VicK磷酸酶活性调控变异链球菌主要毒力的转录组学分析

Transcriptomic Analysis of VicK Phosphatase Activity Regulating Major Virulence in Streptococcus Mutans

目的探究VicK磷酸酶活性对变异链球菌(S.mutans)主要毒力因子调控的全貌。方法采用RNA-seq技术对VicK^(P222A)突变株和野生对照株进行全基因转录组分析,并将分析所得的差异表达基因归类,推测S.mutans主要毒力表型的变化并进行相关验证。结果与野生对照株相比,VicK^(P222A)中810个基因的转录水平发生了明显变化。除了参与多糖和麦芽糖合成的基因外,细胞凝集、应激耐受、胞外DNA(eDNA)释放和细菌素合成相关基因都发生了变化。对转录组分析发现,与野生对照株相比,VicK^(P222A)在高渗环境中生长较慢,但对渗透压的敏感性较低,面对过氧化氢刺激VicK^(P222A)表现出较弱的抵抗力;VicK^(P222A)突变株中,eDNA的释放高于野生对照株,提示细胞自溶增强;S.mutans与血链球菌(S.sanguinis)拮抗实验证实,当S.mutans作为早期定植者时,VicK^(P222A)对S.sanguinis表现出更强的抑制作用。结论本研究揭示了VicK磷酸酶活性对S.mutans全基因组转录水平的影响,抑制VicK磷酸酶活性会影响S.mutans的渗透压耐受、氧化应激、细胞自溶及细菌素分泌等过程。

Objective To explore the overall picture of the regulation of VicK phosphatase activity on the main virulence factors of Streptococcus mutans(S.mutans).Methods RNA-seq technology was used to analyze the whole genome transcriptome of VicK^(P222A) mutant strain and wild control strain.The differentially expressed genes obtained by the analysis were classified and changes of the main virulence phenotypes of S.mutans were speculated and verified.Results The results showed that transcriptional level of 810 genes were significantly changed in VicK^(P222A) mutant compared with the wild control strain UASpec.In addition to genes involved in polysaccharide and maltose synthesis,expressions of genes related to cell agglutination,stress tolerance,extracellular DNA(eDNA)release and bacteriocin production were significantly changed.Analysis of related virulence revealed by transcriptome analysis indicated that although VicK^(P222A) grew more slowly compared with UASpec in hypertonic environment,it displayed lower sensitivity to osmotic stress than UASpec,and showed higher sensitivity to oxidative stress than UASpec.In addition,inhibition of VicK phosphatase activity resulted in higher eDNA release,suggesting enhanced cell autolysis.Competition assays on plates between S.mutans and S.sanguinis showed that VicK^(P222A) showed stronger inhibition to S.sanguinis when S.mutans was the early colonizer.Conclusion This study revealed the effect of VicK phosphatase activity on the transcriptional level of the whole genome of S.mutans.Inhibition of VicK phosphatase activity significantly changes the osmotic tolerance,oxidative stress tolerance,cell autolysis,and secretion of bacteriocin in S.mutans.