脂磷壁酸合成相关基因<i>dltD</i>对高致龋力变异链球菌株耐酸能力的影响

杜景云; 吴敏婧; 李艺君; 黄珊; 江山; 陈帅; 黄晓晶

doi:10.12182/20220360102

脂磷壁酸合成相关基因dltD对高致龋力变异链球菌株耐酸能力的影响

Effect of Lipoteichoic Acid Synthesis-Related Gene dltD on Acid Tolerance of Highly Cariogenic Strains of Streptococcus mutans

摘要

摘要:
目的通过构建变异链球菌593（Streptococcus mutans 593, SM593）dltD基因缺失株，探究dltD在SM593耐酸能力中的作用和可能机制，为龋病的生态防治提供理论依据。
方法 ①同源重组构建SM593 dltD基因缺失株SM593-ΔdltD；②采用全自动生长曲线分析仪绘制在不同pH培养条件下SM593和SM593-ΔdltD的生长曲线，比较二者耐酸能力；通过SM593和SM593-ΔdltD不同时间点的菌落计数（colony forming unit, CFU）计算其生存率并比较二者耐酸反应（acid tolerance response, ATR）能力；③通过不同pH条件下的糖酵解能力检测、质子通透性检测、质子移位膜腺苷三磷酸酶（H⁺-ATPase）活性检测初步探究dltD基因缺失影响耐酸的可能机制。
结果 ①PCR及测序结果显示SM593 dltD基因缺失株构建成功；②随着培养基pH逐渐降低，SM593-ΔdltD生长减缓，当培养基pH=5.0时无法生长，与 SM593相比耐酸能力下降；SM593-ΔdltD与SM593相比ATR能力下降；③不同pH条件下，SM593-ΔdltD与SM593相比，糖酵解能力无明显差异，质子通透性增加（P<0.05），H⁺-ATPase活性下降（P<0.05）。
结论 dltD基因缺失株较原始株相比耐酸能力明显下降，可能是由于dltD基因缺失导致质子通透性显著增强、H⁺-ATPase活性显著降低，从而使菌株维持胞内pH稳态能力下降所致。

Abstract:
Objective To study the role and possible mechanism of dltD in the acid tolerance of Streptococcus mutans 593 (SM593), and to provide a theoretical basis for the ecological prevention and control of dental caries by constructing the dltD gene deletion strain of SM593 (SM593-ΔdltD).
Methods 1) SM593-ΔdltD was constructed by homologous recombination. 2) The growth curve of SM593 dltD and SM593-ΔdltD under different pH culture conditions was drawn by the automatic growth curve analyzer to compare their acid tolerance. Colony forming unit (CFU) at different time points was used to calculate the survival rate and to compare the acid tolerance response (ATR) of SM593 and SM593-ΔdltD. 3) Under different pH conditions, glycolysis experiments, proton permeability test and H⁺-ATPase activity test were conducted to make preliminary exploration into the mechanisms of how dltD gene deletion may affect acid tolerance.
Results 1) PCR and sequencing results showed that the SM593-ΔdltD was constructed successfully. 2) With decreasing pH value of the culture medium, the growth of SM593-ΔdltD slowed down. When the pH value of the culture medium was 5.0, SM593-ΔdltD was not allowed to grow, and its acid tolerance was lower than that of SM593. Compared with SM593, the ATR capability of SM593-ΔdltD was decreased. 3) SM593 dltD and SM593-ΔdltD did not show obvious difference in their glycolysis ability under different pH conditions. Compared with SM593 dltD, the proton permeability of SM593-ΔdltD under different pH conditions was increased significantly (P<0.05), and H⁺-ATPase activity decreased significantly (P<0.05).
Conclusion Compared with SM593 dltD, SM593-ΔdltD showed obvious decrease in acid tolerance, which may be caused by the significant increase in proton permeability and significant decrease in the H⁺-ATPase activity induced by the deletion of the dltD gene, hence reducing its ability to maintain intracellular pH homeostasis.

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