Objective: To investigate the changes in the expression level of sRNA SpR19 and its potential target protein GroEL in clinical isolates of Streptococcus mutans with different cariogenicity exposed to different pH conditions and explore the possibility of using these molecules as biomarkers for assessing the cariogenicity of the bacteria.
Methods: The total RNAs were extracted from the clinical isolates of Streptococcus mutans with high (strain 17) and low cariogenicity (strain 5) for high-throughput sequencing for profiling of the differentially expressed sRNAs. The candidate sRNA, SpR19, was selected for further study on the basis of bioinformatics analysis considering the role of its potential target in the cariogenic process. The differential expression levels of SpR19 in the strains exposed to both pH5.5 and pH7 culture conditions were verified by quantitative real-time PCR. The expression of the potential target of SpR19, GroEL, was also investigated at both the protein and mRNA level using Western blotting and quantitative real-time PCR.
Results: Bioinformatic analysis suggested multiple potential target sites of SpR19 both in GroEL mRNA and in the upstream and downstream inter-genic regions. Under different pH conditions, the highly cariogenic strain 17 expressed consistently low levels of SpR19 as compared with the strain 5 with a low cariogenicity; GroEL showed a reverse expression pattern in the 2 strains. An inverse correlation was found between the expressions of SpR19 and GroEL.
Conclusion: The highly cariogenic strain 17 expressed low levels of SpR19 and high levels of GroEL in both acidic and neutral culture conditions. SpR19 may negatively regulate the cariogenicity of Streptococcus mutants by targeting at GroEL.
目的: 研究在不同pH培养条件下，变异链球菌菌株的sRNA SpR19及其潜在靶向的GroEL蛋白在不同致龋力菌株中的表达变化，探讨其作为高致龋变异链球菌的分子鉴别标志物的可能性。
方法: 提取高致龋性变异链球菌的临床分离株（菌株17）和低致龋性变异链球菌的临床分离株（菌株5）的总RNA，建库后进行高通量测序获得差异表达的sRNA；结合生物信息学与文献，挑选关键sRNA与蛋白进行不同致龋菌株表达水平的研究：qRT-PCR验证目的sRNA SpR19在pH5.5和pH7培养条件下不同致龋能力菌株中的表达水平；合成变异链球菌GroEL蛋白抗原多肽并制备多克隆抗体，Western blotting鉴定不同pH培养条件下高、低致龋菌中GroEL的表达水平；qRT-PCR验证不同pH培养条件下不同致龋能力菌株GroEL的mRNA的表达水平。
结果: 生物信息学提示SpR19可能靶向GroEL的mRNA及上下游基因间区；不同pH条件下，相较于低致龋菌，高致龋菌中sRNA SpR19表达下降（P < 0.05），而GroEL蛋白与mRNA高表达（P < 0.05），二者表达趋势相反。
结论: 不同pH条件培养的高致龋性变异链球菌中，sRNA SpR19均低表达，GroEL的蛋白水平与RNA水平均存在高表达，结合生物信息学分析提示sRNA SpR19可能通过靶向GroEL负调控变异链球菌的致龋能力。