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

2019 ◽  
Vol 202 (6) ◽  
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.

scholarly journals Murein Hydrolase LytF of Streptococcus sanguinis and the Ecological Consequences of Competence Development

2017 ◽  
Vol 83 (24) ◽  
Author(s):  
Nyssa Cullin ◽  
Sylvio Redanz ◽  
Kirsten J. Lampi ◽  
Justin Merritt ◽  
Jens Kreth
Keyword(s):  
Dental Caries ◽  
Oral Cavity ◽  
Cell Morphology ◽  
Biofilm Formation ◽  
Oral Bacteria ◽  
Tooth Surface ◽  
Content Type ◽  
Streptococcus Sanguinis ◽  
Murein Hydrolase ◽  
Biofilm Development

ABSTRACT The overall health of the oral cavity is dependent on proper homeostasis between health-associated bacterial colonizers and bacteria known to promote dental caries. Streptococcus sanguinis is a health-associated commensal organism, a known early colonizer of the acquired tooth pellicle, and is naturally competent. We have shown that LytF, a competence-controlled murein hydrolase, is capable of inducing the release of extracellular DNA (eDNA) from oral bacteria. Precipitated LytF and purified LytF were used as treatments against planktonic cultures and biofilms. Larger amounts of eDNA were released from cultures treated with protein samples containing LytF. Additionally, LytF could affect biofilm formation and cellular morphology. Biofilm formation was significantly decreased in the lytF-complemented strain, in which increased amounts of LytF are present. The same strain also exhibited cell morphology defects in both planktonic cultures and biofilms. Furthermore, the LytF cell morphology phenotype was reproducible in wild-type cells using purified LytF protein. In sum, our findings demonstrate that LytF can induce the release of eDNA from oral bacteria, and they suggest that, without proper regulation of LytF, cells display morphological abnormalities that contribute to biofilm malformation. In the context of the oral biofilm, LytF may play important roles as part of the competence and biofilm development programs, as well as increasing the availability of eDNA. IMPORTANCE Streptococcus sanguinis, a commensal organism in the oral cavity and one of the pioneer colonizers of the tooth surface, is associated with the overall health of the oral environment. Our laboratory showed previously that, under aerobic conditions, S. sanguinis can produce H2O2 to inhibit the growth of bacterial species that promote dental caries. This production of H2O2 by S. sanguinis also induces the release of eDNA, which is essential for proper biofilm formation. Under anaerobic conditions, S. sanguinis does not produce H2O2 but DNA is still released. Determining how S. sanguinis releases DNA is thus essential to understand biofilm formation in the oral cavity.

scholarly journals Pleiotropic Regulation of Virulence Genes in Streptococcus mutans by the Conserved Small Protein SprV

2017 ◽  
Vol 199 (8) ◽  
Author(s):  
Manoharan Shankar ◽  
Mohammad S. Hossain ◽  
Indranil Biswas
Keyword(s):  
Dental Caries ◽  
Streptococcus Mutans ◽  
Virulence Factors ◽  
Biofilm Formation ◽  
Normal Growth ◽  
Hypothetical Protein ◽  
Bacteriocin Production ◽  
Transcript Levels ◽  
Content Type ◽  
Tooth Surfaces

ABSTRACT Streptococcus mutans, an oral pathogen associated with dental caries, colonizes tooth surfaces as polymicrobial biofilms known as dental plaque. S. mutans expresses several virulence factors that allow the organism to tolerate environmental fluctuations and compete with other microorganisms. We recently identified a small hypothetical protein (90 amino acids) essential for the normal growth of the bacterium. Inactivation of the gene, SMU.2137, encoding this protein caused a significant growth defect and loss of various virulence-associated functions. An S. mutans strain lacking this gene was more sensitive to acid, temperature, osmotic, oxidative, and DNA damage-inducing stresses. In addition, we observed an altered protein profile and defects in biofilm formation, bacteriocin production, and natural competence development, possibly due to the fitness defect associated with SMU.2137 deletion. Transcriptome sequencing revealed that nearly 20% of the S. mutans genes were differentially expressed upon SMU.2137 deletion, thereby suggesting a pleiotropic effect. Therefore, we have renamed this hitherto uncharacterized gene as sprV (streptococcal pleiotropic regulator of virulence). The transcript levels of several relevant genes in the sprV mutant corroborated the phenotypes observed upon sprV deletion. Owing to its highly conserved nature, inactivation of the sprV ortholog in Streptococcus gordonii also resulted in poor growth and defective UV tolerance and competence development as in the case of S. mutans. Our experiments suggest that SprV is functionally distinct from its homologs identified by structure and sequence homology. Nonetheless, our current work is aimed at understanding the importance of SprV in the S. mutans biology. IMPORTANCE Streptococcus mutans employs several virulence factors and stress resistance mechanisms to colonize tooth surfaces and cause dental caries. Bacterial pathogenesis is generally controlled by regulators of fitness that are critical for successful disease establishment. Sometimes these regulators, which are potential targets for antimicrobials, are lost in the genomic context due to the lack of annotated homologs. This work outlines the regulatory impact of a small, highly conserved hypothetical protein, SprV, encoded by S. mutans. We show that SprV affects the transcript levels of various virulence factors required for normal growth, biofilm formation, stress tolerance, genetic competence, and bacteriocin production.

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

2018 ◽  
Vol 6 (3) ◽  
pp. 38 ◽  
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.

Lemon myrtle extract inhibits lactate production by Streptococcus mutans

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.

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

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.

Disruption of the adh (acetoin dehydrogenase) operon has wide-ranging effects on Streptococcus mutans growth and stress response

2022 ◽  
Author(s):  
Peter Zuber ◽  
Michiko M. Nakano ◽  
Jessica K. Kajfasz ◽  
José A. Lemos
Keyword(s):  
Cell Wall ◽  
Transcription Factor ◽  
Dental Caries ◽  
Streptococcus Mutans ◽  
Stress Responses ◽  
Tooth Enamel ◽  
Human Saliva ◽  
Ph Sensitivity ◽  
Growth Defect ◽  
Carbohydrate Utilization

The agent largely responsible for initiating dental caries, Streptococcus mutans produces acetoin dehydrogenase that is encoded by the adh operon. The operon consists of the adhA and B genes (E1 dehydrogenase), adhC (E2 lipoylated transacetylase), adhD (E3 dihydrolipoamide dehydrogenase), and lplA (lipoyl ligase). Evidence is presented that AdhC interacts with SpxA2, a redox-sensitive transcription factor functioning in cell wall and oxidative stress responses. In-frame deletion mutations of adh genes conferred oxygen-dependent sensitivity to slightly alkaline pH (pH 7.2-7.6), within the range of values observed in human saliva. Growth defects were also observed when glucose or sucrose served as major carbon sources. A deletion of the adhC orthologous gene, acoC gene of Streptococcus gordonii , did not result in pH sensitivity or defective growth in glucose and sucrose. The defects observed in adh mutants were partially reversed by addition of pyruvate. Unlike most 2-oxoacid dehydrogenases, the E3 AdhD subunit bears an N-terminal lipoylation domain nearly identical to that of E2 AdhC. Changing the lipoyl domains of AdhC and AdhD by replacing the lipoate attachment residue, lysine to arginine, caused no significant reduction in pH sensitivity but the adhDK43R mutation eliminating the lipoylation site resulted in an observable growth defect in glucose medium. The adh mutations were partially suppressed by a deletion of rex , encoding an NAD + /NADH-sensing transcription factor that represses genes functioning in fermentation. spxA2 adh double mutants show synthetic growth restriction at elevated pH and upon ampicillin treatment. These results suggest a role for Adh in stress management in S. mutans . IMPORTANCE Dental caries is often initiated by Streptococcus mutans , which establishes a biofilm and a low pH environment on tooth enamel surfaces. The current study has uncovered vulnerabilities of S. mutans mutant strains that are unable to produce the enzyme complex, acetoin dehydrogenase (Adh). Such mutants are sensitive to modest increases in pH to 7.2-7.6, within the range of human saliva, while a mutant of a commensal Streptococcal species is resistant. The S. mutans adh strains are also defective in carbohydrate utilization and are hypersensitive to a cell wall-acting antibiotic. The studies suggest that Adh could be a potential target for interfering with S. mutans colonization of the oral environment.

scholarly journals Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Arpan De ◽  
Sumei Liao ◽  
Jacob P. Bitoun ◽  
Randy Roth ◽  
Wandy L. Beatty ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cell Wall ◽  
Cell Division ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Cell Envelope ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Triple Mutant ◽  
Content Type

ABSTRACTStreptococcus mutansis known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen.S. mutansstrains deficient inrgpG, encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. ThergpGdeficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, thergpGmutant existed primarily in chains of swollen, “squarish” dividing cells. Deficiency ofrgpGalso causes significant reduction in biofilm formation (P< 0.01). Double and triple mutants with deficiency inbrpAand/orpsr, genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using thergpGmutant. There were no major differences in growth rates between the wild-type strain and thergpG brpAandrgpG psrdouble mutants, but the growth rate of thergpG brpA psrtriple mutant was reduced drastically (P< 0.001). Under transmission electron microscopy, both double mutants resembled thergpGmutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, thergpGmutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and thebrpAandpsrsingle mutants. These results suggest that RgpG inS. mutansplays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope.IMPORTANCEStreptococcus mutans, a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation byS. mutans, indicative of a significant role of RGP in cell division and biofilm formation inS. mutans. These results are novel not only inS. mutans, but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr inS. mutansare involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation inS. mutans. This study reveals new potential targets to develop anticaries therapeutics.

scholarly journals A New Small Molecule Specifically Inhibits the Cariogenic Bacterium Streptococcus mutans in Multispecies Biofilms

2011 ◽  
Vol 55 (6) ◽  
pp. 2679-2687 ◽  
Author(s):  
Chang Liu ◽  
Roberta J. Worthington ◽  
Christian Melander ◽  
Hui Wu
Keyword(s):  
Dental Caries ◽  
Small Molecules ◽  
Streptococcus Mutans ◽  
Small Molecule ◽  
Biofilm Formation ◽  
Content Type ◽  
Multispecies Biofilm ◽  
Small Molecule Library ◽  
Cariogenic Bacterium ◽  
Multispecies Biofilms

ABSTRACTStreptococcus mutansis a major cariogenic bacterium. It has adapted to the biofilm lifestyle, which is essential for pathogenesis of dental caries. We aimed to identify small molecules that can inhibit cariogenicS. mutansand to discover lead structures that could give rise to therapeutics for dental caries. In this study, we screened a focused small-molecule library of 506 compounds. Eight small molecules which inhibitedS. mutansat a concentration of 4 μM or less but did not affect cell growth or biofilm formation of commensal bacteria, represented byStreptococcus sanguinisandStreptococcus gordonii, in monospecies biofilms were identified. The active compounds share similar structural properties, which are characterized by a 2-aminoimidazole (2-AI) or 2-aminobenzimidazole (2-ABI) subunit. In multispecies biofilm models, the most active compound also inhibited cell survival and biofilm formation ofS. mutansbut did not affect commensal streptococci. This inhibitor downregulated the expression of six biofilm-associated genes,ftf,pac,relA,comDE,gbpB, andgtfB, in planktonicS. mutanscells, while it downregulated the expression of onlyftf,pac, andrelAin the biofilm cells ofS. mutans. The most potent compound also inhibited production of two key adhesins ofS. mutans, antigen I/II and glucosyltransferase (GTF). However, the compound did not alter the expression of the corresponding genes in bothS. sanguinisandS. gordonii, indicating that it possesses a selective inhibitory activity againstS. 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.

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