Regulatory involvement of the PerR and SloR metalloregulators in the Streptococcus mutans oxidative stress response

ABSTRACTStreptococcus mutans is a commensal of the human oral microbiome that can instigate dental caries under conditions of dysbiosis. This study investigates S. mutans metalloregulators and their involvement in mediating a response to oxidative stress. Oxidative stress in the oral cavity can derive from temporal increases in reactive oxygen species (ROS) after meal consumption, and from endogenous bacterial ROS-producers that colonize the dentition as constituents of dental plaque. We hypothesize that the PerR (SMU.593) and SloR (SMU.186) metalloregulatory proteins in S. mutans contribute to oxidative stress tolerance by regulating the expression of genes responsive to H2O2 challenge. The results of qRT-PCR experiments with S. mutans cultures exposed to 0.5mM H2O2 reveal perR transcription that is responsive to the peroxide stressor, and sloR transcription that is subject to PerR repression. The results of gel shift assays support direct binding of a PerR homolog to the S. mutans sloR promoter at Fur and PerR consensus sequences on the UA159 chromosome. In addition, transcription of the S. mutans tpx and dpr antioxidant genes is upregulated in a perR/sloR double knockout mutant, consistent with heightened resistance of the S. mutans GMS802 perR-deficient strain when challenged with H2O2. Cumulatively, these results reveal a relationship of reciprocity between the PerR and SloR metalloregulators during the S. mutans response to oxidative stress and begin to elucidate the fitness strategies that evolved to foster S. mutans survival and persistence in the transient environments of the human oral cavity.IMPORTANCEIn 2020, untreated dental caries, especially in the permanent dentition, ranked among the most prevalent infectious diseases worldwide. Moreover, caries disproportionately affects children and individuals of low socioeconomic status. Untreated caries can lead to systemic health problems and has been associated with extended school and work absences, inappropriate use of emergency departments, and an inability for military forces to deploy. In combination with public health policy, research aimed at alleviating S. mutans-induced tooth decay is important because it can improve oral health, as well as overall health, especially for underserved populations. This research is focused on the S. mutans SloR and PerR metalloregulatory proteins that can help inform the development of therapeutics aimed at alleviating and potentially preventing dental caries.

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Regulatory involvement of the PerR and SloR metalloregulators in the Streptococcus mutans oxidative stress response

Journal of Bacteriology ◽

10.1128/jb.00678-20 ◽

2021 ◽

Author(s):

Talia R. Ruxin ◽

Julia A. Schwartzman ◽

Cleo R. Davidowitz ◽

Zachary Peters ◽

Andrew Holtz ◽

...

Keyword(s):

Oxidative Stress ◽

Stress Response ◽

Dental Caries ◽

Streptococcus Mutans ◽

Oxidative Stress Response ◽

Reciprocal Relationship ◽

Oral Microbiome ◽

Tooth Decay ◽

Antioxidant Genes ◽

Metalloregulatory Proteins

Streptococcus mutans is a commensal of the human oral microbiome that can promote dental caries under conditions of dysbiosis. This study investigates metalloregulators and their involvement in the S. mutans oxidative stress response. Oxidative stress in the human mouth can derive from temporal increases in reactive oxygen species (ROS) after meal consumption and from endogenous bacterial ROS-producers that colonize the dentition. We hypothesize that the S. mutans PerR (SMU.593) and SloR (SMU.186) metalloregulatory proteins contribute to the regulation of oxidative stress genes and their products. Expression assays with S. mutans UA159 wild type cultures exposed to H2O2 reveal that H2O2 upregulates perR, and that PerR represses sloR transcription upon binding directly to Fur and PerR consensus sequences within the sloR operator. In addition, the results of Western blot experiments implicate the Clp proteolytic system in SloR degradation under conditions of H2O2-stress. To reveal a potential role for SloR in the H2O2-resistant phenotype of S. mutans GMS802 (a perR-deficient strain), we generated a sloR/perR double knockout mutant, GMS1386, where we observed upregulation of the tpx and dpr antioxidant genes. These results are consistent with GMS802 H2O2 resistance and with a role for PerR as a transcriptional repressor. Cumulatively, these findings support a reciprocal relationship between PerR and SloR during the S. mutans oxidative stress response and begin to elucidate the fitness strategies that evolved to foster S. mutans persistence in the transient environments of the human oral cavity. IMPORTANCE In 2020, untreated dental caries, especially in the permanent dentition, ranked among the most prevalent infectious diseases worldwide, disproportionately impacting individuals of low socioeconomic status. Untreated caries can lead to systemic health problems and has been associated with extended school and work absences, inappropriate use of emergency departments, and an inability for military forces to deploy. Together with public health policy, research aimed at alleviating S. mutans-induced tooth decay is important because it can improve oral health (and overall health), especially in underserved populations. This research, focused on S. mutans metalloregulatory proteins and their gene targets, is significant because it can promote virulence gene control in an important oral pathogen, and contribute to the development of an anti-caries therapeutic that can reduce tooth decay.

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Oxidative Stressors Modify the Response of Streptococcus mutans to Its Competence Signal Peptides

Applied and Environmental Microbiology ◽

10.1128/aem.01345-17 ◽

2017 ◽

Vol 83 (22) ◽

Cited By ~ 9

Author(s):

Matthew De Furio ◽

Sang Joon Ahn ◽

Robert A. Burne ◽

Stephen J. Hagen

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Dental Caries ◽

Signaling Pathway ◽

Streptococcus Mutans ◽

Reactive Oxygen ◽

Oxygen Species ◽

Oral Biofilm ◽

Genetic Competence ◽

Content Type

ABSTRACTThe dental caries pathogenStreptococcus mutansis continually exposed to several types of stress in the oral biofilm environment. Oxidative stress generated by reactive oxygen species has a major impact on the establishment, persistence, and virulence ofS. mutans. Here, we combined fluorescent reporter-promoter fusions with single-cell imaging to study the effects of reactive oxygen species on activation of genetic competence inS. mutans. Exposure to paraquat, which generates superoxide anion, produced a qualitatively different effect on activation of expression of the gene for the master competence regulator, ComX, than did treatment with hydrogen peroxide (H2O2), which can yield hydroxyl radical. Paraquat suppressed peptide-mediated induction ofcomXin a progressive and cumulative fashion, whereas the response to H2O2displayed a strong threshold behavior. Low concentrations of H2O2had little effect on induction ofcomXor the bacteriocin genecipB, but expression of these genes declined sharply if extracellular H2O2exceeded a threshold concentration. These effects were not due to decreased reporter gene fluorescence. Two different threshold concentrations were observed in the response to H2O2, depending on the gene promoter that was analyzed and the pathway by which the competence regulon was stimulated. The results show that paraquat and H2O2affect theS. mutanscompetence signaling pathway differently, and that some portions of the competence signaling pathway are more sensitive to oxidative stress than others.IMPORTANCEStreptococcus mutansinhabits the oral biofilm, where it plays an important role in the development of dental caries. Environmental stresses such as oxidative stress influence the growth ofS. mutansand its important virulence-associated behaviors, such as genetic competence.S. mutanscompetence development is a complex behavior that involves two different signaling peptides and can exhibit cell-to-cell heterogeneity. Although oxidative stress is known to influenceS. mutanscompetence, it is not understood how oxidative stress interacts with the peptide signaling or affects heterogeneity. In this study, we used fluorescent reporters to probe the effect of reactive oxygen species on competence signaling at the single-cell level. Our data show that different reactive oxygen species have different effects onS. mutanscompetence, and that some portions of the signaling pathway are more acutely sensitive to oxidative stress than others.

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Cloning, Expression, Purification, and Preliminary Characterization of Single-Crystal X-Ray Diffraction of Glucosyltransferase B of Streptococcus mutans

Natural Product Communications ◽

10.1177/1934578x19849339 ◽

2019 ◽

Vol 14 (5) ◽

pp. 1934578X1984933

Author(s):

Joshua L. Mieher ◽

Norbert Schormann ◽

Manisha Patel ◽

Hui Wu ◽

Champion Deivanayagam

Keyword(s):

Dental Caries ◽

Oral Cavity ◽

Single Crystal ◽

Streptococcus Mutans ◽

Tooth Enamel ◽

Tooth Surface ◽

X Ray Diffraction ◽

Persistent Disease ◽

X Ray

Dental caries characterized by acid damage of tooth enamel is a persistent disease that begins with the formation of biofilms on the tooth surface. The secreted glucosyltransferases enable Streptococcus mutans to synthesize extracellular glucan polymers using ingested starch within the oral cavity, which eventually results in the production of acid, a contributing factor to cariogenesis. In this paper, we report the cloning, expression, purification, crystallization, and preliminary X-ray diffraction characterization of glucosyltransferase B.

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Isolation and characterization of Streptococcus mutans in heart valve and dental plaque specimens from a patient with infective endocarditis

Journal of Medical Microbiology ◽

10.1099/jmm.0.46609-0 ◽

2006 ◽

Vol 55 (8) ◽

pp. 1135-1140 ◽

Cited By ~ 63

Author(s):

Ryota Nomura ◽

Kazuhiko Nakano ◽

Hirotoshi Nemoto ◽

Kazuyo Fujita ◽

Satoko Inagaki ◽

...

Keyword(s):

Infective Endocarditis ◽

Dental Caries ◽

Oral Cavity ◽

Streptococcus Mutans ◽

Heart Valve ◽

Dental Plaque ◽

Biochemical Properties ◽

Oral Streptococci ◽

Isolation And Characterization

Streptococcus mutans, known to be an aetiologic agent of dental caries, also causes infective endocarditis (IE), although a comparison of isolates from the oral cavity and infected heart valve of the same patient has not been reported. In the present study, infected heart valve and dental plaque samples from a patient with IE were analysed. Broad-range PCR with DNA sequencing revealed that 50 clones from the dental plaque isolates were composed of oral streptococci and periodontopathic bacteria, whereas only Streptococcus mutans was detected in 50 clones from the heart valve. Eighteen strains of Streptococcus mutans were isolated from dental plaque and seven from the heart valve, and the biochemical properties of each were in accordance with those of Streptococcus mutans. DNA fingerprinting analysis revealed that all the oral isolates of Streptococcus mutans had similar patterns, which were different from those of the isolates from the infected heart valve. Western blotting using glucosyltransferase (GTF)-specific antiserum showed that the seven strains from the heart valve lacked the three types of intact GTF. In addition, the sucrose-dependent adhesion rates of these isolates were significantly lower than those of the oral isolates (P<0.001). Furthermore, the isolates from the heart valve were less susceptible to erythromycin and kanamycin. These results indicate that the properties of the Streptococcus mutans strains isolated from the infected valve were different from those of typical oral strains, which may be related to the effects of IE.

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Inhibitory Effect of Lactobacillus plantarum CCFM8724 towards Streptococcus mutans- and Candida albicans-Induced Caries in Rats

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/4345804 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Qiuxiang Zhang ◽

Sujia Qin ◽

Xianyin Xu ◽

Jianxin Zhao ◽

Hao Zhang ◽

...

Keyword(s):

Candida Albicans ◽

Dental Caries ◽

Oral Cavity ◽

Lactobacillus Plantarum ◽

Streptococcus Mutans ◽

Microbial Colonization ◽

Control Group ◽

Scoring Method ◽

Treatment And Prevention

Streptococcus mutans is a recognized cariogenic bacterium and a major producer of biofilm matrix. The presence of Candida albicans in dental plaque with S. mutans enhances the virulence leading to the onset of rampant caries which is similar to early childhood caries (ECC). The purpose of this study was to explore the effect of Lactobacillus plantarum CCFM8724 (CCFM8724) on the treatment and prevention of dental caries induced by S. mutans and C. albicans in vivo. Rats were divided into 6 groups: the control group and model group, 2 treatment groups, and 2 prevention groups (0.02% chlorhexidine or CCFM8724). The fluctuation of microbial colonization and the change of bacteria flora in rat oral cavity after sowing of L. plantarum CCFM8724 were investigated by colony-forming units (CFU) and microflora analysis. The caries of rats were assessed by microcomputed tomography (micro-CT) and Keyes scoring method. The results showed that L. plantarum CCFM8724 in both the treatment and prevention groups could significantly decrease the population of S. mutans and C. albicans in the rats’ oral cavity ( p < 0.001 ), the mineral loss of enamel ( p < 0.05 ), and the scores of caries ( p < 0.05 ). Besides, L. plantarum CCFM8724 exhibited better effects than chlorhexidine. Hence, L. plantarum CCFM8724 was proved to be a potential oral probiotic on caries treatment and prevention in vivo and it may have the prospect of application in dental caries (especially ECC) prevention products.

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Green Tea Polyphenol Epigallocatechin-3-Gallate-Stearate Inhibits the Growth of Streptococcus mutans: A Promising New Approach in Caries Prevention

Dentistry Journal ◽

10.3390/dj6030038 ◽

2018 ◽

Vol 6 (3) ◽

pp. 38 ◽

Cited By ~ 10

Author(s):

Amy Melok ◽

Lee Lee ◽

Siti Mohamed Yussof ◽

Tinchun Chu

Keyword(s):

Dental Caries ◽

Oral Cavity ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Morphological Changes ◽

The United States ◽

Chlorhexidine Gluconate ◽

Log Reduction ◽

Colony Forming Units ◽

Green Tea Polyphenol

Streptococcus mutans (S. mutans) is the main etiological bacteria present in the oral cavity that leads to dental caries. All of the S. mutans in the oral cavity form biofilms that adhere to the surfaces of teeth. Dental caries are infections facilitated by the development of biofilm. An esterified derivative of epigallocatechin-3-gallate (EGCG), epigallocatechin-3-gallate-stearate (EGCG-S), was used in this study to assess its ability to inhibit the growth and biofilm formation of S. mutans. The effect of EGCG-S on bacterial growth was evaluated with colony forming units (CFU) and log reduction; biofilm formation was qualitatively determined by Congo red assay, and quantitatively determined by crystal violet assay, fluorescence-based LIVE/DEAD assays to study the cell viability, and scanning electron microscopy (SEM) was used to evaluate the morphological changes. The results indicated that EGCG-S was able to completely inhibit growth and biofilm formation at concentrations of 250 µg/mL. Its effectiveness was also compared with a commonly prescribed mouthwash in the United States, chlorhexidine gluconate. EGCG-S was shown to be equally effective in reducing S. mutans growth as chlorhexidine gluconate. In conclusion, EGCG-S is potentially an anticariogenic agent by reducing bacterial presence in the oral cavity.

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Disruption of L-Rhamnose Biosynthesis Results in Severe Growth Defects in Streptococcus mutans

Journal of Bacteriology ◽

10.1128/jb.00728-19 ◽

2019 ◽

Vol 202 (6) ◽

Cited By ~ 1

Author(s):

Andrew P. Bischer ◽

Christopher J. Kovacs ◽

Roberta C. Faustoferri ◽

Robert G. Quivey

Keyword(s):

Cell Wall ◽

Dental Caries ◽

Oral Cavity ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Etiologic Agent ◽

Growth Defect ◽

Cell Wall Polysaccharide ◽

Content Type ◽

Severe Growth

ABSTRACT The rhamnose-glucose cell wall polysaccharide (RGP) of Streptococcus mutans plays a significant role in cell division, virulence, and stress protection. Prior studies examined function of the RGP using strains carrying deletions in the machinery involved in RGP assembly. In this study, we explored loss of the substrate for RGP, l-rhamnose, via deletion of rmlD (encoding the protein responsible for the terminal step in l-rhamnose biosynthesis). We demonstrate that loss of rhamnose biosynthesis causes a phenotype similar to strains with disrupted RGP assembly (ΔrgpG and ΔrgpF strains). Deletion of rmlD not only caused a severe growth defect under nonstress growth conditions but also elevated susceptibility of the strain to acid and oxidative stress, common conditions found in the oral cavity. A genetic complement of the ΔrmlD strain completely restored wild-type levels of growth, whereas addition of exogenous rhamnose did not. The loss of rhamnose production also significantly disrupted biofilm formation, an important aspect of S. mutans growth in the oral cavity. Further, we demonstrate that loss of either rmlD or rgpG results in ablation of rhamnose content in the S. mutans cell wall. Taken together, these results highlight the importance of rhamnose production in both the fitness and the ability of S. mutans to overcome environmental stresses. IMPORTANCE Streptococcus mutans is a pathogenic bacterium that is the primary etiologic agent of dental caries, a disease that affects billions yearly. Rhamnose biosynthesis is conserved not only in streptococcal species but in other Gram-positive, as well as Gram-negative, organisms. This study highlights the importance of rhamnose biosynthesis in RGP production for protection of the organism against acid and oxidative stresses, the two major stressors that the organism encounters in the oral cavity. Loss of RGP also severely impacts biofilm formation, the first step in the onset of dental caries. The high conservation of the rhamnose synthesis enzymes, as well as their importance in S. mutans and other organisms, makes them favorable antibiotic targets for the treatment of disease.

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Lemon myrtle extract inhibits lactate production by Streptococcus mutans

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab147 ◽

2021 ◽

Vol 85 (10) ◽

pp. 2185-2190

Author(s):

Yukinori Yabuta ◽

Yui Sato ◽

Arisu Miki ◽

Ryuta Nagata ◽

Tomohiro Bito ◽

...

Keyword(s):

Lactate Dehydrogenase ◽

Dental Caries ◽

Oral Cavity ◽

Streptococcus Mutans ◽

Virulence Factors ◽

Biofilm Formation ◽

Tooth Enamel ◽

Lactate Production ◽

Tooth Decay ◽

Lactate Dehydrogenase Activity

ABSTRACT Backhousia citriodora (lemon myrtle) extract has been found to inhibit glucansucrase activity, which plays an important role in biofilm formation by Streptococcus mutans. In addition to glucansucrase, various virulence factors in S. mutans are involved in the initiation of caries. Lactate produced by S. mutans demineralizes the tooth enamel. This study investigated whether lemon myrtle extract can inhibit S. mutans lactate production. Lemon myrtle extract reduced the glycolytic pH drop in S. mutans culture and inhibited lactate production by at least 46%. Ellagic acid, quercetin, hesperetin, and myricetin, major polyphenols in lemon myrtle, reduced the glycolytic pH drop and lactate production, but not lactate dehydrogenase activity. Furthermore, these polyphenols reduced the viable S. mutans cell count. Thus, lemon myrtle extracts may inhibit S. mutans-mediated acidification of the oral cavity, thereby preventing dental caries and tooth decay.

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Green Tea Polyphenol Epigallocatechin-3-Gallate-Sterate Inhibits the Growth of Streptococcus mutans: A Promising New Approach in Caries Prevention

10.20944/preprints201807.0138.v1 ◽

2018 ◽

Author(s):

Amy Lynn Melok ◽

Lee H. Lee ◽

Siti Ayuni Mohamed Yussof ◽

Tinchun Chu

Keyword(s):

Dental Caries ◽

Oral Cavity ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Morphological Changes ◽

The United States ◽

Chlorhexidine Gluconate ◽

Log Reduction ◽

Colony Forming Units ◽

Green Tea Polyphenol

Streptococcus mutans (S. mutans) is the main etiological bacteria present in the oral cavity that leads to dental caries. All of the S. mutans in the oral cavity form biofilms that adheres to the surfaces of teeth. Dental caries are infections facilitated by the development of biofilm. An esterified derivative of epigallocatechin-3-gallate (EGCG), epigallocatechin-3-gallate-sterate (EGCG-S) was used in this study to assess its ability to inhibit the growth and biofilm formation of S. mutans. The effect of EGCG-S on bacterial growth was evaluated with colony forming units (CFU) and log reduction; biofilm formation was qualitatively determined by Congo red assay, and quantitatively determined by crystal violet assay, fluorescence-based LIVE/DEAD assays to study the cell viability, and scanning electron microscopy (SEM) was used to evaluate the morphological changes. The results indicated that EGCG-S was able to completely inhibit growth and biofilm formation at concentrations of 250 µg/ml. Its effectiveness was also compared with a commonly prescribed mouthwash in the United States, chlorhexidine gluconate. EGCG-S was shown to be equally effective in reducing S. mutans growth as chlorhexidine gluconate. In conclusion, EGCG-S is potentially a natural anticariogenic agent by reducing bacterial presence in the oral cavity.

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The NADPH Oxidase A of Verticillium dahliae is Essential for Pathogenicity, Normal Development, and Stress Tolerance, and it Interacts with Yap1 to Regulate Redox Homeostasis

10.1101/2021.08.17.456638 ◽

2021 ◽

Author(s):

Vasileios Vangalis ◽

Ioannis A. Papaioannou ◽

Emmanouil A. Markakis ◽

Michael Knop ◽

Milton A. Typas

Keyword(s):

Oxidative Stress ◽

Nadph Oxidase ◽

Verticillium Dahliae ◽

Plant Pathogens ◽

Redox Homeostasis ◽

Mitochondrial Respiratory Chain ◽

Knockout Mutant ◽

Double Knockout ◽

Antioxidant Genes ◽

Increased Sensitivity

AbstractMaintenance of redox homeostasis is vital for aerobic organisms and particularly relevant to plant pathogens. These need to balance between endogenous ROS production that is required for their development and pathogenicity, and host-derived oxidative stress. Endogenous ROS in fungi are generated by the membrane-bound NADPH oxidase (NOX) complexes and the mitochondrial respiratory chain, while the transcription factor Yap1 is a major regulator of the antioxidant response. Here we investigated the roles of NoxA and Yap1 in fundamental biological processes of the important plant pathogen Verticillium dahliae. Deletion of noxA impaired growth and morphogenesis, compromised formation of hyphopodia, diminished penetration ability and pathogenicity, increased sensitivity against antifungal agents, and dysregulated expression of antioxidant genes. On the other hand, deletion of yap1 resulted in defects in conidial and microsclerotia formation, increased sensitivity against oxidative stress, and down-regulated antioxidant genes. Localized accumulation of ROS was observed before conidial fusion and during the heterokaryon incompatibility reaction upon nonself fusion. The frequency of inviable fusions was not affected by deletion of Yap1. Analysis of a double knockout mutant revealed an epistatic relationship between noxA and yap1. Our results collectively reveal instrumental roles of NoxA and ROS homeostasis in the biology of V. dahliae.

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