Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence.
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ABSTRACT:
SUBMITTER: Ma Q
PROVIDER: S-EPMC8673623 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dental 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 in Streptococcus mutans is 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 inhibiting S. mutans biofilm 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 from S. mutans resulted 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 vitro assays showed that the compound was capable of inhibiting EPS synthesis and biofilm formation in S. mutans by selectively antagonizing Gtfs instead of by killing the bacteria directly. Moreover, the in vivo anti-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 caries in vivo with a concomitant reduction in the percentage of S. mutans in 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 of S. mutans.
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Project description:Lactate produced in glycolysis has recently been discovered to modify lysine residues on eukaryotic proteins, a post-translational modification (PTM) called lysine lactylation (Kla). This modification not only participates in regulating gene expression through lactylation of histones, but also impacts the function of non-histone proteins. However, lactylation in prokaryotes has not been reported. Here, we report the identification of 1869 lysine lactylation sites in 465 proteins in the cariogenic organism Streptococcus mutans using a newly developed 4-dimensional label-free quantitative proteomic approach, representing the first Kla dataset in prokaryotes. Combining quantitative analysis, we report that the modification levels of 282 sites from 124 proteins increased by over 1.5-fold, and 80 sites from 52 proteins decreased by over 0.67-fold in the biofilm growth state. These results provide a systematic perspective of the distribution and function of Kla and improve our understanding of the role of lactate in the metabolic regulation of prokaryotes.
2023-08-24 | PXD037027 | Pride
Project description:Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model.
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Project description:Candida albicans, as the most common fungus in the oral cavity, is often detected in early childhood caries. Streptococcus mutans is the major etiological agent of dental caries, but the role of S. mutans on C. albicans growth and biofilm development remains to be elucidated. Membrane vesicles (MVs) are a cell-secreted subcellular fraction that play an important role in intercellular communication and disease progression. In the present study, we investigated whether MVs from S. mutans augment C. albicans growth and biofilm development. The results indicated that S. mutans MVs augmented C. albicans biofilm development but had no significant effect on C. albicans growth under planktonic conditions. Subsequently, we labeled S. mutans MVs with PKH26 and used confocal laser scanning microscopy (CLSM) to track S. mutans MVs, which were observed to be located in the C. albicans biofilm extracellular matrix. Monosaccharide tests showed that S. mutans MVs contribute to sucrose metabolism in C. albicans. Polysaccharides were significantly enriched in the S. mutans MV-treated group. MVs from ΔgtfBC mutant strains were compared with those from the wild-type S. mutans. The results revealed that MVs from the ΔgtfBC mutant had no effect on C. albicans biofilm formation and exopolysaccharide production. In addition, C. albicans biofilm transcriptional regulators (Ndt80, Als1, Mnn9, Van1, Pmr1, Gca1, and Big1) expression were upregulated in S. mutans MV-treated group. In summary, the results of the present study showed that S. mutans MVs harboring glucosyltransferases involved in exopolysaccharide production augment C. albicans biofilm development, revealing a key role for S. mutans MVs in cross-kingdom interactions between S. mutans and C. albicans.
| S-EPMC7517897 | biostudies-literature
Project description:The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.
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Project description:Streptococcus mutans is 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, in S. mutans capable of modulating dynamic interactions between two key biofilm matrix components, glucan and extracellular DNA (eDNA). The deletion of smu_833 decreases 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 of gtfBC from the smu_833 mutant 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 acidic in vitro Concurrently, the mutant was less virulent in an in vivo rat 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 of S. mutans by 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.IMPORTANCE Tooth decay, a costly and painful disease affecting the vast majority of people worldwide, is caused by the bacterium Streptococcus 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 of S. mutans virulence and also offers a new opportunity to develop effective therapeutics targeting S. 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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Project description:Streptococcus mutans is a representative biofilm-forming bacterium that causes dental caries through glucosyltransferase (GTF) activity. Glucans are synthesized from sucrose by GTFs and provide binding sites for S. mutans to adhere tightly to the tooth enamel. Therefore, if a novel compound that interferes with GTF function is developed, biofilm formation control in S. mutans would be possible. We discovered that raffinose, an oligosaccharide from natural products, strongly inhibited biofilm formation, GTF-related gene expression, and glucan production. Furthermore, biofilm inhibition on saliva-coated hydroxyapatite discs through the reduction of bacterial adhesion indicated the applicability of raffinose in oral health. These effects of raffinose appear to be due to its ability to modulate GTF activity in S. mutans. Hence, raffinose may be considered an antibiofilm agent for use as a substance for oral supplies and dental materials to prevent dental caries. IMPORTANCE Dental caries is the most prevalent infectious disease and is expensive to manage. Dental biofilms can be eliminated via mechanical treatment or inhibited using antibiotics. However, bacteria that are not entirely removed or are resistant to antibiotics can still form biofilms. In this study, we found that raffinose inhibited biofilm formation by S. mutans, a causative agent of dental caries, possibly through binding to GtfC. Our findings support the notion that biofilm inhibition by raffinose can be exerted by interference with GTF function, compensating for the shortcomings of existing commercialized antibiofilm methods. Furthermore, raffinose is an ingredient derived from natural products and can be safely utilized in humans; it has no smell and tastes sweet. Therefore, raffinose, which can control S. mutans biofilm formation, has been suggested as a substance for oral supplies and dental materials to prevent dental caries.
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Project description:Among cariogenic microbes, Streptococcus mutans is considered a major etiological pathogen of dental caries. Lactobacilli strains have been promoted as possible probiotic agents against S. mutans, although the inhibitory effect of Lactobacilli on caries has not yet been properly addressed. The objective of this study was to screen Lactobacillus strains found in traditional Sichuan pickles and to evaluate their antagonistic properties against S. mutans in vitro and in vivo. In the current study, we analyzed 54 Lactobacillus strains isolated from pickles and found that strain L. plantarum K41 showed the highest inhibitory effect on S. mutans growth as well as on the formation of exopolysaccharides (EPS) and biofilm in vitro. Scanning electron microscopy (SEM) and confocal laser scanning microscope (CLSM) revealed the reduction of both EPS and of the network-like structure in S. mutans biofilm when these bacteria were co-cultured with strain L. plantarum K41. Furthermore, when rats were treated with strain L. plantarum K41, there was a significant reduction in the incidence and severity of dental caries. Due to K41's origin in a high salinity environment, it showed a high tolerance to acids and salts. This may give this strain an advantage in harsh oral conditions. Results showed that L. plantarum K41 isolated from traditional Sichuan pickles effectively inhibited S. mutans biofilm formation and thus possesses a potential inhibitory effect on dental caries in vivo.
| S-EPMC7203412 | biostudies-literature
Project description:Streptococcus mutans, found in the human oral cavity, is a significant contributor to the pathogenesis of dental caries. This bacterium expresses three genetically distinct types of glucosyltransferases named GtfB (GTF-I), GtfC (GTF-SI) and GtfD (GTF-S) that play critical roles in the development of dental plaque. The catalytic domains of GtfB, GtfC and GtfD contain conserved active-site residues for the overall enzymatic activity that relate to hydrolytic glycosidic cleavage of sucrose to glucose and fructose, release of fructose and generation of a glycosyl-enzyme intermediate in the reducing end. In a subsequent transglycosylation step, the glucosyl moiety is transferred to the nonreducing end of an acceptor to form a growing glucan polymer chain made up of glucose molecules. It has been proposed that both sucrose breakdown and glucan synthesis occur in the same active site of the catalytic domain, although the active site does not appear to be large enough to accommodate both functions. These three enzymes belong to glycoside hydrolase family 70 (GH70), which shows homology to glycoside hydrolase family 13 (GH13). GtfC synthesizes both soluble and insoluble glucans (α-1,3 and α-1,6 glycosidic linkages), while GtfB and GtfD synthesize only insoluble or soluble glucans, respectively. Here, crystal structures of the catalytic domains of GtfB and GtfD are reported. These structures are compared with previously determined structures of the catalytic domain of GtfC. With this work, apo structures and inhibitor-complex structures with acarbose are now available for the catalytic domains of GtfC and GtfB. The structure of GtfC with maltose allows further identification and comparison of active-site residues. A model of sucrose binding to GtfB is also included. The new structure of the catalytic domain of GtfD affords a structural comparison of the three S. mutans glycosyltransferases. Unfortunately, the catalytic domain of GtfD is not complete since crystallization resulted in the structure of a truncated protein lacking approximately 200 N-terminal residues of domain IV.
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Project description:Amyloid fibrils are important scaffold in bacterial biofilms. Streptococcus mutans is an established cariogenic bacteria dwelling within biofilms, and C123 segment of P1 protein is known to form amyloid fibrils in S. mutans biofilms, among which C3 segment could serve as a promising anti-amyloid target due to its critical role in C123-P1 interactions. Recently, small molecules have been found to successfully inhibit biofilms by targeting amyloid fibrils. Thus, our study aimed to screen small molecules targeting C3 segment with the capacity to influence amyloid fibrils and S. mutans biofilms. In silico screening was utilized to discover promising small molecules, which were evaluated for their effects on bacterial cells and amyloid fibrils. We selected 99 small molecules and enrolled 55 small molecules named D1-D55 for crystal violet staining. Notably, D25 selectively inhibit S. mutans biofilms but had no significant influence on biofilms formed by Streptococcus gordonii and Streptococcus sanguinis, and D25 showed no bactericidal effects and low cytotoxicity. In addition, amyloid fibrils in free-floating bacteria, biofilms and purified C123 were quantified with ThT assays, and the differences were not statistically significant in the presence or absence of D25. Morphological changes of amyloid fibrils were visualized with TEM images, where amorphous aggregates were obvious coupled with long and atypical amyloid fibrils. Moreover, amyloid-related genes were upregulated in response to D25. In conclusion, D25 is a promising antimicrobial agent with the capacity to influence amyloid fibrils and inhibit S. mutans biofilms.
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