Coordinated Regulation of the EIIMan and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways

ABSTRACTThe glucose/mannose-phosphotransferase system (PTS) permease EIIManencoded bymanLMNin the dental caries pathogenStreptococcus mutanshas a dominant influence on sugar-specific, CcpA-independent catabolite repression (CR). Mutations inmanLaffect energy metabolism and virulence-associated traits, including biofilm formation, acid tolerance, and competence. Using promoter::reporter fusions, expression of themanLMNand thefruRKIoperons, encoding a transcriptional regulator, a fructose-1-phosphate kinase and a fructose-PTS permease EIIFru, respectively, was monitored in response to carbohydrate source and in mutants lacking CcpA, FruR, and components of EIIMan. Expression of genes for EIIManand EIIFruwas directly regulated by CcpA and CR, as evinced byin vivoandin vitromethods. Unexpectedly, not only was thefruRKIoperon negatively regulated by FruR, but also so wasmanLMN. Carbohydrate transport by EIIManhad a negative influence on expression ofmanLMNbut notfruRKI. In agreement with the proposed role of FruR in regulating these PTS operons, loss offruRorfruKsubstantially altered growth on a number of carbohydrates, including fructose. RNA deep sequencing revealed profound changes in gene regulation caused by deletion offruKorfruR. Collectively, these findings demonstrate intimate interconnection of the regulation of two major PTS permeases inS. mutansand reveal novel and important contributions of fructose metabolism to global regulation of gene expression.IMPORTANCEThe ability ofStreptococcus mutansand other streptococcal pathogens to survive and cause human diseases is directly dependent upon their capacity to metabolize a variety of carbohydrates, including glucose and fructose. Our research reveals that metabolism of fructose has broad influences on the regulation of utilization of glucose and other sugars, and mutants with changes in certain genes involved in fructose metabolism display profoundly different abilities to grow and express virulence-related traits. Mutants lacking the FruR regulator or a particular phosphofructokinase, FruK, display changes in expression of a large number of genes encoding transcriptional regulators, enzymes required for energy metabolism, biofilm development, biosynthetic and degradative processes, and tolerance of a spectrum of environmental stressors. Since fructose is a major component of the modern human diet, the results have substantial significance in the context of oral health and the development of dental caries.

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Symbiotic Relationship between Streptococcus mutans and Candida albicans Synergizes Virulence of Plaque BiofilmsIn Vivo

Infection and Immunity ◽

10.1128/iai.00087-14 ◽

2014 ◽

Vol 82 (5) ◽

pp. 1968-1981 ◽

Cited By ~ 216

Author(s):

Megan L. Falsetta ◽

Marlise I. Klein ◽

Punsiri M. Colonne ◽

Kathleen Scott-Anne ◽

Stacy Gregoire ◽

...

Keyword(s):

Candida Albicans ◽

Dental Caries ◽

Streptococcus Mutans ◽

Bacterial Pathogen ◽

Single Species ◽

Content Type ◽

3 Dimensional ◽

Biofilm Development

ABSTRACTStreptococcus mutansis often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC).S. mutansmay not act alone;Candida albicanscells are frequently detected along with heavy infection byS. mutansin plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhancedin vitroandin vivo. The presence ofC. albicansaugments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viableS. mutanscells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeableS. mutansmicrocolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Ourin vitrodata also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence withC. albicansinduces the expression of virulence genes inS. mutans(e.g.,gtfB,fabM). We also found thatCandida-derived β1,3-glucans contribute to the EPS matrix structure, while fungal mannan and β-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.

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Synergism of Streptococcus mutans and Candida albicans Reinforces Biofilm Maturation and Acidogenicity in Saliva: An In Vitro Study

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.623980 ◽

2021 ◽

Vol 10 ◽

Author(s):

Hye-Eun Kim ◽

Yuan Liu ◽

Atul Dhall ◽

Marwa Bawazir ◽

Hyun Koo ◽

...

Keyword(s):

Candida Albicans ◽

Dental Caries ◽

Streptococcus Mutans ◽

Critical Role ◽

In Vitro Study ◽

Acidic Ph ◽

Acidic Environment ◽

Symbiotic Interaction ◽

Biofilm Development

Early childhood caries, a virulent-form of dental caries, is painful, difficult, and costly to treat that has been associated with high levels of Streptococcus mutans (Sm) and Candida albicans (Ca) in plaque-biofilms on teeth. These microorganisms appear to develop a symbiotic cross-kingdom interaction that amplifies the virulence of plaque-biofilms. Although biofilm studies reveal synergistic bacterial-fungal association, how these organisms modulate cross-kingdom biofilm formation and enhance its virulence in the presence of saliva remain largely unknown. Here, we compared the properties of Sm and Sm-Ca biofilms cultured in saliva by examining the biofilm structural organization and capability to sustain an acidic pH environment conducive to enamel demineralization. Intriguingly, Sm-Ca biofilm is rapidly matured and maintained acidic pH-values (~4.3), while Sm biofilm development was retarded and failed to create an acidic environment when cultured in saliva. In turn, the human enamel slab surface was severely demineralized by Sm-Ca biofilms, while there was minimal damage to the enamel surface by Sm biofilm. Interestingly, Sm-Ca biofilms exhibited an acidic environment regardless of their hyphal formation ability. Our data reveal the critical role of symbiotic interaction between S. mutans and C. albicans in human saliva in the context of pathogenesis of dental caries, which may explain how the cross-kingdom interaction contributes to enhanced virulence of plaque-biofilm in the oral cavity.

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Amino Sugars Enhance the Competitiveness of Beneficial Commensals with Streptococcus mutans through Multiple Mechanisms

Applied and Environmental Microbiology ◽

10.1128/aem.00637-16 ◽

2016 ◽

Vol 82 (12) ◽

pp. 3671-3682 ◽

Cited By ~ 17

Author(s):

Lin Zeng ◽

Tanaz Farivar ◽

Robert A. Burne

Keyword(s):

Dental Caries ◽

Streptococcus Mutans ◽

Sugar Metabolism ◽

Amino Sugar ◽

Amino Sugars ◽

Mrna Levels ◽

Oral Streptococci ◽

Streptococcus Gordonii ◽

Carbohydrate Source ◽

Content Type

ABSTRACTBiochemical and genetic aspects of the metabolism of the amino sugarsN-acetylglucosamine (GlcNAc) and glucosamine (GlcN) by commensal oral streptococci and the effects of these sugars on interspecies competition with the dental caries pathogenStreptococcus mutanswere explored. MultipleS. mutanswild-type isolates displayed long lag phases when transferred from glucose-containing medium to medium with GlcNAc as the primary carbohydrate source, but commensal streptococci did not. Competition in liquid coculture or dual-species biofilms betweenS. mutansandStreptococcus gordoniishowed thatS. gordoniiwas particularly dominant when the primary carbohydrate was GlcN or GlcNAc. Transcriptional and enzymatic assays showed that the catabolic pathway for GlcNAc was less highly induced inS. mutansthan inS. gordonii. Exposure to H2O2, which is produced byS. gordoniiand antagonizes the growth ofS. mutans, led to reduced mRNA levels ofnagAandnagBinS. mutans. When the gene for the transcriptional regulatory NagR was deleted inS. gordonii, the strain produced constitutively high levels ofnagA(GlcNAc-6-P deacetylase),nagB(GlcN-6-P deaminase), andglmS(GlcN-6-P synthase) mRNA. Similar to NagR ofS. mutans(NagRSm), theS. gordoniiNagR protein (NagRSg) could bind to consensus binding sites (dre) in thenagA,nagB, andglmSpromoter regions ofS. gordonii. Notably, NagRSgbinding was inhibited by GlcN-6-P, but G-6-P had no effect, unlike for NagRSm. This study expands the understanding of amino sugar metabolism and NagR-dependent gene regulation in streptococci and highlights the potential for therapeutic applications of amino sugars to prevent dental caries.IMPORTANCEAmino sugars are abundant in the biosphere, so the relative efficiency of particular bacteria in a given microbiota to metabolize these sources of carbon and nitrogen might have a profound impact on the ecology of the community. Our investigation reveals that several oral commensal bacteria have a much greater capacity to utilize amino sugars than the dental pathogenStreptococcus mutansand that the ability of the model commensalStreptococcus gordoniito compete againstS. mutansis substantively enhanced by the presence of amino sugars commonly found in the oral cavity. The mechanisms underlying the greater capacity and competitive enhancements of the commensal are shown to depend on how the genes for the catabolic enzymes are regulated, the role of the allosteric modulators affecting such regulation, and the ability of amino sugars to enhance certain activities of the commensal that are antagonistic toS. mutans.

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l-Arginine Modifies the Exopolysaccharide Matrix and Thwarts Streptococcus mutans Outgrowth within Mixed-Species Oral Biofilms

Journal of Bacteriology ◽

10.1128/jb.00021-16 ◽

2016 ◽

Vol 198 (19) ◽

pp. 2651-2661 ◽

Cited By ~ 41

Author(s):

Jinzhi He ◽

Geelsu Hwang ◽

Yuan Liu ◽

Lizeng Gao ◽

LaTonya Kilpatrick-Liverman ◽

...

Keyword(s):

Dental Caries ◽

Streptococcus Mutans ◽

Biological Properties ◽

Vehicle Control ◽

Microbial Interactions ◽

Mixed Species ◽

Matrix Assembly ◽

Content Type ◽

Biofilm Development ◽

Biofilm Matrix

ABSTRACTl-Arginine, a ubiquitous amino acid in human saliva, serves as a substrate for alkali production by arginolytic bacteria. Recently, exogenousl-arginine has been shown to enhance the alkalinogenic potential of oral biofilm and destabilize its microbial community, which might help control dental caries. However,l-arginine exposure may inflict additional changes in the biofilm milieu when bacteria are growing under cariogenic conditions. Here, we investigated how exogenousl-arginine modulates biofilm development using a mixed-species model containing both cariogenic (Streptococcus mutans) and arginolytic (Streptococcus gordonii) bacteria in the presence of sucrose. We observed that 1.5% (wt/vol)l-arginine (also a clinically effective concentration) exposure suppressed the outgrowth ofS. mutans, favoredS. gordoniidominance, and maintainedActinomyces naeslundiigrowth within biofilms (versus vehicle control). In parallel, topicall-arginine treatments substantially reduced the amounts of insoluble exopolysaccharides (EPS) by >3-fold, which significantly altered the three-dimensional (3D) architecture of the biofilm. Intriguingly,l-arginine repressedS. mutansgenes associated with insoluble EPS (gtfB) and bacteriocin (SMU.150) production, whilespxBexpression (H2O2production) byS. gordoniiincreased sharply during biofilm development, which resulted in higher H2O2levels in arginine-treated biofilms. These modifications resulted in a markedly defective EPS matrix and areas devoid of any bacterial clusters (microcolonies) on the apatitic surface, while thein situpH values at the biofilm-apatite interface were nearly one unit higher in arginine-treated biofilms (versus the vehicle control). Our data reveal new biological properties ofl-arginine that impact biofilm matrix assembly and the dynamic microbial interactions associated with pathogenic biofilm development, indicating the multiaction potency of this promising biofilm disruptor.IMPORTANCEDental caries is one of the most prevalent and costly infectious diseases worldwide, caused by a biofilm formed on tooth surfaces. Novel strategies that compromise the ability of virulent species to assemble and maintain pathogenic biofilms could be an effective alternative to conventional antimicrobials that indiscriminately kill other oral species, including commensal bacteria.l-Arginine at 1.5% has been shown to be clinically effective in modulating cariogenic biofilms via alkali production by arginolytic bacteria. Using a mixed-species ecological model, we show new mechanisms by whichl-arginine disrupts the process of biofilm matrix assembly and the dynamic microbial interactions that are associated with cariogenic biofilm development, without impacting the bacterial viability. These results may aid in the development of enhanced methods to control biofilms usingl-arginine.

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Sucrose- and Fructose-Specific Effects on the Transcriptome of Streptococcus mutans, as Determined by RNA Sequencing

Applied and Environmental Microbiology ◽

10.1128/aem.02681-15 ◽

2015 ◽

Vol 82 (1) ◽

pp. 146-156 ◽

Cited By ~ 19

Author(s):

Lin Zeng ◽

Robert A. Burne

Keyword(s):

Gene Expression ◽

Streptococcus Mutans ◽

Regulation Of Gene Expression ◽

Etiologic Agent ◽

Sucrose Transport ◽

Carbohydrate Source ◽

Content Type ◽

Specific Effects ◽

Genome Scale ◽

The Impact

ABSTRACTRecent genome-scale studies have begun to establish the scope and magnitude of the impacts of carbohydrate source and availability on the regulation of gene expression in bacteria. The effects of sugars on gene expression are particularly profound in a group of lactic acid bacteria that rely almost entirely on their saccharolytic activities for energy production and growth. ForStreptococcus mutans, the major etiologic agent of human dental caries, sucrose is the carbohydrate that contributes in the most significant manner to establishment, persistence, and virulence of the organism. However, because this organism produces multiple extracellular sucrolytic enzymes that can release hexoses from sucrose, it has not been possible to study the specific effects of sucrose transport and metabolism on gene expression in the absence of carbohydrates that by themselves can elicit catabolite repression and induce expression of multiple genes. By employing RNA deep-sequencing (RNA-Seq) technology and mutants that lacked particular sucrose-metabolizing enzymes, we compared the transcriptomes ofS. mutansbacteria growing on glucose, fructose, or sucrose as the sole carbohydrate source. The results provide a variety of new insights into the impact of sucrose transport and metabolism byS. mutans, including the likely expulsion of fructose after sucrose internalization and hydrolysis, and identify a set of genes that are differentially regulated by sucrose versus fructose. The findings significantly enhance our understanding of the genetics and physiology of this cariogenic pathogen.

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Glycosyltransferase-Mediated Biofilm Matrix Dynamics and Virulence ofStreptococcus mutans

Applied and Environmental Microbiology ◽

10.1128/aem.02247-18 ◽

2018 ◽

Vol 85 (5) ◽

Cited By ~ 13

Author(s):

Katherine Rainey ◽

Suzanne M. Michalek ◽

Zezhang T. Wen ◽

Hui Wu

Keyword(s):

Dental Caries ◽

Streptococcus Mutans ◽

Virulence Factor ◽

Membrane Vesicles ◽

Tooth Decay ◽

Content Type ◽

Matrix Components ◽

Biofilm Development ◽

Biofilm Matrix ◽

Matrix Dynamics

ABSTRACTStreptococcus mutansis a key cariogenic bacterium responsible for the initiation of tooth decay. Biofilm formation is a crucial virulence property. We discovered a putative glycosyltransferase, SMU_833, inS. mutanscapable of modulating dynamic interactions between two key biofilm matrix components, glucan and extracellular DNA (eDNA). The deletion ofsmu_833decreases glucan and increases eDNA but maintains the overall biofilm biomass. The decrease in glucan is caused by a reduction in GtfB and GtfC, two key enzymes responsible for the synthesis of glucan. The increase in eDNA was accompanied by an elevated production of membrane vesicles, suggesting that SMU_833 modulates the release of eDNA via the membrane vesicles, thereby altering biofilm matrix constituents. Furthermore, glucan and eDNA were colocalized. The complete deletion ofgtfBCfrom thesmu_833mutant significantly reduced the biofilm biomass despite the elevated eDNA, suggesting the requirement of minimal glucans as a binding substrate for eDNA within the biofilm. Despite no changes in overall biofilm biomass, the mutant biofilm was altered in biofilm architecture and was less acidicin vitro. Concurrently, the mutant was less virulent in anin vivorat model of dental caries, demonstrating that SMU_833 is a new virulence factor. Taken together, we conclude that SMU_833 is required for optimal biofilm development and virulence ofS. mutansby modulating extracellular matrix components. Our study of SMU_833-modulated biofilm matrix dynamics uncovered a new target that can be used to develop potential therapeutics that prevent and treat dental caries.IMPORTANCETooth decay, a costly and painful disease affecting the vast majority of people worldwide, is caused by the bacteriumStreptococcus mutans. The bacteria utilize dietary sugars to build and strengthen biofilms, trapping acids onto the tooth’s surface and causing demineralization and decay of teeth. As knowledge of our body’s microbiomes increases, the need for developing therapeutics targeted to disease-causing bacteria has arisen. The significance of our research is in studying and identifying a novel therapeutic target, a dynamic biofilm matrix that is mediated by a new virulence factor and membrane vesicles. The study increases our understanding ofS. mutansvirulence and also offers a new opportunity to develop effective therapeutics targetingS. mutans. In addition, the mechanisms of membrane vesicle-mediated biofilm matrix dynamics are also applicable to other biofilm-driven infectious diseases.

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Different Roles of EIIABMan and EIIGlc in Regulation of Energy Metabolism, Biofilm Development, and Competence in Streptococcus mutans

Journal of Bacteriology ◽

10.1128/jb.00169-06 ◽

2006 ◽

Vol 188 (11) ◽

pp. 3748-3756 ◽

Cited By ~ 95

Author(s):

Jacqueline Abranches ◽

Melissa M. Candella ◽

Zezhang T. Wen ◽

Henry V. Baker ◽

Robert A. Burne

Keyword(s):

Energy Metabolism ◽

Streptococcus Mutans ◽

Energy Levels ◽

Sugar Metabolism ◽

Oral Streptococci ◽

Phosphotransferase System ◽

Exogenous Dna ◽

Biofilm Development ◽

Metabolism Gene

ABSTRACT The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major carbohydrate transport system in oral streptococci. The mannose-PTS of Streptococcus mutans, which transports mannose and glucose, is involved in carbon catabolite repression (CCR) and regulates the expression of known virulence genes. In this study, we investigated the role of EIIGlc and EIIABMan in sugar metabolism, gene regulation, biofilm formation, and competence. The results demonstrate that the inactivation of ptsG, encoding a putative EIIGlc, did not lead to major changes in sugar metabolism or affect the phenotypes of interest. However, the loss of EIIGlc was shown to have a significant impact on the proteome and to affect the expression of a known virulence factor, fructan hydrolase (fruA). JAM1, a mutant strain lacking EIIABMan, had an impaired capacity to form biofilms in the presence of glucose and displayed a decreased ability to be transformed with exogenous DNA. Also, the lactose- and cellobiose-PTSs were positively and negatively regulated by EIIABMan, respectively. Microarrays were used to investigate the profound phenotypic changes displayed by JAM1, revealing that EIIABMan of S. mutans has a key regulatory role in energy metabolism, possibly by sensing the energy levels of the cells or the carbohydrate availability and, in response, regulating the activity of transcription factors and carbohydrate transporters.

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Uptake and Metabolism ofN-Acetylglucosamine and Glucosamine by Streptococcus mutans

Applied and Environmental Microbiology ◽

10.1128/aem.00820-14 ◽

2014 ◽

Vol 80 (16) ◽

pp. 5053-5067 ◽

Cited By ~ 39

Author(s):

Zachary D. Moye ◽

Robert A. Burne ◽

Lin Zeng

Keyword(s):

Streptococcus Mutans ◽

De Novo ◽

Dominant Role ◽

Acid Tolerance ◽

Amino Sugars ◽

Phosphotransferase System ◽

Pathogenic Potential ◽

Content Type

ABSTRACTGlucosamine andN-acetylglucosamine are among the most abundant sugars on the planet, and their introduction into the oral cavity via the diet and host secretions, and through bacterial biosynthesis, provides oral biofilm bacteria with a source of carbon, nitrogen, and energy. In this study, we demonstrated that the dental caries pathogenStreptococcus mutanspossesses an inducible system for the metabolism ofN-acetylglucosamine and glucosamine. These amino sugars are transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS), with the glucose/mannose enzyme II permease encoded bymanLMNplaying a dominant role. Additionally, a previously uncharacterized gene product encoded downstream of themanLMNoperon, ManO, was shown to influence the efficiency of uptake and growth onN-acetylglucosamine and, to a lesser extent, glucosamine. A transcriptional regulator, designated NagR, was able to bind the promoter regionsin vitro, and repress the expressionin vivo, of thenagAandnagBgenes, encodingN-acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase, respectively. The binding activity of NagR could be inhibited by glucosamine-6-phosphatein vitro. Importantly, in contrast to the case with certain otherFirmicutes, the gene forde novosynthesis of glucosamine-6-phosphate inS. mutans,glmS, was also shown to be regulated by NagR, and NagR could bind theglmSpromoter regionin vitro. Finally, metabolism of these amino sugars byS. mutansresulted in the production of significant quantities of ammonia, which can neutralize cytoplasmic pH and increase acid tolerance, thus contributing to enhanced persistence and pathogenic potential.

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Novel Antibiofilm Chemotherapy Targets Exopolysaccharide Synthesis and Stress Tolerance in Streptococcus mutans To Modulate Virulence ExpressionIn Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01381-12 ◽

2012 ◽

Vol 56 (12) ◽

pp. 6201-6211 ◽

Cited By ~ 41

Author(s):

Megan L. Falsetta ◽

Marlise I. Klein ◽

José A. Lemos ◽

Bruno B. Silva ◽

Senyo Agidi ◽

...

Keyword(s):

Dental Caries ◽

Streptococcus Mutans ◽

Positive Control ◽

Sprague Dawley ◽

Content Type ◽

Treatment Regimens ◽

Novel Target ◽

Underlying Mechanisms

ABSTRACTFluoride is the mainstay of dental caries prevention, and yet current applications offer incomplete protection and may not effectively address the infectious character of the disease. Therefore, we evaluated the effectiveness of a novel combination therapy (CT; 2 mM myricetin, 4 mMtt-farnesol, 250 ppm of fluoride) that supplements fluoride with naturally occurring, food-derived, antibiofilm compounds. Treatment regimens simulating those experienced clinically (twice daily for ≤60 s) were used bothin vitroover a saliva-coated hydroxyapatite biofilm model andin vivowith a rodent model of dental caries. The effectiveness of CT was evaluated based on the incidence and severity of carious lesions (compared to fluoride or vehicle control). We found that CT was superior to fluoride (positive control,P< 0.05); topical applications dramatically reduced caries development in Sprague-Dawley rats, all without altering theStreptococcus mutansor total populations within the plaque. We subsequently identified the underlying mechanisms through which applications of CT modulate biofilm virulence. CT targets expression of keyStreptococcus mutansgenes during biofilm formationin vitroandin vivo. These are associated with exopolysaccharide matrix synthesis (gtfB) and the ability to tolerate exogenous stress (e.g.,sloA), which are essential for cariogenic biofilm assembly. We also identified a unique gene (SMU.940) that was severely repressed and may represent a potentially novel target; its inactivation disrupted exopolysaccharide accumulation and matrix development. Altogether, CT may be clinically more effective than current anticaries modalities, targeting expression of bacterial virulence associated with pathogenesis of the disease. These observations may have relevance for development of enhanced therapies against other biofilm-dependent infections.

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Effects of Carbohydrate Source on Genetic Competence in Streptococcus mutans

Applied and Environmental Microbiology ◽

10.1128/aem.01205-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4821-4834 ◽

Cited By ~ 27

Author(s):

Zachary D. Moye ◽

Minjun Son ◽

Ariana E. Rosa-Alberty ◽

Lin Zeng ◽

Sang-Joon Ahn ◽

...

Keyword(s):

Dental Caries ◽

Signaling Pathways ◽

Streptococcus Mutans ◽

Fluorescent Protein ◽

Single Cell Analysis ◽

Virulence Determinants ◽

Pathogenic Potential ◽

Carbohydrate Source ◽

Genetic Competence ◽

Content Type

ABSTRACTThe capacity to internalize and catabolize carbohydrates is essential for dental caries pathogens to persist and cause disease. The expression of many virulence-related attributes byStreptococcus mutans, an organism strongly associated with human dental caries, is influenced by the peptide signaling pathways that control genetic competence. Here, we demonstrate a relationship between the efficiency of competence signaling and carbohydrate source. A significant increase in the activity of the promoters forcomX,comS, andcomYAafter exposure to competence-stimulating peptide (CSP) was observed in cells growing on fructose, maltose, sucrose, or trehalose as the primary carbohydrate source, compared to cells growing on glucose. However, only cells grown in the presence of trehalose or sucrose displayed a significant increase in transformation frequency. Notably, even low concentrations of these carbohydrates in the presence of excess glucose could enhance the expression ofcomX, encoding a sigma factor needed for competence, and the effects on competence were dependent on the cognate sugar:phosphotransferase permease for each carbohydrate. Using green fluorescent protein (GFP) reporter fusions, we observed that growth in fructose or trehalose resulted in a greater proportion of the population activating expression ofcomXandcomS, encoding the precursor ofcomX-inducing peptide (XIP), after addition of CSP, than growth in glucose. Thus, the source of carbohydrate significantly impacts the stochastic behaviors that regulate subpopulation responses to CSP, which can induce competence inS. mutans.IMPORTANCEThe signaling pathways that regulate development of genetic competence inStreptococcus mutansare intimately intertwined with the pathogenic potential of the organism, impacting biofilm formation, stress tolerance, and expression of known virulence determinants. Induction of the gene for the master regulator of competence, ComX, by competence-stimulating peptide (CSP) occurs in a subpopulation of cells. Here, we show that certain carbohydrates that are common in the human diet enhance the ability of CSP to activate transcription ofcomXand that a subset of these carbohydrates stimulates progression to the competent state. The cognate sugar:phosphotransferase permeases for each sugar are needed for these effects. Interestingly, single-cell analysis shows that the carbohydrates that increasecomgene expression do so by enhancing the proportion of cells that respond to CSP. A mathematical model is developed to explain how carbohydrates modulate bistable behavior in the system via the ComRS pathway and ComX stability.

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