AgrIV调控PSM表达的分子机制及其在金葡菌致病中的作用研究

Phenol-soluble modulins (PSMs) are recently discovered key virulence determinants of Staphylococcus aureus. PSMs may cause lysis of many human cell types including leukocytes and erythrocytes, stimulate inflammatory responses, and contribute to a large extent to the pathogenic success of S. aureus. The expression of PSMs is under the strict control of accessory gene regulator (Agr) system by the direct binding of AgrA protein to the psm promoter regions. However, the impact of AgrB, AgrD and AgrC proteins in this process is still unclear and need to be further investigated. XQ is a highly virulent S. aureus strain originally isolated from a Chinese young patient with serious S. aureus skin infection and bacteriemia. It is found that the pathogenicity of XQ strain is positively correlated with the transcriptional level of psm gene. So we presumed that the high PSM expression regulated by the specific fourth Agr system plays an important role in the pathogegesis of virulent S. aureus. In this study, the gene-deletion mutants, gene-complementary and over-expressed strains of psm genes will be constructed and used to evaluate the roles of PSMs in the pathogenesis of virulent XQ strain. To assess the impact of AgrB, AgrD, and AgrC proteins on the Agr-regulated PSM expression, we attempt to replace the agrB-agrD-agrC gene of XQ Agr system (belongs to AgrⅣ type) with the relative gene locus of AgrⅠ, AgrⅡ or AgrⅢ type, and to investigate the changes in PSM expression and pathogenicity of XQ strain. The DNA binding site of AgrA protein in psm gene promotor of XQ strain will also be identified to illuminate the molecular mechanism of AgrⅣ regulation on PSM expression. Results of the present project will not only lead to a better comprehension on the control of Agr system to PSM expression and its impact on the pathogenesis of S. aureus, but also provide a potential target for the development of anti-staphylococcal drug against widespread serious S. aureus infections.

酚溶调控蛋白(PSM)具有杀白细胞、溶血、致炎等多种功能，是金葡菌重要的毒力因子。PSM的表达受Agr系统调控，通过AgrA蛋白与psm基因启动子的直接结合发挥调控作用，但Agr其他组分对PSM表达的影响尚不清楚。课题组前期分离鉴定了一株强致病性金葡菌（XQ株），属AgrⅣ型，其PSM表达水平与致病性强弱成正相关，推测特定的AgrⅣ调控着PSM的高表达，并在XQ株致病中发挥重要作用。本课题拟通过构建psm基因敲除株、互补株及过表达株，深入研究PSM在金葡菌强致病性中的作用；再用Ⅰ、Ⅱ或Ⅲ型Agr系统的agrB-agrD-agrC基因替换XQ株中相应基因，观察不同型别Agr前体蛋白对PSM表达的影响，并结合AgrA识别位点分析，阐明AgrⅣ调控PSM高表达的分子机制。研究结果不仅对理解AgrⅣ调控PSM高表达在金葡菌强致病性中的作用有重要意义，也可为临床强致病性金葡菌感染的防治提供新的靶标。

酚溶调控蛋白(PSM)是金葡菌重要的毒力因子，具有杀白细胞、溶血、致炎等多种功能；同时，PSMs还具转录调控功能，能调节α毒素、Pmt系统等的表达，在金葡菌致病过程中发挥着重要作用。本项目通过构建金葡菌Newman菌株的psmα缺失突变株，却发现psmα的缺失并不会引起Newman的溶血活性、生长分裂能力发生明显改变（这与国外研究相悖，可能是由于菌株背景不同所致），但导致了细菌上清蛋白表达谱的变化（尤AtlA蛋白表达显著降低）、毒力减弱、RNAseq分析提示11个基因表达上调，20个表达下降。.PSM的表达受Agr系统调控，通过AgrA蛋白与psm基因启动子的直接结合发挥调控作用，但Agr其他组分对PSM表达的影响尚不清楚。本项目分别以金葡菌Newman菌株（AgrⅠ型）及N315菌株（AgrⅡ型）为研究对象，通过构建Newman的Ⅱ/Ⅲ/Ⅳ型agr系统基因组替换株及pLi50-Ⅱ/Ⅲ/Ⅳ型agr质粒回补株，以及N315的Ⅰ/Ⅲ/Ⅳ型agr基因组替换株及pLi50-Ⅰ/Ⅲ/Ⅳ型agr质粒回补株，利用不同型别Agr系统替换（包括基因组替换及质粒回补）Newman及N315菌株原有Agr系统，以研究不同Agr型别对金葡菌溶血活性、细菌分裂生长、生物膜形成、蛋白表达、基因转录、色素形成等生物学活性的影响，结果显示Newman及N315的不同Agr型别基因组替换株及质粒回补株均表现出与其野生株及agr系统敲除株不同的生物学活性，与四种Agr型别金葡菌野生株的生物学特性也迥然不同。但四种不同Agr型别替换株及质粒回补株彼此之间的生物学活性却极度相似，可能是处于同样菌株背景下，受相同调控机制影响弱化了细菌的基因组成差异，使菌株表现出相似活性，但具体机制有待进一步深入研究。共发表论文13篇，其中SCI收录3篇，中文期刊4篇，参加全国、地区性学术会议论文6篇，协助培养硕士研究生2名。