prkC等重要基因在金黄色葡萄球菌小菌落变异体（SCVs）形成中的作用及其调控机制研究

金黄色葡萄球菌小菌落变异体（SCVs）经常引起持续感染和复发感染。我们前期比较基因组研究发现1株临床分离的金葡菌SCVs基因组中prkC、plsY、deoC、sstD等基因发生突变。因此，我们提出"prkC、plsY、deoC、sstD等基因突变（氨基酸改变）在临床分离金葡菌SCVs形成中发挥重要作用、蛋白激酶prkC遗传变异通过引起下游信号通路的变化影响SCVs的形成"这一科学假设。本项目拟采用等位基因替换构建prkC、plsY、deoC、sstD等基因的单基因和多基因突变子；通过细胞ATP测定、电镜观察细胞结构、小鼠致死性试验、体外竞争生长试验和细胞内持留试验等验证以上基因突变在金葡菌SCVs形成中的作用；通过DNA-蛋白质pull-down等研究遗传变异的PrkC调控的精细分子机制，进而明确其对SCVs形成的影响。最终阐明临床分离金葡菌SCVs形成的新的分子机制。

Staphylococcus aureus small colony variants (SCVs) often lead to persistent infections and recurrent infections. Our previous comparative genomic study revealed that mutations in genes such as prkC, plsY, deoC, sstD in a clinical S. aureus SCVs strain. Therefore, we propose the scientific hypothesis that mutations in genes such as prkC, plsY, deoC, sstD lead to the formation of S. aureus SCVs, and protein kinase PrkC genetic variation affects the formation of SCVs by causing changes in downstream signal pathways. Firstly, allelic replacement is used to construct single-gene and multi-gene mutants of genes such as prkC, plsY, deoC, sstD. Then, ATP measurement, electron microscopic observation of cell structure, mouse lethality test, in vitro competitive growth test and intracellular retention test, etc. are performed to verify the role of above gene mutations in the formation of S. aureus SCVs. Effect of amino acid mutation of protein kinase PrkC on the regulation mechanism of SCVs formation are analyzed using DNA-protein pull-down assay etc. Finally this study will clarify the novel molecular mechanism of clinical SCVs formation.