Effect Of Htra On The Expression And Biological Activity Of Gtfs Of Streptococcus Mutans Isolated From Children With High Cario-susceptibility

Objective:The cariogenicity of Streptococcus mutans(S.mutans)is closely related to dental plaque biofilm on the tooth surface,and the formation of plaque biofilm is associated with the extracellular polysaccharide(EPS).Glucosyl transferases(GTFs),the key enzyme in the process of EPS synthesis,has been proved to be the important virulence factor of S.mutans.Water soluble glucan and water insoluble glucan,which synthesized by GTFs,are not only the important part of EPS,but also play the key role in adhesion to tooth surface and the formation of plaque biofilm.High temperature requirement serine proteinase A(HtrA),a kind of serine protein on the surface of Streptococcus,has functions of molecular chaperone and proteolytic activity.It plays an important role in the growth,pressure feeling,biodegrading of fault structural protein,normal protein processing and regulating the protein maturity of Streptococcus.HtrA genes also exist in S.mutans.Whether the HtrA genes have influence on GTFs,and how to influence are not clear yet.The aim of this research was to compare the amount of messenger RNA(mRNA),protein of GTFs and protein biological activities between the HtrA high virulence strains and HtrA deficiency strains of S.mutans isolated from deciduous teeth to explore the function and mechanism of HtrA gene in the process of dental caries,so as to find new targets for prevention and control of dental caries.Method: HtrA deficiency strains and HtrA high virulence strains,which have been obtained at the early study by our group.Revived S.mutans strains and inoculated to Mitis Salivarius(MS)medium respectively,contrast two strains with morphological identification and biochemical identification.Inoculated single colony of HtrA deficiency strains and HtrA high virulence strains to the Brain Heart Infusion Broth(BHI)medium after identification respectively.Cultivated each strain to index period 10 hours in anaerobic environment(37?),collect bacteria liquid,adjust the fluid to the same concentration.Take same volume of two bacteria to extract total RNA and GTFs.Use quantitative real-time reverse transcription polymerase chain reaction to detect the content of gtfb,gtfc,gtfd and polyacrylamide gelelectrophoresis(SDS-PAGE)to isolate protein.Use sulfuric acid anthrone method to detect GTFs' biological activity of two bacteria and western Blot to detect the content of Glucosyl transferaseB(GTFB),Glucosyl transferaseC(GTFC),Glucosyl transferaseD(GTFD).Results: The GTFs mRNA and protein expression of HtrA deficiency strains were higher than that of HtrA high virulence strains,but the GTFs biological activities of HtrA deficiency strains are lower than that of HtrA high virulence strains.There were significant differences between two groups(P<0.05).Conclusion: For deciduous teeth,the HtrA gene of S.mutans plays an important role in the process of regulating GTFs expression.It not only participate in regulating the expression of GTFs,but also important in the transporting process of GTFs.Its mechanism is not clear yet and needs further research.