The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways.

The assignment of functions to uncharacterized proteins discovered in genome projects requires easily accessible tools and computational resources for large-scale, user-friendly leveraging of the protein, genome, and metagenome databases by experimentalists. This article describes the web resource developed by the Enzyme Function Initiative (EFI; accessed at https://efi.igb.illinois.edu/ ) that provides "genomic enzymology" tools ("web tools") for (1) generating sequence similarity networks (SSNs) for protein families (EFI-EST); (2) analyzing and visualizing genome context of the proteins in clusters in SSNs (in genome neighborhood networks, GNNs, and genome neighborhood diagrams, GNDs) (EFI-GNT); and (3) prioritizing uncharacterized SSN clusters for functional assignment based on metagenome abundance (chemically guided functional profiling, CGFP) (EFI-CGFP). The SSNs generated by EFI-EST are used as the input for EFI-GNT and EFI-CGFP, enabling easy transfer of information among the tools. The networks are visualized and analyzed using Cytoscape, a widely used desktop application; GNDs and CGFP heatmaps summarizing metagenome abundance are viewed within the tools. We provide a detailed example of the integrated use of the tools with an analysis of glycyl radical enzyme superfamily (IPR004184) found in the human gut microbiome. This analysis demonstrates that (1) SwissProt annotations are not always correct, (2) large-scale genome context analyses allow the prediction of novel metabolic pathways, and (3) metagenome abundance can be used to identify/prioritize uncharacterized proteins for functional investigation.

Soluble urokinase-type plasminogen activator receptor is associated with signs of periodontitis in adolescents.

Owing to its molecular stability in body fluids, soluble urokinase-type plasminogen activator receptor (suPAR) is used as a biomarker for the level of systemic inflammation. This study compares the suPAR levels in serum with those in the saliva of adolescents and evaluates their association with the periodontal conditions. Adolescents identified as screen positive (n = 87) or screen negative (n = 73) for periodontitis had saliva and serum samples taken, along with subgingival plaque samples. The concentrations of suPAR were determined in saliva and serum, and 18 microbial species and the immunoglobulin response to them was evaluated. Factor analyses were used to reduce the number of variables within each of the domains of clinical, microbiological, and immunological findings. The median salivary suPAR concentration was 13.18 [(interquartile range (IQR): 6.20-23.36] µg l-1 and was not associated with the serum suPAR levels (median 2.05; IQR: 1.62-2.46 µg l-1 ). Linear regression analysis showed that the log10 (salivary suPAR concentration) was statistically significantly positively associated with the clinical phenotype 'Periodontitis Extent' (ß = 0.28; 95% CI: 0.16-0.39) along with 'Putative periodontopathogens' (ß = 0.65; 95% CI: 0.51-0.79). The study represents the first determination of salivary suPAR concentration in a larger well-defined adolescent population. Our results suggest the potential for clinical use of suPAR in saliva as an inflammatory risk indicator/biomarker of periodontitis.

Periodontal disease and Helicobacter pylori infection: a community-based study using serology and rapid urease test.

AIMS: The aims of the present study were to assess the prevalence of periodontal disease and Helicobacter pylori (H. pylori) infection and their associations within a predefined Indian population. METHODS: A community-based cross-sectional study of 500 selected individuals using a questionnaire, oral examination, rapid urease testing of dental plaque, and serological examination for immunoglobulin G antibody to H. pylori was carried out. RESULTS: Periodontal disease and H. pylori infection were prevalent in more than 50% of the population. Age, smoking, and diabetic status of the individuals were risk factors for periodontal disease after multivariate analysis, and a lack of proper sewage and waste disposal facilities were found to increase the risk of H. pylori infection. Although there was no association between periodontal disease and H. pylori seropositivity in the community, a highly-significant association was found between periodontal disease and colonization of H. pylori in dental plaque. CONCLUSIONS: Because periodontal disease is associated with the increased colonization of H. pylori, new treatment modalities, such as plaque control measures, should be employed for the complete management of H. pylori-associated gastric disease.

Site-specific dental plaque pH in 13-year-old Thai schoolchildren.

OBJECTIVE: The aim of this paper was to study pH conditions between dental sites, taking account the presence of caries, calculus, and microbial composition and alkali production. MATERIALS AND METHODS: One hundred 13-year-old Thai schoolchildren were recorded for caries experience (DMFT, DT), calculus, plaque, and gingivitis. Ex vivo urease activity was measured on 11, 26, 31, and 46 (distal aspect) with the rapid urease test and pH at baseline and after rinse with 0.25 % urea solution on mesial site in vivo. Interproximal plaque from contralateral teeth was microbiological analysed with the checkerboard technique. RESULTS: Thirty-four children were caries free. Plaque and calculus were abundant; all children showed a high resting plaque pH and the mandibular incisor showed significantly (p < 0.01) higher pH at baseline, max pH and AOC7.0 after urea challenge, ex vivo urease activity and calculus but lower caries experience than other teeth. A significant inverse correlation (p < 0.02) was found between caries frequency and ex vivo urease activity for tooth 11. Anaerobes predominated over streptococci, but no significant differences between dental sites were found. CONCLUSIONS: The study group had a high baseline plaque pH, in vivo and ex vivo urease activity, and calculus but low caries experience, which was best reflected in the lower incisor region. CLINICAL RELEVANCE: Urease activity and pH on site level may be important determinants for individuals at caries risk.

Caries-free subjects have high levels of urease and arginine deiminase activity.

OBJECTIVES: This study investigated the relationship between urease and arginine deiminase system (ADS) activities and dental caries through a cross-sectional study. MATERIAL AND METHODS: Urease and ADS activities were measured in saliva and plaque samples from 10 caries-free subjects and 13 caries-active. Urease activity was obtained from the ammonia produced by incubation of plaque and saliva samples in urea. ADS activity was obtained from the ammonia generated by the arginine-HCl and Tris-maleate buffer. Specific activity was defined as micromoles of ammonia per minute per milligram of protein. Shapiro-Wilk statistical test was used to analyze the distribution of the data, and Mann-Whitney test was used to determine the significance of the data. RESULTS: The specific urease activity in saliva and plaque was significantly higher in individuals with low DMFT scores. ADS activity in saliva (6.050 vs 1.350, p=0.0154) and plaque (8.830 vs 1.210, p=0.025) was also higher in individuals with low DMFT scores. CONCLUSIONS: Caries-free subjects had a higher ammonia generation activity by urease and arginine deiminase system for both saliva and plaque samples than low caries-active subjects. High levels of alkali production in oral environment were related to caries-free subjects.

Caries-free subjects have high levels of urease and arginine deiminase activity

Objectives: This study investigated the relationship between urease and arginine deiminase system (ADS) activities and dental caries through a cross-sectional study. Material and Methods: Urease and ADS activities were measured in saliva and plaque samples from 10 caries-free subjects and 13 caries-active. Urease activity was obtained from the ammonia produced by incubation of plaque and saliva samples in urea. ADS activity was obtained from the ammonia generated by the arginine-HCl and Tris-maleate buffer. Specific activity was defined as micromoles of ammonia per minute per milligram of protein. Shapiro-Wilk statistical test was used to analyze the distribution of the data, and Mann-Whitney test was used to determine the significance of the data. Results: The specific urease activity in saliva and plaque was significantly higher in individuals with low DMFT scores. ADS activity in saliva (6.050 vs 1.350, p=0.0154) and plaque (8.830 vs 1.210, p=0.025) was also higher in individuals with low DMFT scores. Conclusions: Caries-free subjects had a higher ammonia generation activity by urease and arginine deiminase system for both saliva and plaque samples than low caries-active subjects. High levels of alkali production in oral environment were related to caries-free subjects. .

Oral arginine metabolism may decrease the risk for dental caries in children.

Arginine metabolism by oral bacteria via the arginine deiminase system (ADS) increases the local pH, which can neutralize the effects of acidification from sugar metabolism and reduce the cariogenicity of oral biofilms. To explore the relationship between oral arginine metabolism and dental caries experience in children, we measured ADS activity in oral samples from 100 children and correlated it with their caries status and type of dentition. Supragingival dental plaque was collected from tooth surfaces that were caries-lesion-free (PF) and from dentinal (PD) and enamel (PE) caries lesions. Regardless of children's caries status or type of dentition, PF (378.6) had significantly higher ADS activity compared with PD (208.4; p < .001) and PE (194.8; p = .005). There was no significant difference in the salivary arginolytic activity among children with different caries status. Mixed-model analysis showed that plaque caries status is significantly associated with ADS activity despite children's age, caries status, and dentition (p < .001), with healthy plaque predicting higher ADS activity compared with diseased plaque. Plaque arginine metabolism varies greatly among children and tooth sites, which may affect their susceptibility to caries.

Progress toward understanding the contribution of alkali generation in dental biofilms to inhibition of dental caries.

Alkali production by oral bacteria is believed to have a major impact on oral microbial ecology and to be inhibitory to the initiation and progression of dental caries. A substantial body of evidence is beginning to accumulate that indicates the modulation of the alkalinogenic potential of dental biofilms may be a promising strategy for caries control. This brief review highlights recent progress toward understanding molecular genetic and physiologic aspects of important alkali-generating pathways in oral bacteria, and the role of alkali production in the ecology of dental biofilms in health and disease.

Urease activity in dental plaque and saliva of children during a three-year study period and its relationship with other caries risk factors.

UNLABELLED: Bacterial urease activity in dental plaque and in saliva generates ammonia, which can increase the plaque pH and can protect acid-sensitive oral bacteria. Recent cross-sectional studies suggest that reduced ability to generate ammonia from urea in dental plaque can be an important caries risk factor. In spite of this proposed important clinical role, there is currently no information available regarding important clinical aspects of oral ureolysis in children. OBJECTIVE: The objective of this study was to evaluate the distribution and pattern of urease activity in the dental plaque and in the saliva of children during a three-year period, and to examine the relationship of urease with some important caries risk factors. METHODS: A longitudinal study was conducted with repeated measures over a three-year period on a panel of 80 children, aged 3-6 years at recruitment. The dynamics of change in urease activity were described and associated with clinical, biological, and behavioural caries risk factors. RESULTS: Urease activity in plaque showed a trend to remain stable during the study period and was negatively associated with sugar consumption (P<0.05). Urease activity in unstimulated saliva increased with age, and it was positively associated with the levels of mutans streptococci in saliva and with the educational level of the parents (P<0.05). CONCLUSIONS: The results of this study reveal interesting and complex interactions between oral urease activity and some important caries risk factors. Urease activity in saliva could be an indicator of mutans infection in children.

Could alkali production be considered an approach for caries control?

This study investigated the relationship of arginine deiminase (ADS) and urease activities with dental caries through a case-control study. ADS and urease activities were measured in dental smooth-surface supragingival plaque and whole saliva samples from 93 subjects, who were in three different groups: caries-free (n = 31), caries-active (n = 30), and caries-experienced (n = 32). ADS activity was measured by quantification of the ammonia generated from the incubation of plaque and saliva samples in a mixture containing 50 mM arginine-HCl and 50 mM Tris-maleate buffer, pH 6.0. ADS-specific activity was defined as nanomoles of ammonia generated per minute per milligram of protein. Urease activity was determined by quantification of ammonia produced from 50 mM urea. For bacterial identification and enumeration real-time qPCR analysis was used. Groups were compared using Kruskal-Wallis tests. Spearman correlations were used to analyze plaque metabolic activity and bacterial relationships. The results revealed significantly higher ammonia production from arginine in saliva (1.06 vs. 0.18; p < 0.0001) and plaque samples (1.74 vs. 0.58; p < 0.0001) from caries-free subjects compared to caries-active subjects. Urease levels were about 3-fold higher in the plaque of caries-free subjects (p < 0.0001). Although higher urease activity in saliva of caries-experienced and caries-free subjects was evident, no significant difference was found between the groups.

Periodontal disease activity measured by the benzoyl-DL-arginine-naphthylamide test is associated with preterm births.

BACKGROUND: Infection is a risk factor for preterm birth. This study was conducted in the field and addressed the link between periodontal pathogens measured with the benzoyl-DL-arginine-naphthylamide (BANA) test and preterm birth. METHODS: This prospective study was performed in Changhua, Taiwan. Periodontal examinations included the plaque index, papillary bleeding scores, and measurement of the BANA enzyme in plaque samples at the second and third trimesters. Independent variables included maternal demographic characteristics, previous pregnancy histories, risk factors, plaque and gingivitis scores, and current pregnancy outcomes. RESULTS: There were 19 (7%) preterm deliveries among the 268 subjects. A history of a previous preterm birth and low birth weight, frequency of prenatal visits, preterm uterine contractions, antepartum hemorrhages, placenta previae, and preterm premature rupture of membranes were significantly related to preterm birth (P = 0.035, 0.027, <0.001, 0.025, 0.006, 0.014, and <0.001, respectively). Maternal weight gain was higher with a normal term delivery (P = 0.003). Multivariable logistic regression analyses showed that the number of BANA-infected sites in the third trimester (odds ratio [OR]: 5.89; 95% confidence interval [CI]: 1.5 to 31.6), maternal weight gain (OR: 0.78; 95% CI: 0.65 to 0.91), antepartum hemorrhages (OR: 10.0; 95% CI: 2.2 to 46.9), and preterm premature rupture of membranes (OR: 12.6; 95% CI: 3.97 to 42.71) had significant influences on preterm-birth outcomes. CONCLUSIONS: BANA-positive plaque in the third trimester was associated with preterm births after controlling for other risk factors. The BANA test can be used to screen pregnant women at chairside and/or bedside to apply suitable intervention tactics.

Denitrification in human dental plaque.

BACKGROUND: Microbial denitrification is not considered important in human-associated microbial communities. Accordingly, metabolic investigations of the microbial biofilm communities of human dental plaque have focused on aerobic respiration and acid fermentation of carbohydrates, even though it is known that the oral habitat is constantly exposed to nitrate (NO3-) concentrations in the millimolar range and that dental plaque houses bacteria that can reduce this NO3- to nitrite (NO2-). RESULTS: We show that dental plaque mediates denitrification of NO3- to nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2) using microsensor measurements, 15N isotopic labelling and molecular detection of denitrification genes. In vivo N2O accumulation rates in the mouth depended on the presence of dental plaque and on salivary NO3- concentrations. NO and N2O production by denitrification occurred under aerobic conditions and was regulated by plaque pH. CONCLUSIONS: Increases of NO concentrations were in the range of effective concentrations for NO signalling to human host cells and, thus, may locally affect blood flow, signalling between nerves and inflammatory processes in the gum. This is specifically significant for the understanding of periodontal diseases, where NO has been shown to play a key role, but where gingival cells are believed to be the only source of NO. More generally, this study establishes denitrification by human-associated microbial communities as a significant metabolic pathway which, due to concurrent NO formation, provides a basis for symbiotic interactions.

The effect of sucrose on plaque and saliva urease levels in vivo.

OBJECTIVE: Dietary sugar exposures induce an immediate drop of the plaque pH. Based on in vitro observations, it was hypothesized that oral bacteria may rapidly respond to this environmental change by increasing the activity or expression of alkali-generating pathways, such as the urease pathway. The objective of this exploratory in vivo study was to determine the short-term effect of a brief sucrose exposure on plaque and saliva urease activity and expression, and to relate this effect to caries experience. METHODS: Urease activity levels were measured in plaque and saliva samples collected from 20 children during fasting conditions and 30 min after rinsing with a sucrose solution. Streptococcus salivarius ureC-specific mRNA in saliva was quantified using real-time RT-PCR. The impact of host-related factors, such as age, gender, sugar consumption, salivary mutans streptococci levels and caries status on urease activity was evaluated. RESULTS: Plaque urease activity under fasting conditions was higher in subjects with low caries and mutans streptococci levels. This difference was not observed after the sucrose exposure. The response of urease to sucrose in vivo did not depend on caries experience or salivary mutans levels. Significant increase in urease activity of plaque and saliva after exposure to sucrose was observed only in the subjects who had low urease levels at baseline. CONCLUSIONS: The findings of this exploratory study suggest that plaque urease activity may have an important long-term influence in caries development but not during a cariogenic challenge.

Fluoride, triclosan and organic weak acids as modulators of the arginine deiminase system in biofilms and suspension cells of oral streptococci.

INTRODUCTION: The arginine deiminase system (ADS) of oral bacteria is a major generator of alkali (ammonia) in dental plaque and is considered to have anticaries effects. However, many of the antimicrobial agents used in oral care products may reduce alkali production by the ADS. The objective of our work was to assess the sensitivity of the ADS of oral streptococci to commonly used antimicrobials, fluoride, triclosan and organic weak acids. METHODS: Streptococcus sanguinis NCTC 10904 and Streptococcus ratti FA-1 were grown in suspension cultures and mono-organism biofilms. ADS activity at pH values of 4, 5 and 6 was assessed, and the actions of the agents was determined in terms of reduced production of alkali from arginine, inhibition of ADS enzymes and changes in uptake of arginine. RESULTS: ADS activity was not greatly affected by pH changes between 4 and 6 and was greater per unit of biomass for cell suspensions than for biofilms. NaF was a poor inhibitor, while triclosan was highly effective with a 50% inhibitory dose for the two organisms between 0.03 and 0.05 and between 0.10 and 0.15 mm-h for suspension cells and biofilms, respectively. The weak acid indomethacin was nearly as potent at pH 4.0 as triclosan, while capric and lauric acids were less potent, especially for biofilms. The methyl ester of lauric acid was slightly stimulatory. The major targets for the inhibitors appeared to be transport systems for arginine uptake, although carbamate kinase was a secondary target. CONCLUSION: Triclosan, indomethacin, caprate and laurate can reduce ADS activity in dental plaque.

Proteolytic degradation of human salivary MUC5B by dental biofilms.

The degradation of complex substrates, like salivary mucins, requires an arsenal of glycosidases and proteases to sequentially degrade the oligosaccharides and polypeptide backbone. The mucin MUC5B is a complex oligomeric glycoprotein, heterogeneous in molecular mass (14-40 x 10(6) Da), with a diverse repertoire of oligosaccharides, differing in composition and charge. The aim of this study was to investigate whether proteolytic degradation of the mucin polypeptide backbone could be identified and if cooperation of dental biofilm bacteria was required. Cooperative bacteria-mediated proteolysis of MUC5B was determined by comparing individual species and mixed consortia of strains isolated from supragingival plaque, and freshly harvested supragingival plaque. Proteolytic activity was analysed using fluorescent labelled substrate and by visualizing mucin degradation by SDS-PAGE. Dental plaque degraded the polypeptide backbone of the salivary MUC5B mucin. The mucin was also degraded by a specific consortium of isolated species from supragingival plaque, although individual species and other consortia did not. Certain bacteria in supragingival dental plaque therefore cooperate as a consortium to proteolyse human salivary MUC5B and hydrolyse glycosides.

Correlations of oral bacterial arginine and urea catabolism with caries experience.

BACKGROUND/AIM: Alkali generation by oral bacteria plays a key role in plaque pH homeostasis and may be a major impediment to the development of dental caries. To determine if the capacity of oral samples to produce ammonia from arginine or urea was related to caries experience, the arginine deiminase system (ADS) and urease activity in saliva and dental plaque samples were measured in 45 adult subjects. METHODS: The subjects were divided into three groups according to caries status; 13 caries-free (CF) individuals (decayed, missing, and filled teeth = 0); 21 caries-active (CA) individuals (decayed teeth >or= 4); and 11 caries-experienced (CE) individuals (decayed teeth = 0; missing and filled teeth > 0). Real-time polymerase chain reaction was used to quantify the proportion of certain acid- or alkali-producing organisms in the samples. RESULTS: The amount of ammonia generated from the test substrates by plaque samples was generally higher than that produced by salivary samples in all groups. Significantly higher levels of salivary ADS activity and plaque urease activity were observed in CF subjects compared to CA subjects (P = 0.0004 and P = 0.014, respectively). The proportions of Streptococcus mutans from saliva and dental plaque of CA subjects were significantly higher than those from the CF group (P = 0.0153 and P = 0.0009, respectively). In the CA group, there was an inverse relationship between urease activity and the levels of S. mutans (P < 0.0001). CONCLUSION: This study supports the theory that increased caries risk is associated with reduced alkali-generating capacity of the bacteria colonizing the oral cavity; providing compelling evidence to further our understanding of oral alkali-generation in health and disease.

Inorganic chemistry of defensive peroxidases in the human oral cavity.

The innate host response system is comprised of various mechanisms for orchestrating host response to microbial infection of the oral cavity. The heterogeneity of the oral cavity and the associated microenvironments that are produced give rise to different chemistries that affect the innate defense system. One focus of this review is on how these spatial differences influence the two major defensive peroxidases of the oral cavity, salivary peroxidase (SPO) and myeloperoxidase (MPO). With hydrogen peroxide (H(2)O(2)) as an oxidant, the defensive peroxidases use inorganic ions to produce antimicrobials that are generally more effective than H(2)O(2) itself. The concentrations of the inorganic substrates are different in saliva vs. gingival crevicular fluid (GCF). Thus, in the supragingival regime, SPO and MPO work in unison for the exclusive production of hypothiocyanite (OSCN(-), a reactive inorganic species), which constantly bathes nascent plaques. In contrast, MPO is introduced to the GCF during inflammatory response, and in that environment it is capable of producing hypochlorite (OCl(-)), a chemically more powerful oxidant that is implicated in host tissue damage. A second focus of this review is on inter-person variation that may contribute to different peroxidase function. Many of these differences are attributed to dietary or smoking practices that alter the concentrations of relevant inorganic species in the oral cavity (e.g.: fluoride, F(-); cyanide, CN(-); cyanate, OCN(-); thiocyanate, SCN(-); and nitrate, NO(3)(-)). Because of the complexity of the host and microflora biology and the associated chemistry, it is difficult to establish the significance of the human peroxidase systems during the pathogenesis of oral diseases. The problem is particularly complex with respect to the gingival sulcus and periodontal pockets (where the very different defensive stratagems of GCF and saliva co-mingle). Despite this complexity, intriguing in vitro and in vivo studies are reviewed here that reveal the interplay between peroxidase function and associated inorganic chemistry.

Salivary gel-forming mucin MUC5B--a nutrient for dental plaque bacteria.

INTRODUCTION: Model systems with oral bacteria from dental plaque have demonstrated that the utilization of complex glycoproteins as a food source cannot be undertaken by single species but requires concerted degradation by a multi-species consortium, with each member contributing one or a few hydrolytic enzymes. Unlike previous studies, the aim of the present investigation was to explore the ability of fresh dental plaque to degrade salivary mucin, MUC5B, isolated by methods designed to retain intact the natural polymeric structure and physiological conformation, in an attempt to mimic the naturally occurring interaction between the oral microbiota and salivary mucins. METHODS: Human salivary MUC5B was isolated from whole saliva by density-gradient centrifugation and incubated with freshly isolated supragingival dental plaque with samples subjected to fluorescent staining for viability and metabolic activity. In addition, the degradation of MUC5B oligosaccharide side chains was studied using a lectin assay, recognizing three different carbohydrate epitopes commonly found on mucin oligosaccharide side chains. RESULTS: The addition of purified salivary MUC5B elicited a strong metabolic response from the biofilm cells, whereas individual strains of Streptococcus oralis and Streptococcus gordonii isolated from the same plaque were not able to utilize the MUC5B. The degradation of terminal saccharide moieties on the MUC5B was demonstrated by a marked decrease in both sialic acid and fucose reactivity. CONCLUSION: These results have shown that dental plaque is capable of utilizing human salivary MUC5B as a nutrient source, a process possibly requiring the synergistic degradation of the molecule by a consortium of oral bacteria in the plaque community.

Effect of Porphyromonas gingivalis PrtC on cytokine expression in ECV304 endothelial cells and its level in subgingival plaques from patients with chronic periodontitis.

AIM: To investigate the effect of the collagenase gene (prtC) product of Porphyromonas gingivalis on inducing host cells to secrete inflammatory cytokines, and to discuss the correlation between the PrtC level in subgingival plaque samples and clinical parameters. METHODS: A prokaryotic expression system pET32a-prtC-Escheria coli BL21DE3 was constructed. Antigenicity and immunoreactivity of the recombinant PrtC protein (rPrtC) was identified by Western blotting. ELISA was applied to detect interleukin (IL)-1alpha, IL-8, and TNF-alpha levels in supernatants from rPrtC-induced human umbilical vein endothelial cells (HUVEC) originated ECV304 cells. Clinical parameters recorded at baseline and after treatment included bleeding on probing (BOP), probing depth (PD), and attachment loss (AL). ELISA was established to measure the PrtC level in 196 subgingival plaque samples from 49 patients with chronic periodontitis. RESULTS: After coincubation with 1 microg/mL rPrtC for 24 h and with 5 or 10 microg/mL rPrtC for 12 h, the levels of IL-1 alpha, IL-8, and TNF-alpha secreted by the ECV304 cells increased significantly (P<0.05). The PrtC level in the BOP-positive or the > or =5 mm AL or > or = 6 mm PD sites was higher than that in the BOP-negative or the < or =2 mm AL or < or =6 mm PD sites (P<0.05), respectively. Compared with baseline, the PrtC levels in different AL sites or in the < or =6 mm PD pockets decreased remarkably after treatment (P<0.01), but in the BOP-positive or in the > 6 mm PD sites, the PrtC levels changed insignificantly (P>0.05). CONCLUSION: rPrtC is able to directly induce host cells to synthesize and secrete IL-1 alpha, IL-8, and TNF-alpha. The PrtC level in subgingival samples is correlated with BOP, AL, and PD.