Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells

Background: Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells. Results: By employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, MIP-1β) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity. Conclusion: A. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory responses in human whole blood. Our findings therefore suggest that release of surface components from live bacterial cells could constitute a mechanism for systemic stimulation and be of particular importance in chronic localized infections, such as periodontitis

Peptidoglycan-associated lipoprotein of Aggregatibacter actinomycetemcomitans induces apoptosis and production of proinflammatory cytokines via TLR2 in murine macrophages RAW 264.7 in vitro

Peptidoglycan-associated lipoprotein (PAL) is a conserved pro-inflammatory outer membrane lipoprotein in Gram-negative bacteria. Compared to systemic pathogens, little is known about the virulence properties of PAL in Aggregatibacter actinomycetemcomitans (AaPAL). The aims of this study were to investigate the cytolethality of AaPAL and its ability to induce pro-inflammatory cytokine production in macrophages. Mouse macrophages were stimulated with AaPAL, and the production of IL-1β, IL-6, TNF-α, and MCP-1 was measured after 6, 24, and 48 h. To investigate which receptor AaPAL employs for its interaction with macrophages, anti-toll-like receptor (TLR)2 and anti-TLR4 antibodies were used to block respective TLRs on macrophages. Metabolic activity and apoptosis of the macrophages were investigated after stimulation with AaPAL. AaPAL induced the production of MCP-1, TNF-α, IL-6, and IL-1β from mouse macrophages in order of decreasing abundance. The pre-treatment of macrophages with an anti-TLR2 antibody significantly diminished cytokine production. Under AaPAL stimulation, the metabolic activity of macrophages decreased in a dose- and time-dependent manner. Furthermore, AaPAL induced apoptosis in 56% of macrophages after 48 h of incubation. Our data suggest that AaPAL can kill macrophages by apoptosis. The results also emphasize the role of AaPAL as a potent pro-inflammatory agent in A. actinomycetemcomitans-associated infections.</p

Porphyromonas gingivalis may interfere with conception in women

In this observational and prospective study, we investigated if microbiological and serological markers of periodontitis associated with conception in 256 non-pregnant women (Mage = 29.2 years; range 19-42 years). Clinical oral and gynecological examinations were performed, major periodontal pathogens in the saliva were detected, and serum and saliva antibodies against major periodontal pathogens were analyzed. The follow-up period for becoming pregnant was 12 months. Porphyromonas gingivalis was significantly (p = 0.032) more frequently detected in the saliva among those who did not become pregnant (8.3%) than among those who became pregnant (2.1%). The median levels of salivary P. gingivalis immunoglobulin A (IgA; p = 0.006) and IgG (p = 0.007) antibodies were higher among those who did not become pregnant compared to those who became pregnant. Hazard ratios (HR) for not becoming pregnant were HR = 3.75 (95% confidence interval [CI] 1.01-13.9; p = 0.048) if the subject was polymerase chain reaction-positive for P. gingivalis with high salivary antibodies against it, and HR = 1.62 (95% CI 1.03-2.54; p = 0.035) if she had high levels of serum P. gingivalis IgA and signs of periodontal infection. P. gingivalis associated with no success in getting pregnant.Peer reviewe

Proteomic Analysis and Virulence Assessment of Granulicatella adiacens Secretome

Despite reports on the occurrence of Granulicatella adiacens in infective endocarditis, few mechanistic studies on its virulence characteristics or pathogenicity are available. Proteins secreted by this species may act as determinants of host-microbe interaction and play a role in virulence. Our aim in this study was to investigate and functionally characterize the secretome of G. adiacens. Proteins in the secretome preparation were digested by trypsin and applied to nanoLC-ESI-MS/MS. By using a combined mass spectrometry and bioinformatics approach, we identified 101 proteins. Bioinformatics tools predicting subcellular localization revealed that 18 of the secreted proteins possessed signal sequence. More than 20% of the secretome proteins were putative virulence proteins including serine protease, superoxide dismutase, aminopeptidase, molecular chaperone DnaK, and thioredoxin. Ribosomal proteins, molecular chaperones, and glycolytic enzymes, together known as “moonlighting proteins,” comprised fifth of the secretome proteins. By Gene Ontology analysis, more than 60 proteins of the secretome were grouped in biological processes or molecular functions. KEGG pathway analysis disclosed that the secretome consisted of enzymes involved in biosynthesis of antibiotics. Cytokine profiling revealed that secreted proteins stimulated key cytokines, such as IL-1β, MCP-1, TNF-α, and RANTES from human PBMCs. In summary, the results from the current investigation of the G. adiacens secretome provide a basis for understanding possible pathogenic mechanisms of G. adiacens

Specified Species in Gingival Crevicular Fluid Predict Bacterial Diversity

BACKGROUND: Analysis of gingival crevicular fluid (GCF) samples may give information of unattached (planktonic) subgingival bacteria. Our study represents the first one targeting the identity of bacteria in GCF. METHODOLOGY/PRINCIPAL FINDINGS: We determined bacterial species diversity in GCF samples of a group of periodontitis patients and delineated contributing bacterial and host-associated factors. Subgingival paper point (PP) samples from the same sites were taken for comparison. After DNA extraction, 16S rRNA genes were PCR amplified and DNA-DNA hybridization was performed using a microarray for over 300 bacterial species or groups. Altogether 133 species from 41 genera and 8 phyla were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, per patient. Projection to latent structures by means of partial least squares (PLS) was applied to the multivariate data analysis. PLS regression analysis showed that species of genera including Campylobacter, Selenomonas, Porphyromonas, Catonella, Tannerella, Dialister, Peptostreptococcus, Streptococcus and Eubacterium had significant positive correlations and the number of teeth with low-grade attachment loss a significant negative correlation to species diversity in GCF samples. OPLS/O2PLS discriminant analysis revealed significant positive correlations to GCF sample group membership for species of genera Campylobacter, Leptotrichia, Prevotella, Dialister, Tannerella, Haemophilus, Fusobacterium, Eubacterium, and Actinomyces. CONCLUSIONS/SIGNIFICANCE: Among a variety of detected species those traditionally classified as Gram-negative anaerobes growing in mature subgingival biofilms were the main predictors for species diversity in GCF samples as well as responsible for distinguishing GCF samples from PP samples. GCF bacteria may provide new prospects for studying dynamic properties of subgingival biofilms

Phosphorylcholine is located in Aggregatibacter actinomycetemcomitans fimbrial protein Flp 1

Phosphorylcholine (ChoP) is covalently incorporated into bacterial surface structures, contributing to host mimicry and promoting adhesion to surfaces. Our aims were to determine the frequency of ChoP display among Aggregatibacter actinomycetemcomitans strains, to clarify which surface structures bear ChoP, and whether ChoP-positivity relates to serum killing. The tested oral (N = 67) and blood isolates (N = 27) represented 6 serotypes. Mab TEPC-15 was used for immunoblotting of cell lysates and fractions and for immunofluorescence microscopy of cell surface-bound ChoP. The lysates were denatured with urea for hidden ChoP or treated with proteinase K to test whether it binds to a protein. Three ChoP-positive and two ChoP-negative strains were subjected to serum killing in the presence/absence of CRP and using Ig-depleted serum as complement source. Cell lysates and the first soluble cellular fraction revealed a < 10 kDa band in immunoblots. Among 94 strains, 27 were ChoP positive. No difference was found in the prevalence of ChoP-positive oral (21/67) and blood (6/27) strains. Immunofluorescence microscopy corresponded to the immunoblot results. Proteinase K abolished ChoP reactivity, whereas urea did not change the negative result. The TEPC-15-reactive protein was undetectable in Δflp1 mutant strain. The survival rate of serotype-b strains in serum was 100% irrespective of ChoP, but that of serotype-a was higher in ChoP-positive (85%) than ChoP-negative (71%) strains. The results suggest that a third of rough-colony strains harbor ChoP and that ChoP is attached to fimbrial subunit protein Flp1. It further seems that ChoP-positivity does not enhance but may reduce A. actinomycetemcomitans susceptibility to serum killing

Periodontal disease and bacterial vaginosis increase the risk for adverse pregnancy outcome.

OBJECTIVES: To determine whether periodontal disease or bacterial vaginosis (BV) diagnosed before pregnancy increase the risk for adverse pregnancy outcome. METHODS: We enrolled a total of 252 women who had discontinued contraception in order to become pregnant. The first 130 pregnant women were included in the analyses. RESULTS: Multivariate analysis showed a strong association between periodontal disease and adverse pregnancy outcome (OR 5.5, 95% confidence interval 1.4-21.2; p = 0.014), and a borderline association between BV and adverse pregnancy outcome (OR 3.2, 95% confidence interval 0.9-10.7; p = 0.061). CONCLUSION: Our study suggests that pre-pregnancy counseling should include both oral and vaginal examinations to rule out periodontal disease and BV. This may ultimately have an impact on antenatal healthcare, and decrease the risk for adverse pregnancy outcome

Identification of the Pangenome and Its Components in 14 Distinct Aggregatibacter actinomycetemcomitans Strains by Comparative Genomic Analysis

Aggregatibacter actinomycetemcomitans is genetically heterogeneous and comprises distinct clonal lineages that may have different virulence potentials. However, limited information of the strain-to-strain genomic variations is available.The genome sequences of 11 A. actinomycetemcomitans strains (serotypes a-f) were generated de novo, annotated and combined with three previously sequenced genomes (serotypes a-c) for comparative genomic analysis. Two major groups were identified; serotypes a, d, e, and f, and serotypes b and c. A serotype e strain was found to be distinct from both groups. The size of the pangenome was 3,301 genes, which included 2,034 core genes and 1,267 flexible genes. The number of core genes is estimated to stabilize at 2,060, while the size of the pangenome is estimated to increase by 16 genes with every additional strain sequenced in the future. Within each strain 16.7-29.4% of the genome belonged to the flexible gene pool. Between any two strains 0.4-19.5% of the genomes were different. The genomic differences were occasionally greater for strains of the same serotypes than strains of different serotypes. Furthermore, 171 genomic islands were identified. Cumulatively, 777 strain-specific genes were found on these islands and represented 61% of the flexible gene pool.Substantial genomic differences were detected among A. actinomycetemcomitans strains. Genomic islands account for more than half of the flexible genes. The phenotype and virulence of A. actinomycetemcomitans may not be defined by any single strain. Moreover, the genomic variation within each clonal lineage of A. actinomycetemcomitans (as defined by serotype grouping) may be greater than between clonal lineages. The large genomic data set in this study will be useful to further examine the molecular basis of variable virulence among A. actinomycetemcomitans strains