scholarly journals Raffinose Induces Biofilm Formation by Streptococcus mutans in Low Concentrations of Sucrose by Increasing Production of Extracellular DNA and Fructan

2017 ◽  
Vol 83 (15) ◽  
Author(s):  
Ryo Nagasawa ◽  
Tsutomu Sato ◽  
Hidenobu Senpuku
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Cell Surface Hydrophobicity ◽  
Extracellular Dna ◽  
Regulatory Function ◽  
Extracellular Polysaccharides ◽  
Oral Flora ◽  
Content Type ◽  
Positive Effects ◽  
Low Concentrations

ABSTRACT Streptococcus mutans is the primary etiological agent of dental caries and causes tooth decay by forming a firmly attached biofilm on tooth surfaces. Biofilm formation is induced by the presence of sucrose, which is a substrate for the synthesis of extracellular polysaccharides but not in the presence of oligosaccharides. Nonetheless, in this study, we found that raffinose, which is an oligosaccharide with an intestinal regulatory function and antiallergic effect, induced biofilm formation by S. mutans in a mixed culture with sucrose, which was at concentrations less than those required to induce biofilm formation directly. We analyzed the possible mechanism behind the small requirement for sucrose for biofilm formation in the presence of raffinose. Our results suggested that sucrose contributed to an increase in bacterial cell surface hydrophobicity and biofilm formation. Next, we examined how the effects of raffinose interacted with the effects of sucrose for biofilm formation. We showed that the presence of raffinose induced fructan synthesis by fructosyltransferase and aggregated extracellular DNA (eDNA, which is probably genomic DNA released from dead cells) into the biofilm. eDNA seemed to be important for biofilm formation, because the degradation of DNA by DNase I resulted in a significant reduction in biofilm formation. When assessing the role of fructan in biofilm formation, we found that fructan enhanced eDNA-dependent cell aggregation. Therefore, our results show that raffinose and sucrose have cooperative effects and that this induction of biofilm formation depends on supportive elements that mainly consist of eDNA and fructan. IMPORTANCE The sucrose-dependent mechanism of biofilm formation in Streptococcus mutans has been studied extensively. Nonetheless, the effects of carbohydrates other than sucrose are inadequately understood. Our findings concerning raffinose advance the understanding of the mechanism underlying the joint effects of sucrose and other carbohydrates on biofilm formation. Since raffinose has been reported to have positive effects on enterobacterial flora, research on the effects of raffinose on the oral flora are required prior to its use as a beneficial sugar for human health. Here, we showed that raffinose induced biofilm formation by S. mutans in low concentrations of sucrose. The induction of biofilm formation generally generates negative effects on the oral flora. Therefore, we believe that this finding will aid in the development of more effective oral care techniques to maintain oral flora health.

scholarly journals Molecule Targeting Glucosyltransferase Inhibits Streptococcus mutans Biofilm Formation and Virulence

2015 ◽  
Vol 60 (1) ◽  
pp. 126-135 ◽  
Author(s):  
Zhi Ren ◽  
Tao Cui ◽  
Jumei Zeng ◽  
Lulu Chen ◽  
Wenling Zhang ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Dental Plaque ◽  
Biofilm Formation ◽  
Extracellular Polysaccharides ◽  
Content Type ◽  
Oral Infections ◽  
Concomitant Reduction ◽  
New Strategies

ABSTRACTDental plaque biofilms are responsible for numerous chronic oral infections and cause a severe health burden. Many of these infections cannot be eliminated, as the bacteria in the biofilms are resistant to the host's immune defenses and antibiotics. There is a critical need to develop new strategies to control biofilm-based infections. Biofilm formation inStreptococcus mutansis promoted by major virulence factors known as glucosyltransferases (Gtfs), which synthesize adhesive extracellular polysaccharides (EPS). The current study was designed to identify novel molecules that target Gtfs, thereby inhibitingS. mutansbiofilm formation and having the potential to prevent dental caries. Structure-based virtual screening of approximately 150,000 commercially available compounds against the crystal structure of the glucosyltransferase domain of the GtfC protein fromS. mutansresulted in the identification of a quinoxaline derivative, 2-(4-methoxyphenyl)-N-(3-{[2-(4-methoxyphenyl)ethyl]imino}-1,4-dihydro-2-quinoxalinylidene)ethanamine, as a potential Gtf inhibitor.In vitroassays showed that the compound was capable of inhibiting EPS synthesis and biofilm formation inS. mutansby selectively antagonizing Gtfs instead of by killing the bacteria directly. Moreover, thein vivoanti-caries efficacy of the compound was evaluated in a rat model. We found that the compound significantly reduced the incidence and severity of smooth and sulcal-surface cariesin vivowith a concomitant reduction in the percentage ofS. mutansin the animals' dental plaque (P< 0.05). Taken together, these results represent the first description of a compound that targets Gtfs and that has the capacity to inhibit biofilm formation and the cariogenicity ofS. mutans.

scholarly journals Transcriptional Profiling Reveals the Importance of RcrR in the Regulation of Multiple Sugar Transportation and Biofilm Formation in Streptococcus mutans

mSystems ◽  
2021 ◽  
Author(s):  
Tao Gong ◽  
Xiaoya He ◽  
Jiamin Chen ◽  
Boyu Tang ◽  
Ting Zheng ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Chronic Disease ◽  
Oral Cavity ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Transcriptional Profiling ◽  
Extracellular Polysaccharides ◽  
Tooth Decay ◽  
Content Type ◽  
Cariogenic Bacterium

The human oral cavity is a constantly changing environment. Tooth decay is a commonly prevalent chronic disease mainly caused by the cariogenic bacterium Streptococcus mutans . S. mutans is an oral pathogen that metabolizes various carbohydrates into extracellular polysaccharides (EPSs), biofilm, and tooth-destroying lactic acid.

scholarly journals Catheter-Associated Urinary Tract Infection by Pseudomonas aeruginosa Is Mediated by Exopolysaccharide-Independent Biofilms

2014 ◽  
Vol 82 (5) ◽  
pp. 2048-2058 ◽  
Author(s):  
Stephanie J. Cole ◽  
Angela R. Records ◽  
Mona W. Orr ◽  
Sara B. Linden ◽  
Vincent T. Lee
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Urinary Tract ◽  
Biofilm Formation ◽  
Extracellular Dna ◽  
Extracellular Polysaccharides ◽  
Indwelling Catheters ◽  
Content Type ◽  
Tract Infections

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen that is especially adept at forming surface-associated biofilms.P. aeruginosacauses catheter-associated urinary tract infections (CAUTIs) through biofilm formation on the surface of indwelling catheters.P. aeruginosaencodes three extracellular polysaccharides, PEL, PSL, and alginate, and utilizes the PEL and PSL polysaccharides to form biofilmsin vitro; however, the requirement of these polysaccharides duringin vivoinfections is not well understood. Here we show in a murine model of CAUTI that PAO1, a strain harboringpel,psl, andalggenes, and PA14, a strain harboringpelandalggenes, form biofilms on the implanted catheters. To determine the requirement of exopolysaccharide duringin vivobiofilm infections, we tested isogenic mutants lacking thepel,psl, andalgoperons and showed that PA14 mutants lacking these operons can successfully form biofilms on catheters in the CAUTI model. To determine the host factor(s) that induces the ΔpelDmutant to form biofilm, we tested mouse, human, and artificial urine and show that urine can induce biofilm formation by the PA14 ΔpelDmutant. By testing the major constituents of urine, we show that urea can induce apel-,psl-, andalg-independent biofilm. Thesepel-,psl-, andalg-independent biofilms are mediated by the release of extracellular DNA. Treatment of biofilms formed in urea with DNase I reduced the biofilm, indicating that extracellular DNA supports biofilm formation. Our results indicate that the opportunistic pathogenP. aeruginosautilizes a distinct program to form biofilms that are independent of exopolysaccharides during CAUTI.

scholarly journals AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Chiau-Jing Jung ◽  
Ron-Bin Hsu ◽  
Chia-Tung Shun ◽  
Chih-Chieh Hsu ◽  
Jean-San Chia
Keyword(s):  
Infective Endocarditis ◽  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Rat Model ◽  
Calcium Ions ◽  
Biofilm Formation ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Wild Type ◽  
Content Type

ABSTRACT Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (ΔatlA) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an ΔatlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the ΔatlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in ΔatlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans, which contributes to biofilm formation in infective endocarditis.

scholarly journals Competence-Stimulating-Peptide-Dependent Localized Cell Death and Extracellular DNA Production in Streptococcus mutans Biofilms

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Ryo Nagasawa ◽  
Tatsuya Yamamoto ◽  
Andrew S. Utada ◽  
Nobuhiko Nomura ◽  
Nozomu Obana
Keyword(s):  
Cell Death ◽  
Single Cell ◽  
Streptococcus Mutans ◽  
Structural Stability ◽  
Biofilm Formation ◽  
Extracellular Polymeric Substances ◽  
Imaging Techniques ◽  
Extracellular Dna ◽  
Content Type ◽  
Cariogenic Bacterium

ABSTRACT Extracellular DNA (eDNA) is a biofilm component that contributes to the formation and structural stability of biofilms. Streptococcus mutans, a major cariogenic bacterium, induces eDNA-dependent biofilm formation under specific conditions. Since cell death can result in the release and accumulation of DNA, the dead cells in biofilms are a source of eDNA. However, it remains unknown how eDNA is released from dead cells and is localized within S. mutans biofilms. We focused on cell death induced by the extracellular signaling peptide called competence-stimulating peptide (CSP). We demonstrate that nucleic acid release into the extracellular environment occurs in a subpopulation of dead cells. eDNA production induced by CSP was highly dependent on the lytF gene, which encodes an autolysin. Although lytF expression was induced bimodally by CSP, lytF-expressing cells further divided into surviving cells and eDNA-producing dead cells. Moreover, we found that lytF-expressing cells were abundant near the bottom of the biofilm, even when all cells in the biofilm received the CSP signal. Dead cells and eDNA were also abundantly present near the bottom of the biofilm. The number of lytF-expressing cells in biofilms was significantly higher than that in planktonic cultures, which suggests that adhesion to the substratum surface is important for the induction of lytF expression. The deletion of lytF resulted in reduced adherence to a polystyrene surface. These results suggest that lytF expression and eDNA production induced near the bottom of the biofilm contribute to a firmly attached and structurally stable biofilm. IMPORTANCE Bacterial communities encased by self-produced extracellular polymeric substances (EPSs), known as biofilms, have a wide influence on human health and environmental problems. The importance of biofilm research has increased, as biofilms are the preferred bacterial lifestyle in nature. Furthermore, in recent years it has been noted that the contribution of phenotypic heterogeneity within biofilms requires analysis at the single-cell or subpopulation level to understand bacterial life strategies. In Streptococcus mutans, a cariogenic bacterium, extracellular DNA (eDNA) contributes to biofilm formation. However, it remains unclear how and where the cells produce eDNA within the biofilm. We focused on LytF, an autolysin that is induced by extracellular peptide signals. We used single-cell level imaging techniques to analyze lytF expression in the biofilm population. Here, we show that S. mutans generates eDNA by inducing lytF expression near the bottom of the biofilm, thereby enhancing biofilm adhesion and structural stability.

scholarly journals Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance

2016 ◽  
Vol 198 (7) ◽  
pp. 1087-1100 ◽  
Author(s):  
Gursonika Binepal ◽  
Kamal Gill ◽  
Paula Crowley ◽  
Martha Cordova ◽  
L. Jeannine Brady ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Streptococcus Mutans ◽  
Transport System ◽  
Biofilm Formation ◽  
Cellular Response ◽  
Pathogenic Bacteria ◽  
Transport Systems ◽  
Content Type ◽  
Growth And Survival ◽  
Dental Biofilm

ABSTRACTPotassium (K+) is the most abundant cation in the fluids of dental biofilm. The biochemical and biophysical functions of K+and a variety of K+transport systems have been studied for most pathogenic bacteria but not for oral pathogens. In this study, we establish the modes of K+acquisition inStreptococcus mutansand the importance of K+homeostasis for its virulence attributes. TheS. mutansgenome harbors four putative K+transport systems that included two Trk-like transporters (designated Trk1 and Trk2), one glutamate/K+cotransporter (GlnQHMP), and a channel-like K+transport system (Kch). Mutants lacking Trk2 had significantly impaired growth, acidogenicity, aciduricity, and biofilm formation. [K+] less than 5 mM eliminated biofilm formation inS. mutans. The functionality of the Trk2 system was confirmed by complementing anEscherichia coliTK2420 mutant strain, which resulted in significant K+accumulation, improved growth, and survival under stress. Taken together, these results suggest that Trk2 is the main facet of the K+-dependent cellular response ofS. mutansto environment stresses.IMPORTANCEBiofilm formation and stress tolerance are important virulence properties of caries-causingStreptococcus mutans. To limit these properties of this bacterium, it is imperative to understand its survival mechanisms. Potassium is the most abundant cation in dental plaque, the natural environment ofS. mutans. K+is known to function in stress tolerance, and bacteria have specialized mechanisms for its uptake. However, there are no reports to identify or characterize specific K+transporters inS. mutans. We identified the most important system for K+homeostasis and its role in the biofilm formation, stress tolerance, and growth. We also show the requirement of environmental K+for the activity of biofilm-forming enzymes, which explains why such high levels of K+would favor biofilm formation.

scholarly journals A Novel Gene Involved in the Survival of Streptococcus mutans under Stress Conditions

2013 ◽  
Vol 80 (1) ◽  
pp. 97-103 ◽  
Author(s):  
Dan Li ◽  
Yukie Shibata ◽  
Toru Takeshita ◽  
Yoshihisa Yamashita
Keyword(s):  
Streptococcus Mutans ◽  
Streptococcus Pyogenes ◽  
Biofilm Formation ◽  
Acid Tolerance ◽  
Stress Conditions ◽  
Insertion Mutagenesis ◽  
Trna Modification ◽  
Content Type ◽  
Virulence Proteins ◽  
Random Insertion

ABSTRACTAStreptococcus mutansmutant defective in aciduricity was constructed by random-insertion mutagenesis. Sequence analysis of the mutant revealed a mutation ingidA, which is known to be involved in tRNA modification inStreptococcus pyogenes. Complementation ofgidAbyS. pyogenesgidArecovered the acid tolerance ofS. mutans. Although thegidA-inactivatedS. pyogenesmutant exhibited significantly reduced expression of multiple extracellular virulence proteins, theS. mutansmutant did not. On the other hand, thegidAmutant ofS. mutansshowed reduced ability to withstand exposure to other stress conditions (high osmotic pressure, high temperature, and bacitracin stress) besides an acidic environment. In addition, loss of GidA decreased the capacity for glucose-dependent biofilm formation by over 50%. This study revealed thatgidAplays critical roles in the survival ofS. mutansunder stress conditions, including lower pH.

scholarly journals Essential Roles of thesppRAFructose-Phosphate Phosphohydrolase Operon in Carbohydrate Metabolism and Virulence Expression byStreptococcus mutans

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Lin Zeng ◽  
Robert A. Burne
Keyword(s):  
Dental Caries ◽  
Organic Acids ◽  
Streptococcus Mutans ◽  
Biochemical Characterization ◽  
Constitutive Expression ◽  
Extracellular Dna ◽  
Tooth Surface ◽  
Oral Diseases ◽  
Sugar Phosphate ◽  
Content Type

ABSTRACTThe dental caries pathogenStreptococcus mutanscan ferment a variety of sugars to produce organic acids. Exposure ofS. mutansto certain nonmetabolizable carbohydrates, such as xylitol, impairs growth and can cause cell death. Recently, the presence of a sugar-phosphate stress inS. mutanswas demonstrated using a mutant lacking 1-phosphofructokinase (FruK) that accumulates fructose-1-phosphate (F-1-P). Here, we studied an operon inS. mutans,sppRA, which was highly expressed in thefruKmutant. Biochemical characterization of a recombinant SppA protein indicated that it possessed hexose-phosphate phosphohydrolase activity, with preferences for F-1-P and, to a lesser degree, fructose-6-phosphate (F-6-P). SppA activity was stimulated by Mg2+and Mn2+but inhibited by NaF. SppR, a DeoR family regulator, repressed the expression of thesppRAoperon to minimum levels in the absence of the fructose-derived metabolite F-1-P and likely also F-6-P. The accumulation of F-1-P, as a result of growth on fructose, not only inducedsppAexpression, but it significantly altered biofilm maturation through increased cell lysis and enhanced extracellular DNA release. Constitutive expression ofsppA, via a plasmid or by deletingsppR, greatly alleviated fructose-induced stress in afruKmutant, enhanced resistance to xylitol, and reversed the effects of fructose on biofilm formation. Finally, by identifying three additional putative phosphatases that are capable of promoting sugar-phosphate tolerance, we show thatS. mutansis capable of mounting a sugar-phosphate stress response by modulating the levels of certain glycolytic intermediates, functions that are interconnected with the ability of the organism to manifest key virulence behaviors.IMPORTANCEStreptococcus mutansis a major etiologic agent for dental caries, primarily due to its ability to form biofilms on the tooth surface and to convert carbohydrates into organic acids. We have discovered a two-gene operon inS. mutansthat regulates fructose metabolism by controlling the levels of fructose-1-phosphate, a potential signaling compound that affects bacterial behaviors. With fructose becoming increasingly common and abundant in the human diet, we reveal the ways that fructose may alter bacterial development, stress tolerance, and microbial ecology in the oral cavity to promote oral diseases.

scholarly journals Effects of pH on the Properties of Membrane Vesicles Including Glucosyltransferase in Streptococcus mutans

2021 ◽  
Vol 9 (11) ◽  
pp. 2308
Author(s):  
Yusuke Iwabuchi ◽  
Tomoyo Nakamura ◽  
Yasuka Kusumoto ◽  
Ryoma Nakao ◽  
Tsutomu Iwamoto ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Membrane Vesicles ◽  
Extracellular Dna ◽  
Tooth Surface ◽  
Initial Ph ◽  
Effects Of Ph ◽  
Low Levels ◽  
Quantitative Changes ◽  
Biofilm Development

Streptococcus mutans releases membrane vesicles (MVs) and induces MV-dependent biofilm formation. Glucosyltransferases (Gtfs) are bound to MVs and contribute to the adhesion and glucans-dependent biofilm formation of early adherent bacteria on the tooth surface. The biofilm formation of S. mutans may be controlled depending on whether the initial pH tends to be acidic or alkaline. In this study, the characteristics and effects of MVs extracted from various conditions {(initial pH 6.0 and 8.0 media prepared with lactic acid (LA) and acetic acid (AA), and with NaOH (NO), respectively)} on the biofilm formation of S. mutans and early adherent bacteria were investigated. The quantitative changes in glucans between primary pH 6.0 and 8.0 conditions were observed, associated with different activities affecting MV-dependent biofilm formation. The decreased amount of Gtfs on MVs under the initial pH 6.0 conditions strongly guided low levels of MV-dependent biofilm formation. However, in the initial pH 6.0 and 8.0 solutions prepared with AA and NO, the MVs in the biofilm appeared to be formed by the expression of glucans and/or extracellular DNA. These results suggest that the environmental pH conditions established by acid and alkaline factors determine the differences in the local pathogenic activities of biofilm development in the oral cavity.

scholarly journals Ursolic Acid Targets Glucosyltransferase and Inhibits Its Activity to Prevent Streptococcus mutans Biofilm Formation

2021 ◽  
Vol 12 ◽  
Author(s):  
Yucui Liu ◽  
Yanxin Huang ◽  
Cong Fan ◽  
Zhongmei Chi ◽  
Miao Bai ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Ursolic Acid ◽  
Biofilm Formation ◽  
Natural Compound ◽  
Structural Integrity ◽  
Site Directed Mutagenesis ◽  
Extracellular Polysaccharides ◽  
Virulence Determinants ◽  
Pentacyclic Triterpene ◽  
Catalytic Center

Streptococcus mutans (S. mutans), the prime pathogen of dental caries, can secrete glucosyltransferases (GTFs) to synthesize extracellular polysaccharides (EPSs), which are the virulence determinants of cariogenic biofilms. Ursolic acid, a type of pentacyclic triterpene natural compound, has shown potential antibiofilm effects on S. mutans. To investigate the mechanisms of ursolic acid-mediated inhibition of S. mutans biofilm formation, we first demonstrated that ursolic acid could decrease the viability and structural integrity of biofilms, as evidenced by XTT, crystal violet, and live/dead staining assays. Then, we further revealed that ursolic acid could compete with the inherent substrate to occupy the catalytic center of GTFs to inhibit EPS formation, and this was confirmed by GTF activity assays, computer simulations, site-directed mutagenesis, and capillary electrophoresis (CE). In conclusion, ursolic acid can decrease bacterial viability and prevent S. mutans biofilm formation by binding and inhibiting the activity of GTFs.

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