Glucosyltransferases (Gtfs) play critical roles in the etiology and pathogenesis of Streptococcus mutans (S. mutans)- mediated dental caries including early childhood caries. Gtfs enhance the biofilm formation and promotes colonization of cariogenic bacteria by generating biofilm extracellular polysaccharides (EPSs), the key virulence property in the cariogenic process. Therefore, Gtfs have become an appealing target for effective therapeutic interventions that inhibit cariogenic biofilms. Importantly, targeting Gtfs selectively impairs the S. mutans virulence without affecting S. mutans existence or the existence of other species in the oral cavity. Over the past decade, numerous Gtfs inhibitory molecules have been identified, mainly including natural and synthetic compounds and their derivatives, antibodies, and metal ions. These therapeutic agents exert their inhibitory role in inhibiting the expression gtf genes and the activities and secretion of Gtfs enzymes with a wide range of sensitivity and effectiveness. Understanding molecular mechanisms of inhibiting Gtfs will contribute to instructing drug combination strategies, which is more effective for inhibiting Gtfs than one drug or class of drugs. This review highlights our current understanding of Gtfs activities and their potential utility, and discusses challenges and opportunities for future exploration of Gtfs as a therapeutic target.
Biofilms ; Dental Caries/prevention & control* ; Glucosyltransferases/antagonists & inhibitors* ; Humans ; Streptococcus mutans/enzymology*

OBJECTIVETo determine the physicochemical properties of the mutanase of Trichoderma harzianum isolated from China and to study the influence of mutanase on the adherence of oral Streptococci and the structure of oral biofilms.

METHODSSix fungal strains belonging to Trichoderma were tested for mutanase production in the same cultural condition, the strain producing the highest mutanase activity was studied further and the pH and temperature optimum of the enzyme was determined. The RT-PCR method was used to obtain the gene coding for mutanase and the product was cloned to pMD18-T simple vector for sequencing. Inhibition effects of mutanase on the adherence of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 were studied by adherence test. The optical sectioning of biofilms with or without mutanase supplementation were analyzed by confocal laser scanning microscopy (CLSM).

RESULTSThe highest enzymatic activity was achieved by Trichoderma harzianum Th1, the maximum activity was at pH 5.5 and at 40 degrees C. The nucleotide sequence was 92% homology with that of a known gene coding a mutanase (GenBank accession No. AJ243799). The adherence of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 was significantly inhibited by mutanase. Compared with control, the biofilms with mutanase supplementation had lower height and sparser structure.

CONCLUSIONSThe mutanase from Trichoderma harzianum Th1 can inhibit the adherence of oral Streptococci and had an influence on the structure of oral biofilms.

Bacterial Adhesion ; drug effects ; Biofilms ; Glycoside Hydrolases ; chemistry ; physiology ; Streptococcus mutans ; drug effects ; Streptococcus sobrinus ; drug effects ; Trichoderma ; enzymology ; pathogenicity

OBJECTIVETo compare the gene sequence and protein structure of lactate dehydrogenase (LDH) in Streptococcus oligofermentans with those of other bacteria with different acid generating capacities in oral cavity and to analyze the differences of their LDH.

METHODSLDH gene sequence of Streptococcus oligofermentans was measured by Institute of Microbiology, Chinese Academy of Sciences. LDH gene sequences of four Streptococcus and Lactobacillus casei in the NCBI Genbank was identified and compared among the six bacteria's LDH gene sequences and amino acid sequences by BLAST software. ExPASy database was used to predict the physical-chemical characteristics, secondary structure, trans-membrane regions, and spatial structure of Streptococcus oligofermentans LDH protein, which was compared with those of other bacteria.

RESULTSThe full-length of the LDH gene sequences of Streptococcus oligofermentans was 987 base pairs. The highest similarity was 89% with that of the Streptococcus sanguis, and 81% similarity with Streptococcus mutans, and 70% similarity with Lactobacillus casei. LDH amino acid sequence of Streptococcus oligofermentans was similar to Streptococcus sanguinis, with the highest similitude of 96%, with a similitude of 81% to Streptococcus mutans, but differed greatly from that of Lactobacillus casei, with a similitude of only 66%. Streptococcus oligofermentans LDH protein's physical-chemical characteristics, trans-membrane region's numbers, proportions of secondary structure, structural domain's location resembled those of Streptococcus mutans, Streptococcus sanguinis and Lactobacillus casei. Spatial structure differences between the LDH of Streptococcus oligofermentans and that of Streptococcus mutans and Lactobacillus casei were distinct.

CONCLUSIONSStreptococcus oligofermentans LDH's gene sequence, amino acid sequence, and spatial structure all vary from those of Streptococcus mutans and Lactobacillus casei, and these differeces may be a inherent reason that lead to the changes of its LDH's biological functions and incapacity of producing lactic acid.

Amino Acid Sequence ; Base Sequence ; L-Lactate Dehydrogenase ; genetics ; isolation & purification ; metabolism ; Lactic Acid ; metabolism ; Molecular Weight ; Protein Structure, Secondary ; Species Specificity ; Streptococcus ; enzymology ; genetics ; Streptococcus mutans ; enzymology ; genetics ; Streptococcus sanguis ; enzymology ; genetics

OBJECTIVETo investigate the effect of lemon peel extracts (LPE) on the activity of lactate dehydrogenase and sucrase of Streptococcus mutans (Sm).

METHODSAfter serial dilution with trypticase soy broth (TSB) medium containing 2% glucose, LPE was used as the experimental group, and TSB without LPE as the control group. Sm was added to each group, which was then cultured for 6, 18, 24 and 48 hours in the anaerobic tank. The activity of lactate dehydrogenase(LDH) was measured with the method of oxidation of reduction coenzymeIand the pH value of the culture solution was also detected. The activity of the sucrose was determined with the method of coloration of 3,5-dinitrosalicylic acid.

RESULTSThe activity of LDH, sucrase and the changes of solution pH were decreased with the increase of the concentration of LPE (P < 0.01). The activity of LDH were declined from (0.8025 ± 0.0913) × 10(3) U/L to (0.2099 ± 0.0283) × 10(3) U/L; the activity of sucrase were declined from (-0.0107 ± 0.0003) × 10(3) U/L to (-0.0078 ± 0.0002) × 10(3) U/L; the ΔpH were declined from (2.8067 ± 0.0404) to (2.5033 ± 0.0416) (24 h results). The differences were significant between experimental groups and the control group (P < 0.01), and there were also significant differences among experimental groups with different LPE concentration (P < 0.01). The inhibitory effect of acid generation and lactate dehydrogenas' activity of Sm were positively correlated (P < 0.01).

CONCLUSIONSLPE can inhibit the activity of lactate dehydrogenase, sucrase and the acid production capacity of the Sm in a dose dependent manner. The inhibitory effects in logarithmic phase is stronger than that in other phases of growth cycle.

Citrus ; chemistry ; Glucose ; L-Lactate Dehydrogenase ; metabolism ; Lactic Acid ; Plant Extracts ; pharmacology ; Streptococcus mutans ; enzymology ; Sucrase ; metabolism

OBJECTIVETo study the genetic diversity of F-ATPase alpha subunit gene uncA derived from Streptococcus mutans (S. mutans) clinical isolates and to investigate the relationship between the genetic diversity of acidurance factor and S. mutans aciduric ability, also and the cariogenicity.

METHODSSixty-four S. mutans strains derived from 34 caries-active individuals and 30 caries-free individuals, including 18 strains displaying high acid tolerance and 20 strains displaying low acid tolerance. Gene uncA was amplified with specific primers from S. mutans genomic DNA, then the PCR products were analyzed by RFLP and sequenced.

RESULTSTwo genotypes A and B of PCR-RFLP were revealed when digested with Hph I. Mbo II also produced two different pattern C and D. The distributions of A and B genotype strains with different caries-sensitivity groups were different (P < 0.05), and the proportion of A genotype strains from caries-activity group was higher than that from caries-free one. The distributions of C and D genotype strains with different acidurance strains were different (P < 0.05), and the proportion of C genotype strains from high acid tolerance group was higher than that from low acid tolerance group. These amplified uncA genes from different group were sequenced and there existed variation of Hph I and Mbo II recognized sites.

CONCLUSIONSThis study indicates that uncA gene of S. mutans F-ATPase obviously displayed genetic diversity. The different Hph I-RFLP and Mbo II-RFLP genotypes could be related to the cariogenicity and acid tolerance of S. mutans strains.

Bacterial Proton-Translocating ATPases ; genetics ; Dental Caries ; microbiology ; Genes, Bacterial ; Genotype ; Humans ; Polymorphism, Restriction Fragment Length ; Streptococcus mutans ; enzymology ; genetics

OBJECTIVETo investigate the effects of compounds of Galla chinensis extract (GCE) and Nidus vespae extract-1 (WVE1) on oral bacteria biofilm structure and activity and to determine the possibility of caries prevention by the compounds.

METHODSThe morphology and activity of treated-oral bacterial biofilm and untreated-oral bacterial biofilm were observed by using fluorescence microscope in combination of idio-fluorochrome to label the died and living bacteria. The visible light semiquantitative method was used to measure biomass glucosyltransferase (GTF, A620) values and to determine the effects of active compounds of GCE and NVE1 on GTF of oral bacteria biofilm.

RESULTSThe living bacteria in the untreated 24 h bacterial biofilm was dominant, and only a small number of died bacteria were found, the biofilm structure was regular and clear. GCE, GCE-B and NVE1 could inhibit the bacteria in the dental biofilm, which showed significant difference with the negative control. GCE and NVE1 could also inhibit GTF activity of 24 h bacterial biofilm in comparison with the negative control.

CONCLUSIONSThe traditional Chinese medicine Galla chinensis and Nidus vespae could not only inhibit bacteria growth on oral bacterial biofilm, but also function by adjusting biofilm structure, composition and GTF activity of 24 h bacterial biofilm.

Actinomyces viscosus ; drug effects ; enzymology ; physiology ; Bacteriological Techniques ; Biofilms ; drug effects ; Dental Caries ; microbiology ; Drugs, Chinese Herbal ; pharmacology ; Glucosyltransferases ; metabolism ; Medicine, Chinese Traditional ; Microbial Sensitivity Tests ; Streptococcus mutans ; drug effects ; enzymology ; physiology ; Streptococcus sanguis ; drug effects ; enzymology ; physiology

OBJECTIVETo investigate the antigenicity of the peptide vaccine HDS from Streptococcus mutans glucosyltransferase and its ability to induce protective immune responses in an experimental rat model of dental caries.

METHODSArtificial antigen HDS-KLH, peptide HDS, glycosyltransferase were injected to immunize rats. Measurement of the specific anti-HDS, GTF IgG or IgA concentration in saliva and serum were undertaken by ELISA among the experimental groups. Gnotobiotic rat model was developed when challenged S. mutans and a caries promoting diet. The jaws of the rats were selected and dyed. The Keyes caries score for each jaw were counted.

RESULTSThe level of serum and salivary specific anti-HDS IgG and IgA in the group immunized by HDS-KLH was significantly higher than that in control group (P < 0.05). The Keyes caries score of GTF, HDS and HDS-KLH immunized group were significantly lower than that of control group, especially lower in smooth tooth surface.

CONCLUSIONArtificial antigen HDS-KLH could induce immune response. As a peptide vaccine, HDS-KLH could reduce the caries incidence in experimental rat model.

Animals ; Dental Caries ; prevention & control ; Glucosyltransferases ; genetics ; immunology ; Male ; Peptides ; immunology ; Rats ; Rats, Sprague-Dawley ; Streptococcal Vaccines ; immunology ; Streptococcus mutans ; enzymology ; genetics ; immunology

The objective of this study was to characterize the oxygen dependent regulation of pyruvate oxidase (SpxB) gene expression and protein production in Streptococcus sanguinis (S. sanguinis). SpxB is responsible for the generation of growth-inhibiting amounts of hydrogen peroxide (H2O2) able to antagonize cariogenic Streptococcus mutans (S. mutans). Furthermore, the ecological consequence of H2O2 production was investigated in its self-inhibiting ability towards the producing strain. Expression of spxB was determined with quantitative Real-Time RT-PCR and a fluorescent expression reporter strain. Protein abundance was investigated with FLAG epitope engineered in frame on the C-terminal end of SpxB. Self inhibition was tested with an antagonism plate assay. The expression and protein abundance decreased in cells grown under anaerobic conditions. S. sanguinis was resistant against its own produced H2O2, while cariogenic S. mutans was inhibited in its growth. The results suggest that S. sanguinis produces H2O2 as antimicrobial substance to inhibit susceptible niche competing species like S. mutans during initial biofilm formation, when oxygen availability allows for spxB expression and Spx production.
Antibiosis ; physiology ; Bacterial Proteins ; biosynthesis ; genetics ; Epitopes ; genetics ; Gene Expression Regulation, Bacterial ; Hydrogen Peroxide ; metabolism ; pharmacology ; Oligopeptides ; Oxygen ; metabolism ; Peptides ; genetics ; Pyruvate Oxidase ; biosynthesis ; genetics ; Streptococcus mutans ; drug effects ; Streptococcus sanguis ; enzymology ; genetics ; growth & development ; Transformation, Bacterial

OBJECTIVETo investigate the aciduricity and genetic diversity of ATP synthase subunit gene uncEBF derived from Uyghur children Streptococcus mutans (Sm) clinical isolates and the relationship between the genetic diversity of ATP synthase and Sm aciduric ability and caries susceptibility.

METHODSForty-one Sm strains derived from 24 caries-active individuals and 17 caries-free individuals, including 16 strains displaying high acid tolerance and 17 strains displaying low acid tolerance. Solutions of all isolated Sm with same density were made and cultured at pH 4.0 to 7.0 brain heart infusion (BHI) liquid. Terminal growth situation was compared. Gene uncEBF of these isolates were amplified with specific primers from Sm genomic DNA, and the polymerase chain reaction (PCR) products were analyzed by PCR-restriction fragment length polymorphism (RFLP) and sequenced.

RESULTSAciduric ability of Sm isolated from the high caries-susceptible children were higher than that isolated from caries-free group (P = 0.023). Alu I digested fragments of uncEBF displayed two different patterns A and B. The distributions of A and B genotype strains with different acidurance were different (P = 0.039). A genotype included 7 strains displaying high acid tolerance and 2 strains displaying low acid tolerance;B genotype included 9 strains displaying high acid tolerance and 15 strains displaying low acid tolerance. The distributions of A and B genotype strains in different caries-sensitivity groups were different (P = 0.009). A genotype included 7 high caries-susceptible strains and 12 caries-free strains; B genotype included 17 high caries-susceptible strains and 5 caries-free strain. Some of these amplified uncEBF genes from different genotype were sequenced and testified that there existed variation of Alu I recognized sites.

CONCLUSIONSThe high cariogenecity of Sm strains isolated from caries-active children shows a close relationship with the high aciduric ability of the isolated Sm strains. uncEBF gene of Sm F-ATPase obviously exhibits genetic diversity.

Bacterial Proton-Translocating ATPases ; genetics ; metabolism ; Child, Preschool ; China ; ethnology ; Dental Caries ; microbiology ; Dental Caries Susceptibility ; Genetic Variation ; Genotype ; Humans ; Hydrogen-Ion Concentration ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Streptococcus mutans ; enzymology ; isolation & purification

Antigens, Bacterial ; metabolism ; Bacterial Adhesion ; physiology ; Biofilms ; growth & development ; Dental Caries ; microbiology ; Extracellular Matrix ; physiology ; Glucosyltransferases ; metabolism ; Humans ; Mouth ; microbiology ; Polysaccharides, Bacterial ; physiology ; Streptococcus mutans ; enzymology ; pathogenicity ; physiology ; Virulence ; Virulence Factors ; physiology