Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.

Oral infectious bacteria in dental plaque and saliva as risk factors in patients with esophageal cancer.

BACKGROUND: High levels of periodontopathic bacteria as well as Streptococcus anginosus were detected in cancer tissue from patients with esophageal cancer. An association between oral infectious bacteria and esophageal cancer has been reported. METHODS: Characteristics of the oral microbiota and periodontal conditions were studied as clinicopathologic factors in patients with esophageal cancer. The study included 61 patients with esophageal cancer and 62 matched individuals without any cancers. Samples of subgingival dental plaque and unstimulated saliva were collected to evaluate the prevalence and abundance of the following oral bacteria using a real-time polymerase chain reaction assay: Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema denticola, and S. anginosus. RESULTS: In the cancer group, the prevalence of all bacteria, with the exception of F. nucleatum, in dental plaque; the prevalence of A. actinomycetemcomitans in saliva; the abundance of all bacteria, with the exception of F. nucleatum and P. intermedia, in dental plaque; and the abundance of A. actinomycetemcomitans and S. anginosus in saliva were significantly higher. Furthermore, a logistic regression analysis suggested that the prevalence of T. forsythia and S. anginosus in dental plaque and of A. actinomycetemcomitans in saliva, as well as a drinking habit, were associated with a high risk of esophageal cancer, with a high odds ratio. CONCLUSIONS: The current findings have potential implications for the early diagnosis of esophageal cancer.

Prevotella intermedia produces two proteins homologous to Porphyromonas gingivalis HmuY but with different heme coordination mode.

As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different from that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, PinO and PinA could use one methionine residue to fulfill this function, with potential support of additional methionine residue/s. The P. intermedia proteins sequestered heme only from the host albumin-heme complex under reducing conditions. Our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme coordination modes. The newer data presented in this manuscript on HmuY homologs produced by P. intermedia sheds more light on the novel mechanism of heme uptake, could be helpful in discovering their biological function, and in developing novel therapeutic approaches.

Subgingival Microbial Changes During the First 3 Months of Fixed Appliance Treatment in Female Adult Patients.

Although periodontal diseases during fixed appliance treatment are a common issue, few studies have focused on the clinical and microbial factors associated with orthodontic appliances. Hence, we investigated changes in the subgingival microbial community and their association with periodontal changes at the early stage of fixed appliance treatment. Subgingival plaques from ten female patients with fixed appliances were obtained at three time points: before, 1 month and 3 months after the placement of the brackets (T0, T1 and T2). The 16S rRNA gene sequencing was used to analyze the microbial community of the subgingival plaque. The Plaque Index (PI) and Gingival Bleeding Index (GBI) were also recorded. The GBI significantly increased at T2, and the PI showed a temporary increase without a significant difference. The alpha diversity indices were stable. However, the beta diversity was significantly higher at T2 compared to T0 and T1. The relative abundance of core microbiomes at the genus level was relatively stable. Four periodontal pathogens at the species level, including Prevotella intermedia (Pi), Campylobacer rectus (Cr), Fusobacterium nucleatum (Fn), and Treponema denticola (Td), increased without significant differences. The subgingival microbial community affected by fixed appliance treatment might cause transient mild gingival inflammation.

Quorum sensing molecules regulate epithelial cytokine response and biofilm-related virulence of three Prevotella species.

Quorum sensing (QS) signaling regulates the motility, adhesion, and biofilm formation of bacteria, and at the same time activates immune response in eukaryotic organisms. We recently demonstrated that the QS molecule, dihydroxy-2, 3-pentanedione (DPD), and its analogs significantly inhibit estradiol-regulated virulence of Prevotella aurantiaca, one of the four species in the Prevotella intermedia group. Here, we examined the combined effects of estradiol and QS signaling on 1) cytokine response of human gingival keratinocytes (HMK) against whole cell extract (WCE) of P. intermedia, Prevotella nigrescens, and Prevotella pallens, and 2) biofilm formation of these three Prevotella species. All experiments were performed in the presence or absence of estradiol, and with different QS molecules: DPD and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD). Concentrations of interleukin (IL)-1ß, -6, and -8 were determined by the Luminex multiplex immunoassay, biofilm mass was quantitatively evaluated by measuring protein concentration via the Bradford method, and the microtopography of biofilms was assessed by scanning electron microscopy (SEM) imaging. Concentrations of IL-6 and IL-8 were elevated when HMK cells were incubated with estradiol and WCE of P. intermedia and P. nigrescens, but decreased when incubated with estradiol and WCE of P. pallens. Butyl-DPD neutralized the estradiol- and WCE-induced regulation of HMK interleukin expression and, at the same time, inhibited the biofilm formation of P. intermedia and P. nigrescens. SEM micrographs revealed a decrease in biofilm mass after application of butyl-DPD, which was most detectable among the P. intermedia ATCC 25611 and P. nigrescens ATCC 33563 and AHN 8293 strains. In conclusion, butyl-DPD analog is able to neutralize the WCE-induced epithelial cytokine response and, at the same time, to inhibit the biofilm formation of P. intermedia and P. nigrescens.

A chinese case of prevotella intermedia and streptococcus constellatus intracranial mixed infection.

Streptococcal Species is increasingly recognized as a potentially preventable emerging infection in human's brain with high prevalence around the world. Streptococcus constellatus is one of the most common pathogens. Meanwhile, anaerobic bacteria are the rare causes for intracranial infection. To date, intracranial mixed infection caused by Prevotella intermedia and Streptococcus constellatus has not been reported. We reported a Chinese case to raise the global awareness of severity of the intracranial mixed infection. Here, we illustrated the epidemiological risk factors, clinical manifestations and outcomes of the patient. For patients who suffer from exacerbated brain infection with fetid cerebrospinal fluid, early repeated imaging is urgently needed and empiric antibiotic therapy should consider anaerobic and aerobic bacteria in these situations.

Aggravation of collagen-induced arthritis by orally administered Porphyromonas gingivalis through modulation of the gut microbiota and gut immune system.

Porhyromonas gingivalis, a causative bacterium of periodontitis, is implicated in the etiology of rheumatoid arthritis (RA), mainly because of expressing peptidyl arginine deiminase (PAD) that generates RA-related autoantigens. However, compared with other periodontopathic bacteria, the precise role of P. gingivalis in RA is largely unknown. We found that orally administered P. gingivalis changed the gut microbiome with concomitant elevation of serum endotoxin and inflammatory markers, and impairment of the gut barrier function. Based on findings showing a relationship between gut microbiota and RA, we investigated whether the change of gut microbiota induced by P. gingivalis and Prevotella intermedia, another periodontopathic bacterium without PAD, is associated with collagen-induced arthritis (CIA). DBA/1J mice were orally administered with or without bacteria followed by induction of CIA. P. gingivalis, but not P. intermedia, administration significantly aggravated arthritis with increased interleukin-17 levels in sera and culture supernatants, increased Th17 cell proportions among mesenteric lymphocytes, and a significant change in the gut microbiome. However, P. gingivalis administration did not elevate the level of anti-citrullinated protein antibody. These results suggest a unique role of P. gingivalis in the link between periodontitis and RA by affecting the gut immune system and the gut microbiota composition.

Periodontal Tissue Regeneration Using Syngeneic Adipose-Derived Stromal Cells in a Mouse Model.

Current treatment of periodontitis is still associated with a high degree of variability in clinical outcomes. Recent advances in regenerative medicine by mesenchymal cells, including adipose stromal cells (ASC) have paved the way to improved periodontal regeneration (PD) but little is known about the biological processes involved. Here, we aimed to use syngeneic ASCs for periodontal regeneration in a new, relevant, bacteria-induced periodontitis model in mice. Periodontal defects were induced in female C57BL6/J mice by oral gavage with periodontal pathogens. We grafted 2 × 105 syngeneic mouse ASCs expressing green fluorescent protein (GFP) (GFP+/ASC) within a collagen vehicle in the lingual part of the first lower molar periodontium (experimental) while carrier alone was implanted in the contralateral side (control). Animals were sacrificed 0, 1, 6, and 12 weeks after treatment by GFP+/ASC or vehicle graft, and microscopic examination, immunofluorescence, and innovative bio-informatics histomorphometry methods were used to reveal deep periodontium changes. From 1 to 6 weeks after surgery, GFP+ cells were identified in the periodontal ligament (PDL), in experimental sites only. After 12 weeks, cementum regeneration, the organization of PDL fibers, the number of PD vessels, and bone morphogenetic protein-2 and osteopontin expression were greater in experimental sites than in controls. Specific stromal cell subsets were recruited in the newly formed tissue in ASC-implanted periodontium only. These data suggest that ASC grafting in diseased deep periodontium, relevant to human pathology, induces a significant improvement of the PDL microenvironment, leading to a recovery of tooth-supporting tissue homeostasis. Stem Cells Translational Medicine 2017;6:656-665.

Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg2+ and Ca2+ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs.

[The characteristics of microbiota of periodontal recesses in smoking patients with chronic generalized periodontitis].

The clinical examination of 36 tobacco smokers with chronic generalized periodontitis of light, average and severe degree was carried out. The examination established poor hygienic condition of oral cavity, less expressed inflammatory reaction of tissues of periodont and predominance of occurrences of destruction of alveolar portion of bone as compared with the group of 59 non-smoking patients with chronic generalized periodontitis of light, average and severe degree. The study demonstrated higher rate of detection of T. forsythia in smokers as compared with non-smoking patients at all stages of of development of chronic generalized periodontitis. Under light stage of chronic generalized periodontitis increasing of rate of detection of T. forsythia more than twice was registered. P.gigngivalis and P.intermedia were detected in smoking patients with light stage of chronic generalized periodontitis either in the same values or more rarely as compared with non-smokers. In the group of smokers with average stage of chronic generalized periodontitis increasing of rate of occurrence of association of T. forsythia-P. gigngivalis-P. intermedia occurred more than five times in comparison with non-smokers. The obtained results indicate on relationship between alterations of microbiota and aggressive development of chronic generalized periodontitis in smoking patients and on development in periodontal recesses of smokers of favorable conditions for growth of T. forsythia. The presence of T. forsythia is a significant factor of development of destructive processes in tissues of periodont.

Human dental stem cells suppress PMN activity after infection with the periodontopathogens Prevotella intermedia and Tannerella forsythia.

Periodontitis is characterized by inflammation associated with the colonization of different oral pathogens. We here aimed to investigate how bacteria and host cells shape their environment in order to limit inflammation and tissue damage in the presence of the pathogen. Human dental follicle stem cells (hDFSCs) were co-cultured with gram-negative P. intermedia and T. forsythia and were quantified for adherence and internalization as well as migration and interleukin secretion. To delineate hDFSC-specific effects, gingival epithelial cells (Ca9-22) were used as controls. Direct effects of hDFSCs on neutrophils (PMN) after interaction with bacteria were analyzed via chemotactic attraction, phagocytic activity and NET formation. We show that P. intermedia and T. forsythia adhere to and internalize into hDFSCs. This infection decreased the migratory capacity of the hDFSCs by 50%, did not disturb hDFSC differentiation potential and provoked an increase in IL-6 and IL-8 secretion while leaving IL-10 levels unaltered. These environmental modulations correlated with reduced PMN chemotaxis, phagocytic activity and NET formation. Our results suggest that P. intermedia and T. forsythia infected hDFSCs maintain their stem cell functionality, reduce PMN-induced tissue and bone degradation via suppression of PMN-activity, and at the same time allow for the survival of the oral pathogens.

In Vitro Evaluation of Planktonic Growth on Experimental Cement-Retained Titanium Surfaces.

BACKGROUND The purpose of this study was to compare the effects of selected cements, or their combination with titanium, on the growth of two periodontopathic bacteria: Prevotella intermedia (Pi) and Fusobacterium nucleatum (Fn). MATERIAL AND METHODS This study was comprised of several experimental groups: 1) Dental luting cements (glass ionomer cement, methacrylate-based resin cement, zinc-oxide eugenol cement, eugenol-free zinc oxide cement; 2) titanium discs; and 3) titanium combination cement discs. The disks were submerged in bacterial suspensions of either Fn or Pi. Planktonic bacterial growth within the test media was measured by determining the optical density of the cultures (OD600). Mean and standard deviations were calculated for planktonic growth from three separate experiments. RESULTS Intergroup comparison of all experimental groups revealed increased growth of Pi associated with cement-titanium specimens in comparison with cement specimens. Regarding the comparison of all groups for Fn, there was an increased amount of bacterial growth in cement-titanium specimens although the increase was not statistically significant. CONCLUSIONS The combination of cement with titanium may exacerbate the bacterial growth capacity of Pi and Fn in contrast to their sole effect.

The Comparative Evaluation of the Antimicrobial Effect of Propolis with Chlorhexidine against Oral Pathogens: An In Vitro Study.

This study aimed to compare the antimicrobial effectiveness of ethanolic extract of propolis (EEP) to chlorhexidine gluconate (CHX) on planktonic Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Lactobacillus salivarius subsp. salivarius, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Staphylococcus aureus, Enterococcus faecalis, Actinomyces israelii, Candida albicans, and their single-species biofilms by agar dilution and broth microdilution test methods. Both agents inhibited the growth of all planktonic species. On the other hand, CHX exhibited lower minimum bactericidal concentrations than EEP against biofilms of A. actinomycetemcomitans, S. aureus, and E. faecalis whereas EEP yielded a better result against Lactobacilli and P. intermedia. The bactericidal and fungicidal concentrations of both agents were found to be equal against biofilms of Streptecocci, P. gingivalis, A. israelii, and C. albicans. The results of this study revealed that propolis was more effective in inhibiting Gram-positive bacteria than the Gram-negative bacteria in their planktonic state and it was suggested that EEP could be as effective as CHX on oral microorganisms in their biofilm state.

Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Treponema denticola / Prevotella intermedia Co-Infection Are Associated with Severe Periodontitis in a Thai Population.

Periodontitis is a polymicrobial infection of tooth-supporting tissues. This cross-sectional study aimed to examine the associations between five target species and severe periodontitis in a Thai population. Using the CDC/AAP case definition, individuals diagnosed with no/mild and severe periodontitis were included. Quantitative analyses of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), and Prevotella intermedia (Pi) in subgingival plaque were performed using real-time polymerase chain reaction. The association between target species and severe periodontitis was examined using logistic regression analysis. The study subjects comprised 479 individuals with no/mild periodontitis and 883 with severe periodontitis. Bacterial prevalence and quantity were higher in subjects with severe periodontitis than in those with no/mild disease. In the fully adjusted model, all species except Tf showed a dose-dependent relationship with periodontitis. The mere presence of Pg, even in low amount, was significantly associated with severe periodontitis, while the amount of Aa, Td, and Pi had to reach the critical thresholds to be significantly associated with disease. Compared to individuals with low levels of both Td and Pi, high colonization by either Td or Pi alone significantly increased the odds of having severe periodontitis by 2.5 (95%CI 1.7-3.5) folds. The odds ratio was further increased to 14.8 (95%CI 9.2-23.8) in individuals who were highly colonized by both species. Moreover, the presence of Pg and high colonization by Aa were independently associated with severe periodontitis with odds ratios of 5.6 (95%CI 3.4-9.1) and 2.2 (95%CI 1.5-3.3), respectively. Our findings suggest that the presence of Pg and high colonization by Aa, Td, and Pi play an important role in severe periodontitis in this study population. We also demonstrate for the first time that individuals co-infected with Td and Pi were more likely to have periodontitis than were those infected with a single pathogen.

Comparative genome analysis of Prevotella intermedia strain isolated from infected root canal reveals features related to pathogenicity and adaptation.

BACKGROUND: Many species of the genus Prevotella are pathogens that cause oral diseases. Prevotella intermedia is known to cause various oral disorders e.g. periodontal disease, periapical periodontitis and noma as well as colonize in the respiratory tract and be associated with cystic fibrosis and chronic bronchitis. It is of clinical significance to identify the main drive of its various adaptation and pathogenicity. In order to explore the intra-species genetic differences among strains of Prevotella intermedia of different niches, we isolated a strain Prevotella intermedia ZT from the infected root canal of a Chinese patient with periapical periodontitis and gained a draft genome sequence. We annotated the genome and compared it with the genomes of other taxa in the genus Prevotella. RESULTS: The raw data set, consisting of approximately 65X-coverage reads, was trimmed and assembled into contigs from which 2165 ORFs were predicted. The comparison of the Prevotella intermedia ZT genome sequence with the published genome sequence of Prevotella intermedia 17 and Prevotella intermedia ATCC25611 revealed that ~14% of the genes were strain-specific. The Preveotella intermedia strains share a set of conserved genes contributing to its adaptation and pathogenic and possess strain-specific genes especially those involved in adhesion and secreting bacteriocin. The Prevotella intermedia ZT shares similar gene content with other taxa of genus Prevotella. The genomes of the genus Prevotella is highly dynamic with relative conserved parts: on average, about half of the genes in one Prevotella genome were not included in another genome of the different Prevotella species. The degree of conservation varied with different pathways: the ability of amino acid biosynthesis varied greatly with species but the pathway of cell wall components biosynthesis were nearly constant. Phylogenetic tree shows that the taxa from different niches are scarcely distributed among clades. CONCLUSIONS: Prevotella intermedia ZT belongs to a genus marked with highly dynamic genomes. The specific genes of Prevotella intermedia indicate that adhesion, competing with surrounding microbes and horizontal gene transfer are the main drive of the evolution of Prevotella intermedia.

Non-conventional therapeutics for oral infections.

As our knowledge of host-microbial interactions within the oral cavity increases, future treatments are likely to be more targeted. For example, efforts to target a single species or key virulence factors that they produce, while maintaining the natural balance of the resident oral microbiota that acts to modulate the host immune response would be an advantage. Targeted approaches may be directed at the black-pigmented anaerobes, Porphyromonas gingivalis and Prevotella intermedia, associated with periodontitis. Such pigments provide an opportunity for targeted phototherapy with high-intensity monochromatic light. Functional inhibition approaches, including the use of enzyme inhibitors, are also being explored to control periodontitis. More general disruption of dental plaque through the use of enzymes and detergents, alone and in combination, shows much promise. The use of probiotics and prebiotics to improve gastrointestinal health has now led to an interest in using these approaches to control oral disease. More recently the potential of antimicrobial peptides and nanotechnology, through the application of nanoparticles with biocidal, anti-adhesive and delivery capabilities, has been explored. The aim of this review is to consider the current status as regards non-conventional treatment approaches for oral infections with particular emphasis on the plaque-related diseases.

Detection of periodontal pathogens in the patients with aortic aneurysm.

BACKGROUND: The occurrence and development of aortic aneurysm (AA) are associated with infection. Some researchers have detected the DNA of periodontal pathogens in AA samples in certain populations. However, it has not been done in Chinese population. The objective of this study was to evaluate the prevalence of periodontal pathogens in oral tissue samples and aneurysm samples of AA patients. METHODS: Eighty-nine subjects with AA and 59 subjects without AA were examined. Periodontal clinical parameters were evaluated. Unstimulated saliva and subgingival plaque samples were collected from all subjects. Twenty-six dissected AA samples were obtained. Evidence of eight periodontal pathogens including Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa), Prevotella intermedia (Pi), Tannerella forsythensis (Tf), Treponema denticola (Td), Campylobacter rectus (Cr), Fusobacterium nucleatum (Fn), and Prevotella nigrescens (Pn) was ascertained in all samples by 16S rRNA-based polymerase chain reaction (PCR) assay. RESULTS: The periodontal indexes including plaque index (PLI), probing depth (PD), bleeding index (BI), and clinical attachment loss (CAL), of the six Ramfjord index teeth were significantly higher in the AA group than those in the control group (P < 0.01). Eight periodontal pathogens in subgingival plaque samples were more frequently detected in the AA group than in control group. The difference in prevalence between the groups was significant for six (out of eight) periodontal pathogens assayed (Pg, Pi, Fn, Pn, Tf, and Td, P < 0.01). Additionally, all eight periodontal pathogens were more frequently detected in saliva samples of the AA group than in those of the control group, again with six (out of eight) (Pg, Pi, Fn, Cr, Tf, and Td) displaying significant differences in prevalence between the two groups (P < 0.01). Out of 26 aneurysm samples examined, Pg, Pi, Fn, Cr and Tf were detected in 6 (23.1%), 2 (7.7%), 3 (11.5%), 1 (3.8%), 2 (7.7%), respectively, and Aa, Pn, and Td were not detected in dissected aneurysm samples. CONCLUSION: Results of this study suggested that periodontal infection is associated with the occurrence of AA.

Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

Prevotella intermedia induces severe bacteremic pneumococcal pneumonia in mice with upregulated platelet-activating factor receptor expression.

Streptococcus pneumoniae is the leading cause of respiratory infection worldwide. Although oral hygiene has been considered a risk factor for developing pneumonia, the relationship between oral bacteria and pneumococcal infection is unknown. In this study, we examined the synergic effects of Prevotella intermedia, a major periodontopathic bacterium, on pneumococcal pneumonia. The synergic effects of the supernatant of P. intermedia (PiSup) on pneumococcal pneumonia were investigated in mice, and the stimulation of pneumococcal adhesion to human alveolar (A549) cells by PiSup was assessed. The effects of PiSup on platelet-activating factor receptor (PAFR) transcript levels in vitro and in vivo were analyzed by quantitative real-time PCR, and the differences between the effects of pneumococcal infection induced by various periodontopathic bacterial species were verified in mice. Mice inoculated with S. pneumoniae plus PiSup exhibited a significantly lower survival rate, higher bacterial loads in the lungs, spleen, and blood, and higher inflammatory cytokine levels in the bronchoalveolar lavage fluid (macrophage inflammatory protein 2 and tumor necrosis factor alpha) than those infected without PiSup. In A549 cells, PiSup increased pneumococcal adhesion and PAFR transcript levels. PiSup also increased lung PAFR transcript levels in mice. Similar effects were not observed in the supernatants of Porphyromonas gingivalis or Fusobacterium nucleatum. Thus, P. intermedia has the potential to induce severe bacteremic pneumococcal pneumonia with enhanced pneumococcal adhesion to lower airway cells.

The influence of oral bacteria on epithelial cell migration in vitro.

Oral ulcerations often arise as a side effect from chemo- and radiation therapy. In a previous clinical study, Porphyromonas gingivalis was identified as a positive predictor for oral ulcerations after hematopoetic stem cell transplantation, possibly incriminating P. gingivalis in delayed healing of the ulcerations. Therefore, it was tested whether P. gingivalis and its secreted products could inhibit the migration of oral epithelial cells in an in vitro scratch assay. To compare, the oral bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia, and Streptococcus mitis were included. A standardized scratch was made in a confluent layer of human oral epithelial cells. The epithelial cells were challenged with bacterial cells and with medium containing secretions of these bacteria. Closure of the scratch was measured after 17 h using a phase contrast microscope. P. gingivalis, P. nigrescens, and secretions of P. gingivalis strongly inhibited cell migration. A challenge with 1000 heat-killed bacteria versus 1 epithelial cell resulted in a relative closure of the scratch of 25% for P. gingivalis and 20% for P. nigrescens. Weaker inhibitory effects were found for the other bacteria. The results confirmed our hypothesis that the oral bacteria may be involved in delayed wound healing.