. These data represent the first molecular evidence for pili in E. corrodens.

Duplication and differential expression of hemagglutinin genes in Porphyromonas gingivalis.

A third hemagglutinin gene, defined as hagC, was cloned from Porphyromonas gingivalis 381 and sequenced. This gene was found to encode a protein highly homologous (98.6%) to the previously reported HagB hemagglutinin protein. The upstream and downstream regions of hagB and hagC were found to share less than 40% homology compared with 99% for their open reading frames. The antigenic relationship between the two hemagglutinins was demonstrated by Western blot analysis. When expressed in an in vitro transcription-translation system, both genes encoded a protein with a molecular mass of 49 kDa. As determined by reverse transcription polymerase chain reaction, the steady-state levels of hagB and hagC mRNAs were found to vary according to the growth phase and hemin concentration. The amount of transcripts decreased in hemin-limited conditions or in the absence of hemin. Furthermore, hagB mRNAs were detected in the early logarithmic growth phase compared with the hagC transcripts, which were detected only in the mid-exponential phase of growth.

Development of a genetic system for Eikenella corrodens: transfer of plasmids pFM739 and pLES2.

Eikenella corrodens is a Gram-negative microaerophilic rod which is emerging as an important human pathogen. Elucidation of the mechanisms by which it causes disease require efficient methods for the transfer of DNA to E. corrodens. Plasmids pFM739 and pLES2 have been transferred by conjugation from Escherichia coli S17-1 to E. corrodens ATCC 23834 at frequencies of 2.5 x 10(-7) and 2.42 x 10(-7), respectively. In addition, both plasmids could be transferred to four additional, clinical strains of E. corrodens at a similar frequency. The use of bacteriophage T4 as a counterselecting agent is also described.

Identification and sequence analysis of a methylase gene in Porphyromonas gingivalis.

A gene from the periodontal organism Porphyromonas gingivalis has been identified as encoding a DNA methylase. The gene, referred to as pgiIM, has been sequenced and found to contain a reading frame of 864 basepairs. The putative amino acid sequence of the encoded methylase was 288 amino acids, and shared 47% and 31% homology with the Streptococcus pneumoniae DpnII and E. coli Dam methylases, respectively. The activity and specificity of the pgi methylase (M.PgiI) was confirmed by cloning the gene into a dam- strain of E. coli (JM110) and performing a restriction analysis on the isolated DNA with enzymes whose activities depended upon the methylation state of the DNA. The data indicated that M.PgiI, like DpnII and Dam, methylated the adenine residue within the sequence 5'-GATC-3'.

Systemic and mucosal immune responses in mice orally immunized with avirulent Salmonella typhimurium expressing a cloned Porphyromonas gingivalis hemagglutinin.

Porphyromonas gingivalis produces a variety of virulence factors that may have a function in the periodontal disease process. Determination of the role of these various factors in pathogenesis and identification of a means for protecting the host from the destructive effects of this organism are areas of vigorous investigation. In this study we demonstrate the potential of avirulent Salmonella typhimurium strains to stimulate a specific systemic and mucosal immune response to a cloned P. gingivalis hemagglutinin (HagB). An avirulent strain of S. typhimurium, chi 4072, expressing the hagB gene of P. gingivalis 381 on the plasmid pDMD1 was intragastrically administered to BALB/c mice. These mice mounted a serum immunoglobulin G (IgG) and IgA primary response against the hagB gene product and a mucosal immune response as measured by evaluation of saliva. IgA antibodies were also detected in bile. These results demonstrate the feasibility of using attenuated S. typhimurium strains as carriers of P. gingivalis virulence factors for subsequent evaluation of the systemic and mucosal immune response against these antigens. This system will provide a means for evaluating the virulence factors of P. gingivalis for their suitability in the construction of potential vaccines.