| Dental caries seriously harms the oral cavity health of humans. It is a biofilm-dependent infectious disease in which Streptococcus mutans (S.mutans) are the major pathogenic bacteria, especially the serotype c S.mutans. The cariogenic mechanism of the pathogen is involved in three important functions, which are adherence, acidogenicity and aciduricity. In biofilm, S.mutans depend on the glycolysis action to survive and generate a great quantity of acidic ending products. Dental caries exactly results from the decalcification of tooth enamel at critical pH value that is induced by these acids produced from sugar by the bacteria in dental plaque. Lactate dehydrogenase (LDH) has been known to be a major acidogenic factor being called "lactate gate" of S.mutans. At the same time, the acidogenicity of the bacteria at low pH, particularly at the values lower than the critical pH for demineralization of enamel, is considered to be one of the major determinants of its cariogenicity. To protect the glycolysis enzymes and the continues production of acid at low pH, S.mutans must maintain ΔpH across the membrane for cells in acid environments with the internal pH alkaline relative to the external pH. Amembrane-bound proton-translocating ATPase (FoFiATPase, F-ATPase) excretes proton to the outside of streptococcal cells during acid production, and it is called "acid tolerance engine" of S.mutans. Therefore, LDH and F-ATPase are important virulence factors of S.mutans and play key roles in the occurrence and development of dental caries.It is known that the biological function of the bacteria is determined by its genetic nature. Recent studies have showed not only that genetic diversity in S.mutans genome DNA and its virulent factors DNA existed definitely, but also the close relationships between these heterogenicity characteristics and the cariogenicity or the virulence of the bateria have been testified. Therefore, the virulent factor genovariation is one of the material bases of S.mutans cariogenicicy variation. However, until now study on the genetic diversity within LDH and F-ATPase has not been reported.Our research group studied the genetic diversity in genome DNA, surface protein PI DNA and glucosyl transferaseDNA of S.mutans clinical isolated from different caries-sensitivy individuals in the previous experiments. This study investigated the genetic polymorphisms in LDH(encoding gene Idh) and F-ATPase (cab, a, 3 and v subunits encoding gene uncEBF, uncA, uncD and uncG)of S.mutans strains isolated from caries-free and caries-active individuals, also the relationships between the genetic polymorphisms and the bacteria cariogenic virulences (acidogenicity and aciduricity) as follows.The distributions of high, middle, and low LDH enzyme activity strains in difference caries-sensitive groups were different. LDH activity of S.mutans is correlated to the initiation of dental caries to some extent. Coomassie brilliant blue G250 staining and pyruvate-dependent oxidation of NADH-with and without FDP methods can be applied in preliminary quantification of LDH activity and in the screening of S.mutans strains. It is testified that the distributions of two Myel-RFLPgenotypes with different LDH activity strains were different. It is deduced that genetic diversity in Idh would be closely correlated to the differences of LDH enzyme activity of S.mutans strains. Gene transcript quantities of different genotype Idh were variable, and genotype B Idh was significantly increased, while the expressions difference of LDH gene derived from caries-active and caries-free S.mutans strains had no significance. That indicated that LDH gene of S.mutans clinical strains existed polymorphism at mRNA expression level.It is testified that two genotypes A and B of PCR-RFLP were revealed when uncEBF was digested with Alul, and Ddel digested fragments of uncEBF showed two different patterns C and D. The distributions of A and B genotype strains with different acidurance were different, and the proportion of A genotype strains from high acidurance group was higher than that from low acidurance one, also the expression level of genotype A uncEBF was higher than that of genotype B. Meanwhile the expressions difference of uncEBF gene derived from difference acidurance S.mutans strains were significant, and the mRNA expression of high acid tolerance strains was increased. Sequencing testified that the variation of Alul and Ddel recognized sites existed in b subunit encoding gene. It is suggested that the different Alul-KFLP genotypes and the increased expression level of uncEBF would be related to different acid tolerance of S.mutans strains.A, B, C and D genotype uncG were revealed by Alul -RFLP and fisrl-RFLP, but the distribution differences of the four genotypes with different acidurance strains had no significance. uncG transcription quantities derived from different genotype or different aciduranc strains were not shown difference. These results indicated that although uncG of S.mutans F-ATPase obviously showed genetic diversity, the variety would not be associated with the acid tolerance difference of S.mutans strains. The homo logy of amino acid sequences translated from different genotype uncG achieved 100%, suggesting that the relative conservation of Y subunit gene provides stablebasis for the realization of its central rotation function and ATP catalysis.Two genotypes A and B uncA of PCR-RFLP were displayed when digested with Hph I, and Mbo II digested fragments of uncA were also shown two different pattern C and D. Pearson Chi-square (2-sided) Test showed that the distributions of A and B genotype strains with different caries-sensitivity groups were different, and the proportion of A genotype strains from caries-activity group was higher than that from caries-free one. Also the distributions of C and D genotype strains with different acidurance strains were different, and the proportion of C genotype strains from high acid tolerance group was higher than that from low acid tolerance one. Meanwhile the transcriptive quantities of C and D genotype uncA were variable, and genotype C uncA were significantly increased. The expression differences of uncA derived from different genotypes or with different acidurance strains were significant, and the mRNA expressions in uncA of high acid tolerance strains and in C genotype were increased. From the results it can be deduced that uncA genetic heterogeneity of RPLP genotypes and at mRNA expession level were concerned with the acidurance and cariogenicicy of S.mutans strains.Alul -RPLP of uncD exsisted genotypes A and B. The distributions of A and B genotype strains with different acidurance were different, and the detection of A genotype strains from high acidurance group was higher than that from low acidurance one. The gene expression quantities at mRNA level of uncD with A and B genotype strains had no difference, but that with high and low acid tolerance strains were different. It suggested that the diversity of uncD AluI-RFLP genotypes and the specific sites mutation could be related to the different acid tolerance of S.mutans strains, furthermore those strains with high acid tolerance ability would increase the expression level of subunit gene uncD.It is thus evident that S.mutans LDH and F-ATPase exsit obviously genetic diversity in genotype and at mRNA expression level. This diversity characteristic isclosely associated with the acidogenic ability and aciduric ability of S.mutans strains. In the dental plaque biofilm and at low pH value, in order to increase the acidogenic and aciduric ability of strains, the bacteria would mutate the gene nucleotides and regulate the transcriptive levels of the two virulent factors gene, furthermore to adapt low pH environment and enhance the cariogenicity. In case of the adaptive regulation of F-ATPase, the subunits Y , c and a are relatively conservative, while the subunits b> a and P are actively up-regulative.
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