Effect Of HtrA On The Expression Of GbpC Of Streptococcus Mutans Isolated From Children With High Cario-susceptibility

Objective:The purpose of the research was to study the difference between high temperature requirement serine proteinase A（HtrA）gene-deficient strains and high-virulent strains of Streptococcus mutans（S.mutans）isolated from children with high cario-susceptibility about the expression of glucan-binding protein C（gbpC）in vitro on non-stressful environment and acquire the experimental evidence for the regulatory role and mechanism of HtrA on the cariogenicity of Streptococcus mutans in children.Methods:In this study,choosed the HtrA gene-deficient strains which constructed and the high virulent HtrA strains picked out by the previous study group.1.Bacteria resuscitation were carried out respectively,and configurated MS solid medium.After plate streak,put them into the anaerobic incubator at 37℃for 48h.The monoclonal colonies were selected and inoculated into the medium of Brain Heart Infusion Broth（BHI）for enrichment.Until the 12h of the exponent period,use the biochemical identification and Gram stain identification to identify them.2.The two strains discussed above were enriched,then we used turbidimetry to decect the OD600 values of HtrA-high virulent strains and HtrA deficient strains on 1h,2h,3h,4h,5h,6h,7h,8h,9h,10h,11h,12h,13h,14h,15h and 16h under non-stressed environment,and drew the growth curve of bacteria according to the results.3.Selected the bacterial broth of HtrA high virulence and HtrA deficient strains in the exponent period with the same volume and concentration,then used the RNA extraction kit to lyse bacterial cells and collect RNA.Respectively,two groups of bacterial RNA concentration were measured and the RNA were reverse transcribed into c DNA according to the kit instructions.The expression of gbpC gene in two strains was detected by Real-time Quantitative PCR Detecting System（RT-q PCR）.The amplification efficiency and relative expression of gbpC gene in the two groups were calculated to observe the condition how the gene expressed.4.Select the bacterial broth of HtrA high virulence and HtrA deficient strains in the exponent period with the same volume and concentration,then the bactieria were collected through high-speed centrifugation.Bacteria were sonicated and N-acetylmuramidase was used to lyse the bacterial cells for extracting the bacterial cell wall proteins.The expression of gbpC protein was detected by Western Blot and the differences of gbpC protein expression between the two groups were observed.Results:1.The colony morphology was dark blue incarceration,the texture was slightly hard,the surface was rough and the edge was not homogeneous.The biochemical identification results showed that the blank control group did not ferment biochemical reagents and the reagent did not change its color.Both experimental strains could decompose mannitol,sorbitol,secretodextran,raffinose,and turned the indicator yellow;Also,they could hydrolysis aescinate and produced the black precipitate in the indicator.Howerver,they did not hydrolyze arginine.The Gram staining microscopy showed bacterial cells were oval,paired,into a chain or short chain arrangement and the Gram stain showed positive results with purple color,which accorded with standard biological straits of Streptococcus mutans.It indicated that the HtrA gene of Streptococcus mutans had little influence on its biological characteristics and genetic characteristics.2.For the two groups of S.mutans strains above grew in the non-stress environment in vitro,from 0 to4h were their adaptive phase;from 4 to 8h were their logarithmic phase;from8 to 12h,bacterial proliferation began to slow down,and at 12h they came into the stable period.The trend of growth curves of two S.mutans strains were basically the same under non-stress environment,but the total bacterial saturation of HtrA-high virulent strains were higher than that of HtrA-deficient strains after 4h(calculated by the value of OD₆₀₀).The difference between the two groups is statistically significant（p<0.05）.3.The results of RT-q PCR indicated that the efficiency of gbpC gene amplification in HtrA-deficient strains was lower than that of HtrA-high virulent strains.Theβ-actin was selected as internal reference gene to calculate the relative experssion of the gbpC gene.The results was:HtrA-high virulent strains 1.782±0.011;HtrA-deficient strains 1.203±0.008,and the difference between the two groups is statistically significant（p<0.05）.It demonstrated that the expression of gbpC gene in HtrA-high virulent strains was higher than that of HtrA-deficient strains.4.The Western Blot results showed that HtrA-high virulent strains had higher gbpC protein gray value compared with HtrA gene-deficient strains.Theβ-actin was selected as internal reference protein to calculate the relative experssion of the gbpC.The results was:HtrA-high virulent strains 0.494±0.023;HtrA-deficient strains 0.303±0.014,and the difference between the two groups is statistically significant（p<0.05）.It demonstrated that the expression of gbpC in HtrA-high virulent strains was higher than that in HtrA-deficient strains,which was in good agreement with the result of RT-q PCR.It indicates that the absence of the HtrA gene would affect the synthesis of gbpC.Conclusions:HtrA gene may play an important role in the expression of the gbpC expression of S.mutans.HtrA gene may be one of the important factors of high cariogenicity of Streptococcus mutans.This experiment provides a theoretical basis for the hypothesis that HtrA gene would affect the virulence and cariogenicity of Streptococcus mutans of deciduous teeth.And it may become a new direction for the prevention and treatment of dental caries in the future.