RNAⅢ调控γ-溶素的分子机制及其在ST121型金葡菌致病中的作用

γ-hemolysins are β-barrel pore forming toxins produced by Staphylococcus aureus, which assemble into oligomeric pores on the surface of many human cell types, and contribute to a large extent to the pathogenic success of S. aureus. RNAIII is the key effector of the accessory gene regulator (Agr) system. Recent findings indicate that RNAIII regulates many target genes via an antisense base pairing mechanism. However, the effects of RNAIII regulating on γ-hemolysins are still unclear and need to be further investigated. XQ is a highly virulent S. aureus type ST121 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 RNAⅢ andγ-hemolysins genes, and bioinformatics analysis indicate that hlg mRNA, which encodes γ-hemolysin, interacts with sequences of RNAIII. So we presumed that the high-level expression of γ-hemolysin is regulated by RNAIII, which plays an important role in the pathogenesis of virulent S. aureus. In this study, the gene-deletion mutant, gene-complementary and over-expressed strains of hlg (encoding γ-hemolysin) gene will be constructed and used to evaluate the roles of RNAIII in the pathogenesis of virulent XQ strain. Furthermore, the gene-deletion mutants, gene-complementary strains of RNAIII genes will be constructed and used to identify the target site of hlg mRNA interacting with RNAIII. Results of the present project will not only lead to a better comprehension on the control of RNAIII to γ-hemolysins 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.

γ-溶素为一种穿孔毒素，可裂解血细胞，是金葡菌重要的毒力因子，其表达调控机制尚不清楚。RNAⅢ是Agr调节系统的关键效应分子，调控着金葡菌多个毒力因子的表达。课题组前期分离鉴定了一株ST121型强致病性金葡菌（XQ株），其RNAⅢ、γ-溶素的表达水平与菌株致病性成正相关，且生物信息学分析显示RNAⅢ与γ-溶素mRNA存在结合位点，推测RNAⅢ调控着γ-溶素的高表达，并在XQ株致病中发挥重要作用。本课题拟以XQ株为研究对象，通过构建γ-溶素基因敲除株、回补株及过表达菌株，深入研究γ-溶素在ST121型金葡菌强致病性中的作用；再通过RNA结合阻滞实验探讨RNAⅢ调控γ-溶素的关键位点，进一步构建RNAIII位点特异性突变株、回补株，通过基因表达检测阐明RNAⅢ调控γ-溶素表达的分子机制。研究结果对认识γ-溶素在金葡菌致病中的作用有重要意义，还可为临床强致病性金葡菌感染的防治提供新的靶标。

γ-溶素为一种穿孔毒素，可裂解血细胞，是金葡菌重要的毒力因子，其表达调控机制尚不清楚。RNAⅢ是Agr调节系统的关键效应分子，调控着金葡菌多个毒力因子的表达。本项目通过检测γ-溶素在金葡菌临床菌株中的携带率、构建γ-溶素敲除株、比较γ-溶素敲除株与金葡菌野生株的溶血活性差异等实验，证实了γ-溶素是金葡菌致病的重要毒力因子；通过构建金葡菌 RNAIII缺失突变株，RT-PCR实验表明RNAIII缺失突变株的γ-溶血素A亚基和BC亚基转录均降低。进一步研究发现RNAIII的缺失可导致γ-溶素蛋白表达水平显著降低，证实了RNAⅢ对金葡菌γ-溶素表达的调控作用。.此外，通过本项目资助，对ST121型高毒力金葡菌的流行情况进行了调查研究；完成了对西南医院烧伤中心耐甲氧西林金黄色葡萄球菌（MRSA）的耐药与毒力基因分析；完成了对临床高毒力金葡菌菌株的分离鉴定及高毒力研究；首次在中国西部地区发现银葡菌相关感染病例并分离得到两株银葡菌菌株，首次报道了银葡菌小菌落变异体（SCVs）并首次对银葡菌SCV形成机制进行了初步研究。共发表科研论文13篇，其中SCI收录9篇，中文期刊4篇；参加全国、地区性学术会议论文6篇，做大会报告2次；培养博士研究生2名，硕士研究生2名。