Sigrun Eick

Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: Implications for autoimmunity in rheumatoid arthritis

OBJECTIVE To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA). METHODS The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis-knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and α-enolase by P gingivalis was studied by incubating live wild-type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry. RESULTS Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild-type P gingivalis with fibrinogen or α-enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy-terminal arginine residues, which were identified by mass spectrometry. CONCLUSION Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg-X peptide bonds by arginine gingipains, followed by citrullination of carboxy-terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalis-mediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA.

Analysis of neutrophil-derived antimicrobial peptides in gingival crevicular fluid suggests importance of cathelicidin LL-37 in the innate immune response against periodontogenic bacteria

INTRODUCTION During periodontitis, an innate immune response to bacterial challenge is primarily mediated by neutrophils. We compared neutrophilic content and the level of neutrophil-derived antimicrobial peptides in gingival crevicular fluid (GCF) in two clinical forms of severe periodontitis. METHODS GCF was collected from 14 patients with aggressive periodontitis, 17 patients with chronic periodontitis, and nine healthy subjects. Samples were analyzed for periodontopathogen load using real-time polymerase chain reactions. The amounts of myeloperoxidase and alpha-defensins (HNP1-3) were determined by enzyme-linked immunosorbent assay, and the level of cathelicidin (hCAP18/LL-37) was assayed by Western blot. RESULTS Myeloperoxidase concentration was not correlated with levels of LL-37 and HNP1-3 in samples from patients, compared to controls. The amount of HNP1-3 was twofold and fourfold higher in patients with aggressive and chronic periodontitis, respectively. Those with chronic disease had significantly elevated amounts of mature LL-37. The increased concentration of both peptides in chronic periodontitis correlated with the load of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. CONCLUSION The lack of a correlation between LL-37, HNP1-3, and myeloperoxidase content suggests that neutrophils are not the sole source of these bactericidal peptides in the GCF of patients with periodontitis; and that other cells contribute to their local production. The bacterial proteases of P. gingivalis, T. forsythia, and T. denticola might degrade hCAP18/LL-37, because the 11-kDa cathelicidin-derived fragment was present in GCF collected from pockets infected with these bacteria. Collectively, it appears that a local deficiency in LL-37 can be considered as a supporting factor in the pathogenesis of severe cases of periodontitis.

Reversibility of experimental peri-implant mucositis compared with experimental gingivitis in humans.

OBJECTIVE To monitor clinical, microbiological and host-derived alterations occurring around teeth and titanium implants during the development of experimental gingivitis/mucositis and their respective healing sequence in humans. MATERIAL AND METHODS Fifteen subjects with healthy or treated periodontal conditions and restored with dental implants underwent an experimental 3-week period of undisturbed plaque accumulation in the mandible. Subsequently, a 3-week period with optimal plaque control was instituted. At Days 0, 7, 14, 21, 28, 35 and 42, the presence/absence of plaque deposits around teeth and implants was assessed, (plaque index [PlI]) and the gingival/mucosal conditions were evaluated (gingival index[GI]). Subgingival/submucosal plaque samples and gingival/mucosal crevicular fluid (CF) samples were collected from two pre-determined sites around each experimental unit. CF samples were analyzed for matrix-metalloproteinase-8 (MMP-8) and interleukin-1beta (IL-1β). Microbial samples were analyzed using DNA-DNA hybridization for 40 species. RESULTS During 3 weeks of plaque accumulation, the median PlI and GI increased significantly at implants and teeth. Implant sites yielded a greater increase in the median GI compared with tooth sites. Over the 6-week experimental period, the CF levels of MMP-8 were statistically significantly higher at implants compared with teeth (P<0.05). The CF IL-1β levels did not differ statistically significantly between teeth and implants (P>0.05). No differences in the total DNA counts between implant and tooth sites were found at any time points. No differences in the detection frequency were found for putative periodontal pathogens between implant and tooth sites. CONCLUSION Peri-implant soft tissues developed a stronger inflammatory response to experimental plaque accumulation when compared with that of their gingival counterparts. Experimental gingivitis and peri-implant mucositis were reversible at the biomarker level. Clinically, however, 3 weeks of resumed plaque control did not yield pre-experimental levels of gingival and peri-implant mucosal health indicating that longer healing periods are needed.

Effects of tea tree (Melaleuca alternifolia) oil on Staphylococcus aureus in biofilms and stationary growth phase

Tea tree oil (TTO) is known for its antimicrobial activity. In this study, we determined whether TTO is effective against Staphylococcus aureus in biofilms and how TTO activity is affected by the S. aureus growth phase. All clinical strains tested were killed by TTO both as planktonic cells and as biofilms. The minimum biofilm eradication concentration was usually two times higher than the minimum bactericidal concentration, yet it was never higher than 1% v/v. The fastest killing of biofilm occurred during the first 15min of contact with TTO and was not influenced by increasing TTO concentration above 1% v/v. Planktonic stationary phase cells exhibited decreased susceptibility to TTO compared with exponential phase cells. The killing rate for stationary phase cells was also less affected by increasing TTO concentration than that for exponential phase cells. These data show that TTO efficiently kills S. aureus in the stationary growth phase and within biofilms and is therefore a promising tool for S. aureus eradication.

Interpain A, a cysteine proteinase from Prevotella intermedia, inhibits complement by degrading complement factor C3.

Periodontitis is an inflammatory disease of the supporting structures of the teeth caused by, among other pathogens, Prevotella intermedia. Many strains of P. intermedia are resistant to killing by the human complement system, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with recombinant cysteine protease of P. intermedia (interpain A) resulted in a drastic decrease in bactericidal activity of the serum. Furthermore, a clinical strain 59 expressing interpain A was more serum-resistant than another clinical strain 57, which did not express interpain A, as determined by Western blotting. Moreover, in the presence of the cysteine protease inhibitor E64, the killing of strain 59 by human serum was enhanced. Importantly, we found that the majority of P. intermedia strains isolated from chronic and aggressive periodontitis carry and express the interpain A gene. The protective effect of interpain A against serum bactericidal activity was found to be attributable to its ability to inhibit all three complement pathways through the efficient degradation of the α-chain of C3—the major complement factor common to all three pathways. P. intermedia has been known to co-aggregate with P. gingivalis, which produce gingipains to efficiently degrade complement factors. Here, interpain A was found to have a synergistic effect with gingipains on complement degradation. In addition, interpain A was able to activate the C1 complex in serum, causing deposition of C1q on inert and bacterial surfaces, which may be important at initial stages of infection when local inflammatory reaction may be beneficial for a pathogen. Taken together, the newly characterized interpain A proteinase appears to be an important virulence factor of P. intermedia.

The influence of a probiotic milk drink on the development of gingivitis: a pilot study.

AIM The aim of this study was to determine the effect of a probiotic milk drink on gingival health and the development of experimental gingivitis. MATERIAL AND METHODS Fifty volunteer students took part in a parallel-designed non-blinded study. The test group drank a probiotic drink once a day; the control group did not receive any product to drink. After 8 weeks, individual mechanical plaque control was interrupted for 96 h. Papilla bleeding index, interproximal plaque and Turesky plaque index (PI) were recorded at baseline, after 8 weeks and again 96 h later. At the same time points, gingival crevicular fluid had been collected for analysis of polymorphonuclear elastase, myeloperoxidase (MPO) and matrix metalloproteinase-3 (MMP-3). RESULTS Interproximal PI and papillary bleeding were not different between the groups. In the test group, elastase activity and MMP-3 amount were significantly lower after the intake of the probiotic milk drink (p<0.001 and 0.016). There was a significant increase of MPO activity in the control group; both groups were different at the end of the study (p=0.014). CONCLUSIONS The data suggest a beneficial effect of the probiotic milk drink on gingival inflammation.

Comparison of Gingival Crevicular Fluid Sampling Methods in Patients With Severe Chronic Periodontitis

BACKGROUND The analysis of samplings from periodontal pockets is important in the diagnosis and therapy of periodontitis. In this study, three different sampling techniques were compared to determine whether one method yielded samples suitable for the reproducible and simultaneous determination of bacterial load, cytokines, neutrophil elastase, and arginine-specific gingipains (Rgps). Rgps are an important virulence factor of Porphyromonas gingivalis, the exact concentration of which in gingival crevicular fluid (GCF) has not been quantified. METHODS GCF was sampled from four sites per patient (one sample per quadrant using two samples per method) in 36 patients with chronic periodontitis. One week later, the procedure was repeated with alternative methods. Variables determined were loads of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and P. gingivalis, levels of interleukin-6 and -8, activity of neutrophil elastase, and level of Rgps. RESULTS The detected cytokine levels were higher using paper strips compared to paper points. Bacteria were found in similar loads from paper strips and paper points. Rgps were only detectable in high quantities by washing the periodontal pocket. The level of Rgps correlated with the load of P. gingivalis. CONCLUSIONS The use of paper strips was suitable for the simultaneous determination of microbial and immunologic parameters. Obtaining GCF by washing can be useful for special purposes. The gingipain concentration in periodontal pockets was directly determined to be ≤1.5 μM. This value indicated that most of the substrates of these proteases by in vitro assays identified until now can be easily degraded in P. gingivalis-infected sites.

Binding of Complement Inhibitor C4b-Binding Protein Contributes to Serum Resistance of Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the α-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.

Comparison of real-time polymerase chain reaction and DNA-strip technology in microbiological evaluation of periodontitis treatment

The impact of a semiquantitative commercially available test based on DNA-strip technology (microIDent®, Hain Lifescience, Nehren, Germany) on diagnosis and treatment of severe chronic periodontitis of 25 periodontitis patients was evaluated in comparison with a quantitative in-house real-time PCR. Subgingival plaque samples were collected at baseline as well as at 3, 6, and 12 months later. After extracting DNA, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and several other periodontopathogens were determined by both methods. The results obtained by DNA-strip technology were analyzed semiquantitatively and additionally quantitatively by densitometry. The results for the 4 major periodontopathogenic bacterial species correlated significantly between the 2 methods. Samples detecting a high bacterial load by one method and negative by the other were always found in less than 2% of the total samples. Both technologies showed the impact of treatment on microflora. Especially the semiquantitative DNA-strip technology clearly analyzed the different loads of periodontopathogens after therapy and is useful in microbial diagnostics for patients in dental practices.

A Metalloproteinase Karilysin Present in the Majority of Tannerella forsythia Isolates Inhibits All Pathways of the Complement System

Tannerella forsythia is a poorly studied pathogen despite being one of the main causes of periodontitis, which is an inflammatory disease of the supporting structures of the teeth. We found that despite being recognized by all complement pathways, T. forsythia is resistant to killing by human complement, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with karilysin, a metalloproteinase of T. forsythia, resulted in a decrease in bactericidal activity of the serum. T. forsythia strains expressing karilysin at higher levels were more resistant than low-expressing strains. Furthermore, the low-expressing strain was significantly more opsonized with activated complement factor 3 and membrane attack complex from serum compared with the other strains. The high-expressing strain was more resistant to killing in human blood. The protective effect of karilysin against serum bactericidal activity was attributable to its ability to inhibit complement at several stages. The classical and lectin complement pathways were inhibited because of the efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4 by karilysin, whereas inhibition of the terminal pathway was caused by degradation of C5. Interestingly, karilysin was able to release biologically active C5a peptide in human plasma and induce migration of neutrophils. Importantly, we detected the karilysin gene in >90% of gingival crevicular fluid samples containing T. forsythia obtained from patients with periodontitis. Taken together, the newly characterized karilysin appears to be an important virulence factor of T. forsythia and might have several important implications for immune evasion.