副血链球菌调控因子FimR调控网络构建及其在氧化应激中的作用

Streptococcus parasanguinis FW213 (S. parasanguinis) is a member of the viridans streptococci that constitute the major population of the oral microbial ecosystem. In its primary niche, oral cavity, S. parasanguinis is one of the early colonizers of the tooth surface. S. parasanguinis and other viridans streptococci can colonize heart valves and are common causes of native and prosthetic heart valves endocarditis. The infection is often persistent and difficult to treat, and can result in considerable morbidity and mortality despite modern antimicrobial and surgical treatment. Despite the significance of S. parasanguinis in the oral ecosystem and systemic infection, studies on its pathogenic factors have been limited to the fimCBA-tpx operon and the fap1 gene cluster in the past. Although the cellular location of FimA determined by anti-FimA serum is at the tips of the long fimbriae of FW213, very little is known how FimR regulates the expression of fimCBA-tpx operon and other unknown virulence genes and subsequent development of endocarditis. .FimR, a homologue of the iron-regulated DtxR-like protein, regulates a spectrum of genes involved in oxidative stress defense and development of endocarditis. Our preliminary results of electrophoretic mobility shift assay (EMSA) and paraquat killing experiment indicated that FimR is a global regulator protein participating in the oxidative stress responses. To define the complete FimR regulon, ChIP-seq of fragments bound by FimR and RNA-seq of the wild-type and FimR mutant strains will be performed to construct regulatory network of FimR. The consensus binding sequence of FimR will be defined from all promoters of targets that directly regulated by FimR. Once the consensus sequence is determined, a whole genome search for this consensus will be conducted. The search results will be further analyzed by in vitro EMSA. Based on the results of RNA-seq and the global search of FimR consensus, a complete FimR regulon and its downstream network will be established. The impact of the FimR regulon in the survival of S. parasanguinis against oxidative stresses will be investigated by using isogenic mutant's deficient line fimA and some newly identified ORFs. The survival rate against H2O2 and phagocytosis killing will be compared between these mutants and wild-type strain. The result of this proposal will provide basic information for the development of potential therapeutic approaches for controlling infections caused by S. parasanguinis.

副血链球菌在牙菌斑和瓣膜心内膜炎发生过程中发挥着重要作用，但是对副血链球菌毒力基因的研究局限在对fap1基因簇和fimCBA-tpx 操纵子上。而调控因子如何调控毒力基因的表达，还没有报道。序列同源比对表明FimR蛋白是调控蛋白DtxR家族蛋白成员之一，这类蛋白家族在细菌中参与调控生物体内抗氧化体系蛋白表达，从而在心内膜炎发生中发挥着重要作用。前期研究表明FimR除了调控fimCBA-tpx操纵子之外，还可以结合其它一些基因，是全局调控因子，参与抗氧化应激反应。本项目利用ChIP-seq技术和RNA-seq技术相结合，鉴定直接结合基因和次级调控基因，从全基因组水平建立比较完善的FimR调控网络；利用基因突变技术，从分子水平揭示FimR如何调控毒力基因fimA和其它一些靶基因，在抗氧化应激反应中发挥作用，找到可能与心内膜炎发生有关的靶基因，为疾病的治疗提供理论基础。

副血链球菌在牙菌斑和瓣膜心内膜炎疾病发生和发展过程中发挥着重要作用。FimR 蛋白是全局调控蛋白，除了调控毒力基因fimCBA操纵子的表达，还可以结合和调控其它一些毒力基因、转录调控基因，从而在副血链球菌心内膜炎发生和发展中发挥着重要作用。本项目利用染色质免疫共沉淀技术（ChIP-seq）、RNA测序技术（RNA-seq）技术相结合，鉴定和构建FimR的调控网络。利用基因突变技术揭示FimR如何调控毒力基因在抗氧化应激反应中发挥作用，为疾病的治疗提供靶基因。首先，我们利用ChIP-seq技术识别和鉴定出错误发生率（FDR）小于5%的70个区域。对照基因注释信息，结合RNA-seq差异基因分析结果，最终得到了17个可能被调控的基因。同时，利用同源蛋白MntR调控因子的基序信息，进行副血链球菌全基因组序列扫描，最终得到了33个可能被调控基因。对50个调控基因进行功能注释发现14个基因是毒力数据库（VFDB）和文献报道的毒力基因，5个转录调控相关基因，9个是物质转运相关基因和金属离子依赖的酶基因, 其余的大都是代谢相关酶基因。副血链球菌FimR 敲出突变株和野生菌株进行RNA-seq转录组测序和分析。结果显示共有1426个差异基因，这些基因编码蛋白质主要作用在以下途径：核糖体蛋白、ABC转运系统、二元信号转导系统、糖酵解、氨酰tRNA合成、氧化磷酸化、磷酸转移酶系统等。通过RNA-seq差异分析，可以呈现出直接调控的50个基因的表达量变化，还可以识别出间接调控基因及表达量变化。利用50个直接调控基因的表达量和fimR的表达量构建了共表达调控网络。我们用17个直接被调控基因promotor和报告基因氯霉素乙酰基转移酶（CAT）融合并分别构建在野生菌株及FimR突变菌株中，结果显示Spaf_0036 (purD), Spaf_0299 , Spaf_0300 (pepA), Spaf_0344（fimR）和Spaf_0509 (ilvB)表达有显著性变化。利用凝胶迁移实验(EMSA)进一步证实FimR蛋白可以直接和这五个基因结合，调控基因的表达。最后，利用吞噬细胞的吞噬杀伤实验，显示PepA突变株和野生株相比，在吞噬细胞的作用下，生存率明显下降。这个结果证实pepA蛋白参与抗吞噬细胞的吞噬作用，从而推测pepA基因在副血链球菌导致的心内膜炎疾病中发挥着重要作用。

内容获取失败，请点击重试