DexA70, the Truncated Form of a Self-Produced Dextranase, Effectively Disrupts Streptococcus mutans Biofilm.
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ABSTRACT:
SUBMITTER: Liu N
PROVIDER: S-EPMC8505985 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Streptococcus mutans dextranase hydrolyzes the internal ?-1,6-linkages of dextran and belongs to glycoside hydrolase family 66. An N- and C-terminal deletion mutant of S. mutans dextranase was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.6 Å and belonged to space group P2(1), with unit-cell parameters a = 53.2, b = 89.7, c = 63.3 Å, ? = 102.3°. Assuming that the asymmetric unit of the crystal contained one molecule, the Matthews coefficient was calculated to be 4.07 Å(3) Da(-1); assuming the presence of two molecules in the asymmetric unit it was calculated to be 2.03 Å(3) Da(-1).
| S-EPMC3232134 | biostudies-literature
Project description:BackgroundThe accumulation of plaque causes oral diseases. Dental plaque is formed on teeth surfaces by oral bacterial pathogens, particularly Streptococcus mutans, in the oral cavity. Dextranase is one of the enzymes involved in antiplaque accumulation as it can prevent dental caries by the degradation of dextran, which is a component of plaque biofilm. This led to the idea of creating toothpaste containing dextranase for preventing oral diseases. However, the dextranase enzyme must be stable in the product; therefore, encapsulation is an attractive way to increase the stability of this enzyme.MethodsThe activity of food-grade fungal dextranase was measured on the basis of increasing ratio of reducing sugar concentration, determined by the reaction with 3, 5-dinitrosalicylic acid reagent. The efficiency of the dextranase enzyme was investigated based on its minimal inhibitory concentration (MIC) against biofilm formation by S. mutans ATCC 25175. Box-Behnken design (BBD) was used to study the three factors affecting encapsulation: pH, calcium chloride concentration, and sodium alginate concentration. Encapsulation efficiency (% EE) and the activity of dextranase enzyme trapped in alginate beads were determined. Then, the encapsulated dextranase in alginate beads was added to toothpaste base, and the stability of the enzyme was examined. Finally, sensory test and safety evaluation of toothpaste containing encapsulated dextranase were done.ResultsThe highest activity of the dextranase enzyme was 4401.71 unit/g at a pH of 6 and 37 °C. The dextranase at its MIC (4.5 unit/g) showed strong inhibition against the growth of S. mutans. This enzyme at 1/2 MIC also showed a remarkable decrease in biofilm formation by S. mutans. The most effective condition of dextranase encapsulation was at a pH of 7, 20% w/v calcium chloride and 0.85% w/v sodium alginate. Toothpaste containing encapsulated dextranase alginate beads produced under suitable condition was stable after 3 months of storage, while the sensory test of the product was accepted at level 3 (like slightly), and it was safe.ConclusionThis research achieved an alternative health product for oral care by formulating toothpaste with dextranase encapsulated in effective alginate beads to act against cariogenic bacteria, like S. mutants, by preventing dental plaque.
| S-EPMC7678491 | biostudies-literature
Project description:Streptococcus mutans were grown in rich SHI-medium as biofilms anaerobically. Biofilms were starved in minimal medium followed by glucose amendment (at pH 7). Replicate samples for MS/MS-small molecule and transcription analyses were collected at pH 7 and at pH 3.5 (20 hrs after glucose spiking). Only secreted molecules were analyzed with MS/MS (no cell lysis protocol was employed).
2014-04-10 | MSV000078581 | GNPS
Project description:Streptococcus mutans is the primary agent of dental cavities, in large part due to its ability to adhere to teeth and create a molecular scaffold of glucan polysaccharides on the tooth surface. Disrupting the architecture of S. mutans biofilms could help undermine the establishment of biofilm communities that cause cavities and tooth decay. Here we present a synthetic peptide P1, derived from a tick antifreeze protein, which significantly reduces S. mutans biofilm formation. Incubating cells with this peptide decreased biofilm biomass by approximately 75% in both a crystal violet microplate assay and an in vitro tooth model using saliva-coated hydroxyapatite discs. Bacteria treated with peptide P1 formed irregular biofilms with disconnected aggregates of cells and exopolymeric matrix that readily detached from surfaces. Peptide P1 can bind directly to S. mutans cells but does not possess bactericidal activity. Anti-biofilm activity was correlated with peptide aggregation and β-sheet formation in solution, and alternative synthetic peptides of different lengths or charge distribution did not inhibit biofilms. This anti-biofilm peptide interferes with S. mutans biofilm formation and architecture, and may have future applications in preventing bacterial buildup on teeth.
| S-EPMC5364132 | 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.
| S-EPMC3067281 | biostudies-literature
Project description:Dextranase is an enzyme that hydrolyzes dextran ?-1,6 linkages. Streptococcus mutans dextranase belongs to glycoside hydrolase family 66, producing isomaltooligosaccharides of various sizes and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln(100) to Ile(732) of S. mutans dextranase, devoid of its N- and C-terminal variable regions, was determined at 1.6 Å resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like ?-sandwich fold; domain A contained the enzyme's catalytic module, comprising a (?/?)(8)-barrel; and domain C formed a ?-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined, and, in the enzyme-isomaltotriose complex structure, the bound isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites, -4 to -1, in the catalytic cleft. The complexed structure with 4',5'-epoxypentyl-?-d-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp(385) side chain. These structures collectively indicated that Asp(385) was the catalytic nucleophile and that Glu(453) was the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzyme's catalytic machinery.
| S-EPMC3370176 | biostudies-literature
Project description:BackgroundTo investigate the inhibition mechanism of copper ions on Streptococcus mutans-Veillonella parvula dual biofilm.MethodsS. mutans-V. parvula dual biofilm was constructed and copper ions were added at different concentrations. After the biofilm was collected, RNA-seq and qRT-PCR were then performed to get gene information.ResultsThe coculture of S. mutans and V. parvula formed a significantly better dual biofilm of larger biomass than S. mutans mono biofilm. And copper ions showed a more significant inhibitory effect on S. mutans-V. parvula dual biofilm than on S. mutans mono biofilm when copper ions concentration reached 100 µM, and copper ions showed a decreased inhibitory effect on S. gordonii-V. parvula dual biofilm and S. sanguis-V.parvula dual biofilm than on the two mono biofilms as the concentration of copper ions increased. And common trace elements such as iron, magnesium, and zinc showed no inhibitory effect difference on S. mutans-V. parvula dual biofilm. The RNA-seq results showed a significant difference in the expression of a new ABC transporter SMU_651c, SMU_652c, SMU_653c, and S. mutans copper chaperone copYAZ. SMU_651c, SMU_652c, and SMU_653c were predicted to function as nitrite/nitrate transporter-related proteins, which suggested the specific inhibition of copper ions on S. mutans-V. parvula dual biofilm may be caused by the activation of S. mutans reactive nitrogen species.ConclusionsStreptococcus mutans and Veillonella parvula are symbiotic, forming a dual biofilm of larger biomass to better resist the external antibacterial substances, which may increase the virulence of S. mutans. While common trace elements such as iron, magnesium, and zinc showed no specific inhibitory effect on S. mutans-V. parvula dual biofilm, copper ion had a unique inhibitory effect on S. mutans-V. parvula dual biofilm which may be caused by activating S. mutans RNS when copper ions concentration reached 250 µM.
| S-EPMC9883903 | biostudies-literature
Project description:The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci.
| S-EPMC205580 | biostudies-other
Project description:In this experiment we collected small molecule data that represent excreted molecules by Streptococcus mutans growing as a biofilm. The S. mutans biofilms were established and incubated in anaerobic conditions. Samples were collected before and after a drastic pH drop due to glucose amendments. Control samples are included in this folder that represent molecules that were extracted from sterilized growth media only. These peaks should be subtracted from the biofilm samples prior to analyses.
2014-04-10 | MSV000078578 | MassIVE
Project description:Biofilm formation by Streptococcus mutans is considered as its principal virulence factor, causing dental caries. Mutants of S. mutans defective in biofilm formation were generated and analyzed to study the collective role of proteins in its formation. Mutants were characterized on the basis of adherence to saliva-coated surface, and biofilm formation. The confocal laser microscopy and scanning electron microscopy images showed that the control biofilms had cluster of cells covered by layer of exo-polysaccharide while the biofilms of mutants were thin and spaced. Two-dimensional protein electrophoresis data analysis identified 57 proteins that are either up (44 proteins) or down (13 proteins) regulated. These data points to the importance of up and down regulated proteins in the formation of biofilm in Streptococcus mutans.
| S-EPMC3055159 | biostudies-literature