Yuko Ohara-Nemoto

Relationship of quantitative salivary levels of Streptococcus mutans and S. sobrinus in mothers to caries status and colonization of mutans streptococci in plaque in their 2.5-year-old children

OBJECTIVES The aim of this study was to assess the relationships of quantitative salivary levels of Streptococcus mutans and S. sobrinus in mothers with the colonization of mutans streptococci (MS) in plaque and caries status in their 2.5-year-old children. Furthermore, the dynamics of caries status in the children was evaluated in a 2-year follow-up survey. METHODS After oral examination of 54 mother-and-child pairs, the saliva samples from the mothers and the plaque samples from the children were collected. The levels (log DNA copies/ml saliva) of S. mutans and S. sobrinus were quantified using real-time polymerase chain reaction (PCR) assays, while MS in the plaque samples were detected using a cultivation method. In addition, 50 of the 54 children participated in a 2-year follow-up survey of caries prevalence. RESULTS In the 2.5-year-old children, the percentage of dft-positive subjects and mean number of dft were significantly higher in the MS(+) group when compared with the MS(-) group. Findings from the 2-year follow-up survey indicated that MS(+) subjects had a persistently higher mean number of dft at 4.5 years. The 2.5-year-old children were divided into three groups based on the quantitative levels of salivary S. mutans and S. sobrinus in their mothers: those whose mothers had low levels of S. mutans (<4 log DNA copies/ml) and S. sobrinus (<2) (group 1); those whose mothers had a high level of S. mutans (> or = 4) and low level of S. sobrinus (<2) (group 2); and those whose mothers had high levels of both (> or = 4 and > or = 2, respectively) (group 3). Among the three groups, the percentages of MS(+) and dft-positive children were highest in group 3 and lowest in group 1. Furthermore, multiple logistic regression analyses revealed that grouping the mothers based on salivary level of S. mutans and S. sobrinus was an efficient means to predict both MS colonization (OR = 2.96) and prevalence of dental caries (OR = 9.39) in children at 2.5 years of age. CONCLUSIONS In the 54 mother-and-child pairs tested, the maternal salivary levels of S. mutans and S. sobrinus determined by real-time PCR were significantly related to MS colonization in plaque as well as dental caries in their children at 2.5 years of age. Thus, determination of maternal levels of both organisms using the present cut-off values is proposed as an efficient method to indicate the risks of maternal transmission of MS and childhood dental caries.

Infective endocarditis caused by Granulicatella elegans originating in the oral cavity

ABSTRACT We studied the pheno- and genotypes of an oral Granulicatella elegans strain in comparison with those of a blood-derived isolate which caused infective endocarditis. The two isolates exhibited identical biochemical characteristics and had the same drug MICs. Their genotypes were indistinguishable, indicating that these were from the same clone. The transmission of G. elegans from the oral cavity thus should be noted as a possible cause of infective endocarditis.

Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli

V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C‐terminal His6‐tagged proGluSE was successfully expressed from the full‐length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C‐terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I.

Asp- and Glu-specific novel dipeptidyl peptidase 11 of Porphyromonas gingivalis ensures utilization of proteinaceous energy sources.

Background: Dipeptidyl peptidases (DPPs) are required for protein metabolism in Porphyromonas gingivalis. Results: Asp/Glu-specific novel DPP (DPP11) was discovered and characterized. Conclusion: DPP11 ensures efficient degradation of oligopeptide substrates in Gram-negative anaerobic rods. Significance: This observation suggests further variation of substrate specificity in the DPP members. Porphyromonas gingivalis and Porphyromonas endodontalis, asaccharolytic black-pigmented anaerobes, are predominant pathogens of human chronic and periapical periodontitis, respectively. They incorporate di- and tripeptides from the environment as carbon and energy sources. In the present study we cloned a novel dipeptidyl peptidase (DPP) gene of P. endodontalis ATCC 35406, designated as DPP11. The DPP11 gene encoded 717 amino acids with a molecular mass of 81,090 Da and was present as a 75-kDa form with an N terminus of Asp22. A homology search revealed the presence of a P. gingivalis orthologue, PGN0607, that has been categorized as an isoform of authentic DPP7. P. gingivalis DPP11 was exclusively cell-associated as a truncated 60-kDa form, and the gene ablation retarded cell growth. DPP11 specifically removed dipeptides from oligopeptides with the penultimate N-terminal Asp and Glu and has a P2-position preference to hydrophobic residues. Optimum pH was 7.0, and the kcat/Km value was higher for Asp than Glu. Those activities were lost by substitution of Ser652 in P. endodontalis and Ser655 in P. gingivalis DPP11 to Ala, and they were consistently decreased with increasing NaCl concentration. Arg670 is a unique amino acid completely conserved in all DPP11 members distributed in the genera Porphyromonas, Bacteroides, and Parabacteroides, whereas this residue is converted to Gly in all authentic DPP7 members. Substitution analysis suggested that Arg670 interacts with an acidic residue of the substrate. Considered to preferentially utilize acidic amino acids, DPP11 ensures efficient degradation of oligopeptide substrates in these Gram-negative anaerobic rods.

Homologous and heterologous expression and maturation processing of extracellular glutamyl endopeptidase of Staphylococcus epidermidis.

Abstract The extracellular serine endopeptidase GluSE (EC 3.4.21.19) is considered to be one of the virulence factors of Staphylococcus epidermidis. The present study investigated maturation processing of native GluSE and that heterologously expressed in Escherichia coli. In addition to the 28-kDa mature protease, small amounts of proenzymes with molecular masses of 32, 30, and 29 kDa were identified in the extracellular and cell wall-associated fractions. We defined the pre (M1-A27)- and pro (K28-S66)-segments, and found that processing at the E32-S33 and D48-I49 bonds was responsible for production of the 30- and 29-kDa intermediates, respectively. The full-length form of C-terminally His-tagged GluSE was purified as three proenzymes equivalent to the native ones. These molecules possessing an entire or a part of the pro-segment were proteolytically latent and converted to a mature 28-kDa form by thermolysin cleavage at the S66-V67 bond. Mutation of the essential amino acid S235 suggested auto-proteolytic production of the 30- and 29-kDa intermediates. Furthermore, an undecapeptide (I56-S66) of the truncated pro-segment not only functions as an inhibitor of the protease but also facilitates thermolysin processing. These findings could offer clues to the molecular mechanism involved in the regulation of proteolytic activity of pathogenic proteases secreted from S. epidermidis.

Relationship between oral status and prevalence of periodontopathic bacteria on the tongues of elderly individuals

Colonization of periodontopathic bacteria is associated with increased risk of systemic diseases. However, few studies have investigated the relationships between oral status factors and health-related quality of life (HR-QOL) and the prevalence of such bacteria in elderly individuals. This study investigated the prevalence of Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola and Tannerella forsythia in 165 community-dwelling functionally independent 85-year-old Japanese individuals (93 dentate, 72 edentulous) and the relationship to oral status, including oral malodour and HR-QOL. All four of the studied periodontopathic bacteria were found more frequently in tongue coating samples from dentate than edentulous subjects, and the prevalence of Porphyromonas gingivalis, Prevotella intermedia and Treponema denticola was significantly related to the number of teeth with a periodontal pocket depth ≥4 mm. These results suggest the existence of a stable circulation of periodontopathic bacteria between the gingival sulcus and tongue coating over time with teeth. In addition, the presence of teeth with a deep pocket and colonization of Treponema denticola were positively related to the level of CH(3)SH, whilst the number of present teeth contributed positively to HR-QOL, especially with regard to mental health. In conclusion, as the dentate state can retain colonization of periodontopathic pathogens in the oral cavity, both periodontal treatment and tongue care are important for maintaining a healthy oral status in the elderly, and possibly result in avoidance of risk for tooth loss and decline in HR-QOL, as well as protecting from systemic diseases.

Prediction of periodontopathic bacteria in dental plaque of periodontal healthy subjects by measurement of volatile sulfur compounds in mouth air

OBJECTIVES The aim of this study was to determine whether measurements of volatile sulfur compounds (VSCs) are useful to predict colonization of periodontopathic bacteria. For this purpose, we assessed the relationships among distributions of 4 species of periodontopathic bacteria in tongue coating and dental plaque, oral conditions including VSC concentration in mouth air, and smoking habit of periodontal healthy young subjects. METHODS The subjects were 108 young adults (mean age, 23.5±2.56 years) without clinical periodontal pockets. Information regarding smoking habit was obtained by interview. After VSC concentration in mouth, air was measured with a portable sulfide monitor (Halimeter(®)), non-stimulated saliva flow and dental caries status were assessed, and tongue coating and dental plaque samples were collected from the subjects. The tongue coating samples were weighed to determine the amount. The colonization of Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola in both tongue coating and plaque samples was investigated using species-specific polymerase chain reaction assays. RESULTS Significant relationships were observed between the colonization of periodontopathic bacteria in tongue coating and plaque samples, especially that of P. gingivalis. VSC concentration showed the most significant association with colonization of P. gingivalis in both tongue coating and dental plaque. Logistic regression analysis demonstrated that the adjusted partial correlation coefficient [Exp(B)] values for VSC concentration with the colonization of P. gingivalis, P. intermedia, and T. denticola in dental plaque were 135, 35.4 and 10.4, respectively. In addition, smoking habit was also shown to be a significant variable in regression models [Exp(B)=6.19, 8.92 and 2.53, respectively]. Therefore, receiver operating characteristic analysis was performed to predict the colonization of periodontal bacteria in dental plaque in the subjects divided by smoking habit. Based on our results, we found cut-off values that indicated likelihood ratios (LR) within the efficient range for positive findings in both groups. CONCLUSION The present results demonstrated that measurement of VSC concentration in mouth air is a useful method to predict the presence of colonization of some periodontopathic bacteria in dental plaque.

Effects of fosfomycin on Shiga toxin-producing Escherichia coli: quantification of copy numbers of Shiga toxin-encoding genes and their expression levels using real-time PCR

Antibiotic therapy for infection with Shiga toxin-producing Escherichia coli (STEC) is not generally recommended, because it is thought to increase levels of released Shiga toxin (Stx), leading to the severe complication of haemolytic uraemic syndrome (HUS) (Tarr et al., 2005). However, the incidence of HUS in children with STEC infection was found not to be higher in Japan than in other countries, despite the use of antibiotics such as fosfomycin (FOM) (Ikeda et al., 1999; IDSC, 2007). Furthermore, early administration of FOM, as well as a new quinolone, norfloxacin (NFLX), and kanamycin, was recommended in the 1997 guidelines for medical treatment of STEC O157 infection issued by the Ministry of Health and Welfare of Japan. Hence, it remains controversial whether the use of antibiotics for STEC infection is effective or harmful (Wong et al., 2000; Bennish et al., 2006; Panos et al., 2006).

Exopeptidases and gingipains in Porphyromonas gingivalis as prerequisites for its amino acid metabolism

Summary Porphyromonas gingivalis, an asaccharolytic bacterium, utilizes amino acids as energy and carbon sources. Since amino acids are incorporated into the bacterial cells mainly as di- and tri-peptides, exopeptidases including dipeptidyl-peptidase (DPP) and tripeptidyl-peptidase are considered to be prerequisite components for their metabolism. We recently discovered DPP11, DPP5, and acylpeptidyl oligopeptidase in addition to previously reported DPP4, DPP7, and prolyl tripeptidyl peptidase A. DPP11 is a novel enzyme specific for acidic P1 residues (Asp and Glu) and distributed ubiquitously in eubacteria, while DPP5 is preferential for the hydrophobic P1 residue and the first entity identified in prokaryotes. Recently, acylpeptidyl oligopeptidase with a preference for hydrophobic P1 residues was found to release N-terminally blocked di- and tri-peptides. Furthermore, we also demonstrated that gingipains R and K contribute to P1-basic dipeptide production. These observations implicate that most, if not all, combinations of di- and tri-peptides are produced from extracellular oligopeptides even with an N-terminal modification. Here, we review P. gingivalis exopeptidases mainly in regard to their enzymatic characteristics. These exopeptidases with various substrate specificities benefit P. gingivalis for obtaining energy and carbon sources from the nutritionally limited subgingival environment.

Determination of three amino acids causing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases.

Abstract Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE, and GluSW, respectively. The order of their protease activities is GluSE<GluSW<<GluV8. In the present study, we investigated the mechanism that causes these differences. Expression of chimeric proteins between GluV8 and GluSE revealed that the difference is primarily attributed to amino acid residues 170–195, which define the intrinsic protease activity, and additionally to residues 119–169, which affect the proteolytic sensitivity. Among nine substitutions present in residues 170–195 of the three proteases, the substitutions at positions 185, 188, and 189 were responsible for the changes in their activities, and the combination of W185, V188, and P189, which naturally occurs in GluV8, exerts the highest protease activity. W185 and P189 were indispensable for full activity, but V188 could be replaced by hydrophobic amino acids. These three amino acid residues appear to create a substrate-binding pocket together with the catalytic triad and the N-terminal V1, and therefore define the K m values of the proteases. We also describe a method to produce a chimeric form of GluSE and GluV8 that is resistant to proteolysis, and therefore possesses 4-fold higher activity than the wild-type recombinant GluV8.