Comparing The Genomes Between Fluoride-resistant Strains And Their Parental Strains Of Streptococcus Mutans

Objective: The rationale for the caries preventive effect of fluoride has been knownfor many decades. The fact that fluoride can be incorporated into the crystallinelattice of the mineral of the dental hard tissues, resulting in a tissue less soluble inacid environment, has been the scientific cornerstone for caries prevention. Butusing the fluoride for long-term may lead to appearance of the fluoride-resistantstrains. It's an obviously emergent question to how to avoid the occurrence offluoride-resistant strains in using fluoride to prevent caries. To explore thefluoride-resistant mechanism of fluoride-resistant strains is great important. We usedmolecular biological techniques to investigate the genomes and proteins differencebetween the fluoride-resistant strains of Streptococcus mutans and their parentalstrains. The purpose is to determine whether the genomes of the fluoride-resistantstrains of Streptococcus mutans are significantly mutated and whether the strainshave their own characters. Streptococcus mutans is the etiological agent of dentalcaries. Two key virulences attributing to this organism are acid production and acidtolerance, since its acid fermentation by-products can results in thedemineralization of tooth enamel and its ability to maintain a transmembrane pHgradient( Δ pH) at low pH. In order to figure out the reasons for increased acidtolerance of the fluoride-resistant strain of Streptococcus mutans, we determinedand compared the activity and genomes of membrane-bound proton translocatingATPase between Streptococccus mutans fluoride-resistant and its parental strain.Finally, this research provided an advantageous experiment evidence for themechanism of the appearance of the fluoride-resistant strain.Methods: 1. We established the right Arbitrarily Primed Polymerase Chain Reaction(AP-PCR) reaction model for the fluoride-resistant strains and their parental strains. We compared the genomic differences between the strains by the AP-PCR. 2. We established the right Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE) reaction model for the fluoride-resistant strains and their parental strains. We compared the proteins differences between the strains by the SDS-PAGE. 3. The permeabilized cells of the fluoride-resistant strain and its parental strain were prepared by treating them with 10% toluene and then two cycles of freezing and thawing. The permeabilized cells were used for ATPase assay by adding them to the reaction mixture. ATPase activity was assessed by messuring inorganic phosphate released from ATP hydrolysis. 4. We designed the primers of the F-ATPase sequence by Primer 3.0 software and compared the genomic diiferences of F-ATPase of the fluoride-resistant strain and it parental strain by Polymerase Chain Reaction (PCR).Results: 1 The results showed the the DNA amplification bands between the fluoride-resistant strains and their parental strains were completely different. Compared to parental strains, some DNA bands diappeared, but some new bands were observed in fluoride-resistant strains. Their DNA fingerprints were also different. The results suggest that the genomes of the fluoride-resistant strains have changed. 2. Bacteria contain several kinds of proteins, which take part in all kinds of life activities of bacteria. To investigate the difference between the fluoride-resistant strains and their parental strains, proteins of all these strains were analyzed by SDS-PAGE. The results showed the protein files of the fluoride-resistantstrains and their parental strains were completely different. 3. The activity of F-ATPase of the fluoride-resistant strain is significantly higher than it of the parental strain. The enzyme activity was decreasing with timeT4. The results showed the the DNA amplification bands between F-ATPase of the fluoride-resistant strain and its parental strain were different. Compared to parental strains, some DNA bands diappeared, but some new bands were observed in fluoride-resistant strain. The results suggest that the genomes of F-ATPase of the fluoride-resistant strain have changed.Conclusions: The AP-PCR fingerprints of the fluoride-resistant strains of Streptococcus mutatis and their parental strains were significantly different, and the proteins between them were also different. The genomes of the fluoride-resistant strains of Streptococcus mutans have mutated. They have their own characters. These may be the reason for the better acclimatization of fluoride-resistant strains. The higher ATPase activity of the fluoride-resistant strain may account for the increased acid tolerance of this organism, and the increment of ATPase activity and acid tolerance of the fluoride-resistant strain is likely to increase the cariogenic potential of Streptococcus mutans after F-ATPase sequence mutation of fluoride-resistent strain.