Both the unique and repeat regions of the Porphyromonas gingivalis hemagglutin A are involved in adhesion and invasion of host cells.

Porphyromonas gingivalis is one of the major etiologic agents of adult periodontitis and has been associated with cardiovascular diseases. It expresses multiple hemagglutinins that are significant virulence factors and play an important role in bacterial attachment and invasion of host cells. The objective of this study was to determine the impact of P. gingivalis hemagglutinin A (HagA) on the attachment to and invasion of human coronary artery endothelial cells (HCAEC) and gingival epithelial cells (GEC). Bacterial strains expressing the HagA protein (or subunits), including Escherichia coli carrying plasmid pEKS5, E. coli carrying plasmid ST2, and Salmonella enterica serovar Typhimurium with plasmid pNM1.1 were used in this study. The strains were tested for their ability to attach to and invade HCAEC and GEC using antibiotic protection assays. In addition, the unique 5' N-terminal non-repeated segment of HagA was purified in recombinant form and a monoclonal antibody was created against the polypeptide. The monoclonal antibody against the unique portion of HagA was tested for inhibitory activity in these assays. The attachment of both E. coli strains expressing HagA fragment to host cells was significantly increased compared to their respective controls. However, they did not invade GEC or HCAEC. Interestingly, HagA expression in the Salmonella strain increased both adherence to and invasion of HCAEC, which may be due to the presence of the entire hagA ORF. A monoclonal antibody against the unique 5' N-terminal portion of HagA reduced invasion. Further experiments are needed to determine the role of the unique and the repeat segments of P. gingivalis HagA.

Disinfection of toothbrushes contaminated with Streptococcus mutans.

PURPOSE: To determine the most effective method to kill Streptococcus mutans on contaminated toothbrushes. METHODS: Seven toothbrushes (one for each treatment and the control) were contaminated with S. mutans. Toothbrushes were then rinsed in phosphate buffered saline (PBS) and treated as follows: (1) control without treatment; (2) air dry for 4 hours; (3) Crest Pro-Health mouthwash for 20 minutes; (4) Listerine mouthwash for 20 minutes; (5) normal cleaning cycle in a dishwasher; (6) microwave on high power for 5 minutes; and (7) ultraviolet light using the DenTek Toothbrush Sanitizer for 10 minutes. All toothbrushes were rinsed again with PBS. The bristles were cut and vortexed in PBS. Serial dilutions were performed and the number of colonies enumerated after incubation. The experiment was independently repeated seven times. RESULTS: The Crest Pro-Health mouthwash and the dishwasher almost completely eliminated S. mutans. The second most effective treatment was the microwave. The Listerine mouthwash and the air dry groups were not significantly different from each other and ranked third. Although UV light significantly decreased the number of bacteria compared to the control, reduction in the number of S. mutans CFU was significantly lower than that of all the other treatments evaluated. Crest Pro-Health mouthwash for 20 minutes and a normal dishwasher cycle are the most effective methods to eradicate S. mutans from contaminated toothbrushes. Dent

Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext).

Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

Human alpha- and beta-defensins bind to immobilized adhesins from Porphyromonas gingivalis.

Human neutrophil peptide alpha-defensins (HNPs) and human beta-defensins (HBDs) are small well-characterized peptides with broad antimicrobial activities and a diversity of innate immune functions. Although the interactions of defensins with bacteria and their membranes have been well characterized, the interactions of defensins with bacterial adhesins have not. Here we determine if HNPs and HBDs bind to the immobilized adhesins of Porphyromonas gingivalis strain 381, recombinant hemagglutinin B (rHagB) and recombinant fimbrillin A (rFimA), by surface plasmon resonance spectroscopy. Association of HNPs and HBDs with rHagB or rFimA was dose dependent and defensin specific. HBD3, HNP-2, and HNP-1 bound more readily to immobilized rHagB than HBD2 and HBD1 did. HNP-2, HNP-1, and HBD3 bound more readily to immobilized rFimA than HBD1 and HBD2 did. Binding of defensins to adhesins may serve to prevent microbial adherence to tissues, attenuate proinflammatory cytokine responses, and facilitate delivery of bound antigen to antigen-presenting cells with defensin receptors.

Porphyromonas gingivalis and the autophagic pathway: an innate immune interaction?

Autophagy is a mechanism used to maintain several intracellular functions essential to eukaryotic cells. Recently, a role for autophagy in innate and adaptive immunity has also been established including the elimination of invading bacteria. Although some intracellular pathogens are killed by autophagy, several others subvert autophagy to the pathogen's benefit for survival and replication. Porphyromonas gingivalis, an important periodontal pathogen, has been shown to stimulate autophagy in endothelial cells and to use the autophagic pathway to its advantage. In human coronary artery endothelial cells (HCAEC), P. gingivalis localizes within autophagosomes. After intracellular uptake, P. gingivalis transits from early autophagosomes to late autophagosomes and prevents the formation of autolysosomes, either by delaying the autophagosome-lysosome fusion or by redirecting the normal autophagic trafficking. In addition, P. gingivalis was also found to stimulate autophagy in human aortic endothelial cells (HAEC) since co-localization of LC3-II, an autophagosome marker, with P. gingivalis was observed. The trafficking of P. gingivalis into the autophagic pathway appears to be dependent upon the host cell type. Survival of P. gingivalis through the subversion of the host autophagic pathway can be considered a bacterial strategy to evade the innate immune system and persist in the host.

Colonization of maternal and fetal tissues by Porphyromonas gingivalis is strain-dependent in a rodent animal model.

OBJECTIVE: The objective of this study was to develop a rodent model of Porphyromonas gingivalis infection during pregnancy. STUDY DESIGN: Sprague Dawley rats were infected intravenously with 10(5), 10(7), or 10(9) CFU per dam of P gingivalis strain W83, ATCC 33277, or A7436 at gestational day 14 and necropsied at gestational day 18. Maternal organs were cultured to assess the spread of the infection. Six fetal units (placenta, amniotic fluid, membranes, and fetus) per dam were cultured; additional fetal units were examined by histopathology. Polymerase chain reaction was performed on placentas. RESULTS: Colonization rates were dependent on the strain of P gingivalis used and the infection dose. At an infection dose of 10(9) CFU/dam, P gingivalis W83, ATCC 33277, or A7436 was detected in 33%, 83%, or 100% of placentas, respectively. Epithelial hyperplasia, cellular necrosis, and inflammatory infiltrate were observed in infected placental tissues. CONCLUSION: This study demonstrated that P gingivalis can invade both maternal and fetal tissues, resulting in chorioamnionitis and placentitis.

The Porphyromonas gingivalis clpB gene is involved in cellular invasion in vitro and virulence in vivo.

ClpB, a component of stress response in microorganisms, serves as a chaperone, preventing protein aggregation and assisting in the refolding of denatured proteins. A clpB mutant of Porphyromonas gingivalis W83 demonstrated increased sensitivity to heat stress, but not to hydrogen peroxide and extreme pHs. In KB cells, human coronary artery endothelial (HCAE) cells and gingival epithelial cells, the clpB mutant exhibited significantly decreased invasion suggesting that the ClpB protein is involved in cellular invasion. Transmission electron microscopic analysis showed that the clpB mutant was more susceptible to intracellular killing than the wild-type strain in HCAE cells. The global genetic profile of the clpB mutant showed that 136 genes belonging to several different cellular function groups were differentially regulated, suggesting that ClpB is ultimately involved in the expression of multiple P. gingivalis genes. A competition assay in which a mixture of wild-type W83 and the clpB mutant were injected into mice demonstrated that the clpB mutant did not survive as well as the wild type. Additionally, mice treated with the clpB mutant alone survived significantly better than those treated with the wild-type strain. Collectively, these data suggest that ClpB, either directly or indirectly, plays an important role in P. gingivalis virulence.

In vitro activity of amoxicillin, clindamycin, doxycycline, metronidazole, and moxifloxacin against oral Actinomyces.

Actinomyces spp have been increasingly associated with endodontic infections. However, the antimicrobial susceptibility of this genus has not been studied extensively. The objective of this study was to determine the susceptibility of oral isolates of Actinomyces naeslundii, Actinomyces gerencseriae, Actinomyces israelii, Actinomyces viscosus, and Actinomyces odontolyticus to amoxicillin, clindamycin, doxycycline, metronidazole, and moxifloxacin using in vitro assays. The minimum inhibitory concentration (MIC) of each bacterial isolate was determined by using E-test strips (AB Biodisk, Solna, Sweden). The MIC(90) was 0.19 microg/mL for amoxicillin, 0.25 microg/mL for doxycycline, 0.50 microg/mL for moxifloxacin, and 1.00 microg/mL for clindamycin. However, metronidazole was not active against any of the Actinomyces spp tested (MIC(90)>256 microg/mL).

Genome Sequence of Porphyromonas gingivalis Strain 381.

Porphyromonas gingivalis is associated with both oral and systemic diseases. Strain-specific P. gingivalis invasion phenotypes do not reliably predict disease presentation during in vivo studies. Here, we present the genome sequence of 381, a common laboratory strain, with a single contig of 2,378,872 bp and a G+C content of 48.36%.

Genome Sequence of Porphyromonas gingivalis Strain A7A1-28.

Porphyromonas gingivalis is an oral opportunistic pathogen. Sequenced P. gingivalis laboratory strains display limited diversity in antigens that modulate host responses. Here, we present the genome sequence of A7A1-28, a strain possessing atypical fimbrillin and capsule types, with a single contig of 2,249,024 bp and a G+C content of 48.58%.

Antimicrobial efficacy of medicated root canal filling materials.

Bacteria remaining within the root canal system are a significant factor in endodontic failures. The aim of this in vitro study was to evaluate the antimicrobial efficacy of commercially available gutta-percha formulations against common endodontic pathogens. Standard, tetracycline, or iodoform (also called medicated gutta-percha; MGP) containing gutta-percha were tested against Actinomyces israelii, A. naeslundii, Enterococcus faecalis, and Fusobacterium nucleatum using the agar diffusion method. Resilon points and tetracycline disks/E-tests were used as negative and positive controls, respectively. Resilon did not display antimicrobial properties. Standard gutta-percha and MGP inhibited F. nucleatum and A. naeslundii, with MGP also inhibiting A. israelii. Tetracycline containing gutta-percha was found to inhibit growth from all four bacterial species tested. The results of this study suggest that the use of tetracycline containing gutta-percha as an obturation material may be useful as a complement to the cleaning and disinfection phase of root canal procedures.

Dentinal tubule disinfection using three calcium hydroxide formulations.

The objective of this study was to determine the antibacterial efficacy of three calcium hydroxide (CH) formulations using an in vitro model of Enterococcus faecalis dentinal tubule infection. CH mixed with water (CH), CH mixed with iodine-potassium iodide (CH+IKI), and CH mixed with iodoform and silicone oil (Metapex) were tested. Human cylindrical dentin specimens infected with E. faecalis were filled with disinfectants and incubated for 1 week. Dentin powder samples collected with ISO 018 burs showed a statistically significant reduction in E. faecalis for all three experimental groups in comparison with untreated control specimens. Statistically significant differences were also found between the three experimental groups. Metapex was the most effective dentinal tubule disinfectant, followed by CH+IKI and then CH. Similar results were observed at greater dentin tubule depths (ISO 021 burs) with the exception that intracanal treatment with CH resulted in significantly higher numbers of E. faecalis in comparison with untreated control specimens.

Genome Sequence of Porphyromonas gingivalis Strain A7436.

Porphyromonas gingivalis is strongly associated with periodontitis. P. gingivalis strain trafficking and tissue homing differ widely, even among presumptive closely related strains, such as W83 and A7436. Here, we present the genome sequence of A7436 with a single contig of 2,367,029 bp and a G+C content of 48.33%.

Genome Sequence of Porphyromonas gingivalis Strain AJW4.

Porphyromonas gingivalis is associated with oral and systemic diseases. Strain-specific P. gingivalis invasion phenotypes have been correlated with disease presentation in infected laboratory animals. Here, we present the genome sequence of AJW4, a minimally invasive strain, with a single contig of 2,372,492 bp and a G+C content of 48.27%.

Deletion of lipoprotein PG0717 in Porphyromonas gingivalis W83 reduces gingipain activity and alters trafficking in and response by host cells.

P. gingivalis (Pg), a causative agent of chronic generalized periodontitis, has been implicated in promoting cardiovascular disease. Expression of lipoprotein gene PG0717 of Pg strain W83 was found to be transiently upregulated during invasion of human coronary artery endothelial cells (HCAEC), suggesting this protein may be involved in virulence. We characterized the virulence phenotype of a PG0717 deletion mutant of pg W83. There were no differences in the ability of W83Δ717 to adhere and invade HCAEC. However, the increased proportion of internalized W83 at 24 hours post-inoculation was not observed with W83∆717. Deletion of PG0717 also impaired the ability of W83 to usurp the autophagic pathway in HCAEC and to induce autophagy in Saos-2 sarcoma cells. HCAEC infected with W83Δ717 also secreted significantly greater amounts of MCP-1, IL-8, IL-6, GM-CSF, and soluble ICAM-1, VCAM-1, and E-selectin when compared to W83. Further characterization of W83Δ717 revealed that neither capsule nor lipid A structure was affected by deletion of PG0717. Interestingly, the activity of both arginine (Rgp) and lysine (Kgp) gingipains was reduced in whole-cell extracts and culture supernatant of W83Δ717. RT-PCR revealed a corresponding decrease in transcription of rgpB but not rgpA or kgp. Quantitative proteome studies of the two strains revealed that both RgpA and RgpB, along with putative virulence factors peptidylarginine deiminase and Clp protease were significantly decreased in the W83Δ717. Our results suggest that PG0717 has pleiotropic effects on W83 that affect microbial induced manipulation of host responses important for microbial clearance and infection control.

Porphyromonas gingivalis strain specific interactions with human coronary artery endothelial cells: a comparative study.

Both epidemiologic and experimental findings suggest that infection with Porphyromonas gingivalis exacerbates progression of atherosclerosis. As P. gingivalis exhibits significant strain variation, it is reasonable that different strains possess different capabilities and/or mechanisms by which they promote atherosclerosis. Using P. gingivalis strains that have been previously evaluated in the ApoE null atherosclerosis model, we assessed the ability of W83, A7436, 381, and 33277 to adhere, invade, and persist in human coronary artery endothelial (HCAE) cells. W83 and 381 displayed an equivalent ability to adhere to HCAE cells, which was significantly greater than both A7436 and 33277 (P<0.01). W83, 381, and 33277 were more invasive than A7436 (P<0.0001). However, only W83 and A7436 were able to remain viable up to 48 hours in HCAE cell cultures, whereas 381 was cleared by 48 hours and 33277 was cleared by 24 hours. These differences in persistence were in part due to strain specific differences in intracellular trafficking. Both W83 and 381 trafficked through the autophagic pathway, but not A7436 or 33277. Internalized 381 was the only strain that was dependent upon the autophagic pathway for its survival. Finally, we assessed the efficacy of these strains to activate HCAE cells as defined by production of IL-6, IL-8, IL-12p40, MCP-1, RANTES, TNF-α, and soluble adhesion molecules (sICAM-1, sVCAM-1, and sE-selectin). Only moderate inflammation was observed in cells infected with either W83 or A7436, whereas cells infected with 381 exhibited the most profound inflammation, followed by cells infected with 33277. These results demonstrate that virulence mechanisms among different P. gingivalis strains are varied and that pathogenic mechanisms identified for one strain are not necessarily applicable to other strains.

Defensins attenuate cytokine responses yet enhance antibody responses to Porphyromonas gingivalis adhesins in mice.

AIM: Our aim is to assess the ability of human neutrophil peptide alpha-defensins (HNPs) and human beta-defensins (HBDs) to attenuate proinflammatory cytokine responses and enhance antibody responses to recombinant hemagglutinin B (rHagB) or recombinant fimbrillin A (rFimA) from Porphyromonas gingivalis 381 in mice. MATERIALS & METHODS: In the first study, C57BL/6 mice were given 10 microg rHagB or rFimA without and with 1 microg HNP1, HNP2, HBD1, HBD2 or HBD3. At 24 h, mice were euthanized and cytokine concentrations were determined in nasal wash fluid (NWF), bronchoalveolar lavage fluids, saliva and serum. In the second study, C57BL/6 mice were given 10 microg rHagB or rFimA without and with 1 microg HNPs or HBDs similarly on days 0, 7 and 14. At 21 days, mice were euthanized and rHagB- and rFimA-specific antibody responses were determined in NWF, bronchoalveolar lavage fluids, saliva and serum. RESULTS: Mice given rHagB + HNP2, rHagB + HBD1 and rHagB + HBD3 produced significantly lower (p < 0.05) IL-6 responses than mice given rHagB alone. Mice given rHagB + HNP1, rHagB + HNP2, rHagB + HBD1 and rHagB + HBD3 produced significantly lower (p < 0.05) keratinocyte-derived chemokine responses than mice given rHagB alone. Mice given rFimA produced very low levels of IL-6 and only moderate levels of keratinocyte-derived chemokine in NWF that were not attenuated by prior incubation of rFimA with any defensin. Mice given rHagB + HNP1 produced a significantly higher (p < 0.05) serum IgG antibody response than mice given rHagB alone and mice given rFimA + HNP2 produced a higher, but not significant, antibody response. CONCLUSION: The ability of HNPs and HBDs to attenuate proinflammatory cytokine responses in murine NWF and enhance IgG antibody responses in serum was dependent upon both the defensin and antigen of P. gingivalis.

A member of the peptidase M48 superfamily of Porphyromonas gingivalis is associated with virulence in vitro and in vivo.

BACKGROUND: In vivo-induced antigen technology was previously used to identify 115 genes induced in Porphyromonas gingivalis W83 during human infection. One of these, PG2197, a conserved hypothetical protein which has homology to a Zn-dependent protease, was examined with respect to a role in disease. DESIGN: The expression of PG2197 in human periodontitis patients was investigated, but as there is increasing evidence of a direct relationship between P. gingivalis and cardiovascular disease, a mutation was constructed in this gene to also determine its role in adherence, invasion, and persistence within human coronary artery endothelial cells (HCAEC) and neutrophil killing susceptibility. RESULTS: Plaque samples from 20 periodontitis patients were analyzed by real-time PCR, revealing that PG2197 was expressed in 60.0% of diseased sites compared to 15.8% of healthy sites, even though P. gingivalis was detected in equal numbers from both sites. The expression of this gene was also found to be up-regulated in microarrays at 5 and 30 min of invasion of HCAEC. Interestingly, a PG2197 mutant displayed increased adherence, invasion, and persistence within HCAEC when compared to the wild-type strain. CONCLUSION: This gene appears to be important for the virulence of P. gingivalis, both in vivo and in vitro.

Porphyromonas gingivalis htrA is involved in cellular invasion and in vivo survival.

HtrA is a heat-stress protein that functions both as a chaperone and as a serine protease. HtrA has been shown in several organisms to be involved in responses to stressful environmental conditions and involvement of HtrA in virulence has been reported in pathogenic species. A Porphyromonas gingivalis htrA mutant demonstrated no significant difference to the W83 parent strain when subjected to high temperature and pH values from 3 to 11. However, the htrA mutant showed increased sensitivity to H(2)O(2). Cell invasion assays indicated that the total interaction (adherence) with KB cells, human coronary artery endothelial cells and gingival epithelial cells (GEC) was the same for both the wild-type and the htrA mutant. However, the htrA mutant showed increased invasion in KB cells and GEC. Microarray experiments indicated that a total of 253 genes were differentially regulated in the htrA mutant, including a group of stress-related genes, which might be responsible for the observed decreased resistance to H(2)O(2). In animal experiments, a competition assay showed that the htrA mutant did not survive as well as the wild-type. In another in vivo assay, fewer mice infected with the htrA mutant died than mice infected with W83, suggesting that the htrA gene is virulence-related. These data indicate that the htrA gene in P. gingivalis does not relate to stress conditions such as high temperature and pH, but rather to H(2)O(2) stress. The htrA gene also appears to be important for virulence and survival in in vivo animal models.

Human beta-defensin 3 binds to hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis, and attenuates a pro-inflammatory cytokine response.

Regulatory mechanisms in mucosal secretions and tissues recognize antigens and attenuate pro-inflammatory cytokine responses. Here, we asked whether human beta-defensin 3 (HBD3) serves as an upstream suppressor of cytokine signaling that binds and attenuates pro-inflammatory cytokine responses to recombinant hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis strain 381. We found that HBD3 binds to immobilized rHagB and produces a significantly higher resonance unit signal in surface plasmon resonance spectroscopic analysis, than HBD2 and HBD1 that are used as control defensins. Furthermore, we found that HBD3 significantly attenuates (P<0.05) the interleukin (IL)-6, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and tumor-necrosis factor-alpha (TNF-alpha) responses induced by rHagB in human myeloid dendritic cell culture supernatants and the extracellular signal-regulated kinases (ERK 1/2) response in human myeloid dendritic cell lysates. Thus, HBD3 binds rHagB and this interaction may be an important initial step to attenuate a pro-inflammatory cytokine response and an ERK 1/2 response.