丝氨酸/苏氨酸激酶调控猪链球菌2型毒力的分子机制

Like many eukaryotes, the analogous serine/threonine kinases (STKs) is abundantly present in bacterial genomes. The eukaroytic-type signal molecule STK untilize phosphorylation/dephosphorylation of serine/threonine residue on the target proteins to regulate various cellular processes, including growth, metabolism and virulence. In previous studies, an eukaryotic-type serine/threonine kinase (STK) in Streptococcus suis type 2 (SS2) virulent strain was identified as a potential virulent factor and defined the role of STK in SS2 infection. In the STK-deletion mutant strain, the expression levels of putative adhesin genes and virulence genes(gapdh, fbps,ef, mrp sly,sspA and ssnA) were decreased. The virulence of the STK-deletion strain was attenuated in a CD1 murine model. These data suggest that STKis involved in the pathogenesis of SS2.However,the molecular mechanism of regulation virulence by STK in SS2 is unclear. The present study is to gain further insights into the network mediated by STK, microarray is applied to compare the transcription profiles of the parental wildtype strain with that of its STK mutant strain. The comparative proteomics approach is used to distinguish between the 2-D profiles of whole cell proteins in the the parental wildtype strain and ΔStk, with the aim of revealing proteins probably involved in different pathogenicity. In vitro phosphorylation assay is performed to identify the STK substrates from those potential target proteins, including two component regulator CovR, CiaR、SalR、VirR and IRR. The site-directed mutagenesis in the recombinant STK target protein is to confirm phosphorylation positions on the serine /threonine residues.On these basis, this investigation will reveal the characteristic features of STK as a signal transduction molecular that regulates expression of these genes, involving in adherence, virulence and metabolism, to affect SS2 pathogenicity. It also will promote the understanding of the pathogenesis of SS2 infection and provide the theoretical basis for design of molecular vaccines and target drugs.

细菌中存在真核生物型的丝氨酸/苏氨酸激酶(STK)，STK通过对靶蛋白丝氨酸/苏氨酸残基的磷酸化，调控细胞内的各种生命活动，如生长、代谢和毒力等。前期工作表明，猪链球菌2型(SS2)中存在丝氨酸/苏氨酸激酶，STK基因敲除后，毒力因子和黏附素的转录水平下降，对CD1小鼠的致病力明显降低。本项目以强毒株SS2-1和STK基因缺失株△Stk为研究对象，拟通过基因芯片和比较蛋白组学方法，分别从转录水平和蛋白水平，比较缺失株与亲本株差异表达的基因和蛋白，鉴定STK调控的基因和蛋白表达谱；利用体外磷酸化试验鉴定STK磷酸化的候选靶蛋白，如CovR、CiaR、SalR、VirR和IRR等；在体外对重组靶蛋白的氨基酸定点突变，确定STK磷酸化的作用位点。在此基础上，确定STK作为信号分子的基本特点，揭示其调控SS2致病性的分子机制，促进对SS2分子致病机理的认识，为分子疫苗研究和靶标药物的设计提供理论依据。

丝氨酸/苏氨酸激酶(STK)是一种真核生物样的信号传导分子，STK通过对靶蛋白丝氨酸/苏氨酸残基的磷酸化过程，调控基因的表达和蛋白功能，进而调节细胞内的各种生命活动进程。猪链球菌2型(SS2)中存在真核生物型丝氨酸/苏氨酸激酶(ESTK)，在细菌的致病过程中发挥重要作用，但其分子机制尚不清楚。本项目以SS2强毒株SS2-1和STK基因缺失株△Stk为研究对象，通过转录组测序和比较蛋白组学方法，分别从转录水平和蛋白水平，分析缺失株与亲本株差异表达的基因和蛋白，鉴定STK调控的相关基因和蛋白。结果显示STK基因缺失导致了SS2基因组中108个基因表达上调，36 个基因下调。281个菌体蛋白的表达量发生明显变化，147个上调，134个下调，参与细菌分裂、细胞壁合成、氨基酸转运和代谢、 碳水化物运输、DNA修复、应激、信号传导、转录调节和翻译后修饰等过程。同时，利用磷酸化蛋白组学方法鉴定到STK的靶蛋白6个，包括DivIVA、Jag、FabG、 GpsB、YceG和HP(SSUBM407_0364)。本项目研究结果揭示了STK调控SS2致病性的分子机制，在一定程度上推进了SS2分子致病机理的认识，为预防和控制SS2感染进行分子疫苗研究提供了理论指导。.发表相关论文5篇，其中SCI论文2篇.