generation-gene-disrupted-streptococcus-mutans-strain-without-gene

Generation of a Gene-disrupted <em>Streptococcus mutans</em> Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

purification-of-a-high-molecular-mass-protein-in-streptococcus-mutans

Purification of a High Molecular Mass Protein in <em>Streptococcus mutans</em>

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

Tsurumi University School of Dental Medicine

Volatile sulfur compounds (VSCs) in oral air are the only type of gases correlated with the strength of oral malodor. We developed a compact and simple gas chromatograph (GC) equipped with a newly invented indium oxide semiconductor gas sensor (SCS) for measuring the concentrations of VSCs in mouth air. We have assessed the correlation between measurements with a GC-SCS and those with a regular GC.

Development of a compact and simple gas chromatography for oral malodor measurement.

Many food products are claimed to be effective in controlling halitosis. Halitosis is caused mainly by volatile sulfur compounds (VSCs) such as H(2)S and CH(3)SH produced in the oral cavity. Oral microorganisms degrade proteinaceous substrates to cysteine and methionine, which are then converted to VSCs. Most treatments for halitosis focus on controlling the number of microorganisms in the oral cavity. Since tea polyphenols have been shown to have antimicrobial and deodorant effects, we have investigated whether green tea powder reduces VSCs in mouth air, and compared its effectiveness with that of other foods which are claimed to control halitosis. Immediately after administering the products, green tea showed the largest reduction in concentration of both H(2)S and CH(3)SH gases, especially CH(3)SH which also demonstrated a better correlation with odor strength than H(2)S; however, no reduction was observed at 1, 2 and 3 h after administration. Chewing gum, mints and parsley-seed oil product did not reduce the concentration of VSCs in mouth air at any time. Toothpaste, mints and green tea strongly inhibited VSCs production in a saliva-putrefaction system, but chewing gum and parsley-seed oil product could not inhibit saliva putrefaction. Toothpaste and green tea also demonstrated strong deodorant activities in vitro, but no significant deodorant activity of mints, chewing gum or parsley-seed oil product were observed. We concluded that green tea was very effective in reducing oral malodor temporarily because of its disinfectant and deodorant activities, whereas other foods were not effective.

Effect of green tea on volatile sulfur compounds in mouth air.

Previous reports of the reduction of fluoride concentrations in fluoridated water by domestic water treatment systems have indicated that further supplementation with fluoride is required. However, the absorption of fluoride by filters has not yet been directly identified. If these filters do not absorb fluoride, further fluoride supplementation may increase fluorosis. In this study, we determined whether filtering systems absorb fluoride ions.

Neither hollow-fibre membrane filters nor activated-charcoal filters remove fluoride from fluoridated tap water.

Dentistry is a major resource for the treatment of halitosis, therefore dental professionals must also pay attention to their own oral malodour for professional courtesy. However, oral malodour among dental professionals has not yet been investigated. In this study, the diurnal changes in oral malodour in dental-office workers were determined, and preventative measures were assessed.

Diurnal changes in oral malodour among dental-office workers.

This study determined the effect of Hop polyphenols (HPP) on water-insoluble glucan (WIG), which is a major component of dental plaque along with microorganisms, and the effect of HPP-containing tablets on the growth of dental plaque.

Hop polyphenols suppress production of water-insoluble glucan by Streptococcus mutans and dental plaque growth in vivo.

Hydrogen sulfide (H(2)S) is a main cause of physiologic halitosis. H(2)S induces apoptosis in human gingival cells, which may play an important role in periodontal pathology. Recently, it has been reported that H(2)S induced apoptosis and DNA damage in human gingival fibroblasts (HGFs) by increasing the levels of reactive oxygen species. However, the mechanisms of H(2)S-induced apoptosis have not been clarified in HGFs. The objective of this study was to determine the apoptotic pathway activated by H(2)S in HGFs. The HGFs were exposed to 50 ng/mL H(2)S, resulting in 18 ng/mL in the culture medium, which is lower than the concentration in periodontal pockets. The number of apoptotic cells after 24 and 48 h incubation was significantly higher than that in the control cultures (p < 0.05). Mitochondrial membrane depolarization and the release of cytochrome c, and caspase-3, and caspase-9 were also significantly increased after both 24- and 48-h incubation (p < 0.05), whereas caspase-8, a key enzyme in the receptor ligand-mediated pathway causing apoptosis, was not activated. The present study shows that H(2)S triggered the mitochondrial pathway causing apoptosis in HGFs but did not activate the receptor ligand-mediated pathway.

Oral malodorous compound activates mitochondrial pathway inducing apoptosis in human gingival fibroblasts.

Oral malodorous compounds including hydrogen sulfide (H2S) are causative agents of periodontitis because the toxicities are similar to that of cyanate. Previous studies demonstrated that volatile sulfur compounds (VSCs) were highly toxic to periodontal tissues, causing a large reduction in the amount of collagen in human gingival fibroblasts and extracellular matrix as well as, for example, apoptosis, immunologic responses, and matrix metalloproteinase production. The objective of this study was to determine the effect of H2S on the proliferation of osteoblasts and a signaling transduction pathway through the mitogen-activated protein kinase (MAPK).

Oral malodorous compound inhibits osteoblast proliferation.

We report a clinical trial of the effects of test tablets containing bovine lactoferrin and lactoperoxidase on oral malodor and salivary bacteria. Fifteen subjects with volatile sulfur compounds (VSCs) in mouth air above the olfactory threshold (H(2)S >1.5 or CH(3)SH >0.5 ng/10 ml) as detected by gas chromatography were enrolled in the trial. Either a test or a placebo tablet was ingested twice at 1-h intervals in two crossover phases. Mouth air was monitored for VSC levels at the baseline before ingestion of a tablet, 10 min after the first ingestion, 1 h (just before the second ingestion), and 2 h after the first ingestion. Whole saliva was analyzed at the baseline and at 2 h for bacterial numbers. At 10 min, the level of CH(3)SH was significantly lower in the test group (median [interquartile range] = 0.28 [0.00-0.68] ng/10 ml) compared to that in the placebo group (0.73 [0.47-1.00] ng/10 ml; P = 0.011). The median concentration of CH(3)SH in the test group was below the olfactory threshold after 10 min until 2 h, whereas the level in the placebo group was above the threshold during the experimental period. No difference in the numbers of salivary bacteria was detected by culturing or quantitative PCR, but terminal restriction fragment length polymorphism detected one fragment with a significantly lower copy number at 2 h in the test group (mean ± standard error, 4.89 ± 0.11 log(10) copies/10 µl) compared to that in the placebo group (5.38 ± 0.15 log(10) copies/10 µl; P = 0.033). These results indicate a suppressive effect of the test composition on oral malodor and suggest an influence on oral bacteria.

Effects of a composition containing lactoferrin and lactoperoxidase on oral malodor and salivary bacteria: a randomized, double-blind, crossover, placebo-controlled clinical trial.

Hydrogen sulfide (H2S) is a novel gasotransmitter that plays multiple biological roles in various body systems. In addition to its endogenous production, H2S is produced by bacteria colonizing digestive organs, including the oral cavity. H2S was previously shown to enhance pro-apoptotic effects in cancer cell lines, although the mechanisms involved remain unclear. To properly assess the anti-cancer effects of H2S, however, investigations of apoptotic effects in normal cells are also necessary. The aims of this study were (1) to compare the susceptibility to H2S-induced apoptosis between the oral cancer cell line Ca9-22 and oral keratinocytes that were derived from healthy gingiva, and (2) to identify candidate genes involved in the induction of apoptosis by H2S. The susceptibility to H2S-induced apoptosis in Ca9-22 cells was significantly higher than that in keratinocytes. H2S exposure in Ca9-22 cells, but not keratinocytes, enhanced the expression of pleckstrin homology-like domain, family A, member 1 (PHLDA1), which was identified through a differential display method. In addition, PHLDA1 expression increased during actinomycin D-induced apoptosis in Ca9-22 cells. Knockdown of PHLDA1 expression by small interfering RNA in Ca9-22 cells led to expression of active caspase 3, thus indicating apoptosis induction. The tongue cancer cell line SCC-25, which expresses PHLDA1 at a high level, showed similar effects. Our data indicate that H2S is an anti-cancer compound that may contribute to the low incidence of oral cancer. Furthermore, we demonstrated the role of PHLDA1 as an apoptosis suppressor.

Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: role of PHLDA1 as an apoptosis suppressor.

Water-insoluble glucan (WIG) produced by mutans streptococci, an important cariogenic pathogen, plays an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Glucanohydrolases, such as mutanase (α-1,3-glucanase) and dextranase (α-1,6-glucanase), are able to hydrolyze WIG. The purposes of this study were to construct bi-functional chimeric glucanase, composed of mutanase and dextranase, and to examine the effects of this chimeric glucanase on the formation and decomposition of biofilm. The mutanase gene from Paenibacillus humicus NA1123 and the dextranase gene from Streptococcus mutans ATCC 25175 were cloned and ligated into a pE-SUMOstar Amp plasmid vector. The resultant his-tagged fusion chimeric glucanase was expressed in Escherichia coli BL21 (DE3) and partially purified. The effects of chimeric glucanase on the formation and decomposition of biofilm formed on a glass surface by Streptococcus sobrinus 6715 glucosyltransferases were then examined. This biofilm was fractionated into firmly adherent, loosely adherent, and non-adherent WIG fractions. Amounts of WIG in each fraction were determined by a phenol-sulfuric acid method, and reducing sugars were quantified by the Somogyi-Nelson method. Chimeric glucanase reduced the formation of the total amount of WIG in a dose-dependent manner, and significant reductions of WIG in the adherent fraction were observed. Moreover, the chimeric glucanase was able to decompose biofilm, being 4.1 times more effective at glucan inhibition of biofilm formation than a mixture of dextranase and mutanase. These results suggest that the chimeric glucanase is useful for prevention of dental biofilm formation.

Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation.

Genes encoding fluoride transporters have been identified in bacterial and archaeal species. The genome sequence of the cariogenic Streptococcus mutans bacteria suggests the presence of a putative fluoride transporter, which is referred to as a chloride channel permease. Two homologues of this gene (GenBank locus tags SMU_1290c and SMU_1289c) reside in tandem in the genome of S. mutans The aim of this study was to determine whether the chloride channel permeases contribute to fluoride resistance. We constructed SMU_1290c- and SMU_1289c-knockout S. mutans UA159 strains. We also constructed a double-knockout strain lacking both genes. SMU_1290c or SMU_1289c was transformed into a fluoride transporter- disrupted Escherichia coli strain. All bacterial strains were cultured under appropriate conditions with or without sodium fluoride, and fluoride resistance was evaluated. All three gene-knockout S. mutans strains showed lower resistance to sodium fluoride than did the wild-type strain. No significant changes in resistance to other sodium halides were recognized between the wild-type and double-knockout strains. Both SMU_1290c and SMU_1289c transformation rescued fluoride transporter-disrupted E. coli cell from fluoride toxicity. We conclude that the chloride channel permeases contribute to fluoride resistance in S. mutans.

Article Total : 2	Year
Publication title	2017
Publication title	2019

Article	Year
Development of a compact and simple gas chromatography for oral malodor measurement. Journal of periodontology\| PubMed ID: 16805675	2006
Effect of green tea on volatile sulfur compounds in mouth air. Journal of nutritional science and vitaminology\| PubMed ID: 18388413	2008
Neither hollow-fibre membrane filters nor activated-charcoal filters remove fluoride from fluoridated tap water. Journal (Canadian Dental Association)\| PubMed ID: 18538069	2008
Diurnal changes in oral malodour among dental-office workers. International dental journal\| PubMed ID: 18630112	2008
Hop polyphenols suppress production of water-insoluble glucan by Streptococcus mutans and dental plaque growth in vivo. The Journal of clinical dentistry\| PubMed ID: 18763691	2008
Oral malodorous compound activates mitochondrial pathway inducing apoptosis in human gingival fibroblasts. Clinical oral investigations\| PubMed ID: 19548012	2010
Oral malodorous compound inhibits osteoblast proliferation. Journal of periodontology\| PubMed ID: 19961386	2009
Effects of a composition containing lactoferrin and lactoperoxidase on oral malodor and salivary bacteria: a randomized, double-blind, crossover, placebo-controlled clinical trial. Clinical oral investigations\| PubMed ID: 20512389	2011
Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: role of PHLDA1 as an apoptosis suppressor. Experimental cell research\| PubMed ID: 24270013	2014
Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation. Microbiology and immunology\| PubMed ID: 25411090	2015
Contribution of chloride channel permease to fluoride resistance in Streptococcus mutans. FEMS microbiology letters\| PubMed ID: 27190286	2016

Takatoshi Murata

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