金黄色葡萄球菌RNaseH Ⅰ、RNaseH Ⅱ和RNaseH Ⅲ突变菌株的构建及功能研究

Construction and Functional Study of RNaseH Ⅰ, RNaseH Ⅱ and RNaseH Ⅲ mutants in Staphylococcus aureus

目的:在金黄色葡萄球菌中分别构建RNaseHⅠ、RNaseHⅡ和RNaseHⅢ的突变菌株,并研究它们在金黄色葡萄球菌中的生物学功能。方法:利用同源重组的方法在金黄色葡萄球菌中分别构建RNaseHⅠ、RNaseHⅡ和RNaseHⅢ的插入突变菌株,检测突变株生长速度、溶血活性及外分泌蛋白表达水平与野生型的差异,进一步通过流式细胞术检测突变株外分泌蛋白细胞毒性的改变,同时在体外检测突变株在全血中的存活能力。结果:构建了金黄色葡萄球菌RNaseHⅠ、RNaseHⅡ和RNaseHⅢ的插入突变菌株;表型检测结果显示,与野生型菌株相比,3种突变株的生长速度减慢,溶血素和外分泌蛋白明显减少;流式细胞术检测结果显示3种突变株毒性比野生型菌株减弱;全血杀伤实验结果显示3种突变株在全血中的存活率低于野生型菌株。结论:RNaseHⅠ、RNaseHⅡ和RNaseHⅢ在金黄色葡萄球菌生长繁殖、毒素产生和侵袭机体的过程中有重要作用,它们可能与金黄色葡萄球菌的致病性相关。

Objective： To study the function of RNaseHⅠ,RNaseHⅡ and RNaseHⅢ in Staphylococcus aureus by constructing the corresponding mutants in S.aureus 8325-4.Methods： Constructing the insertion mutants by gene homologous recombination.The growth rate,hemolysis activity and the amount of excreted proteins were de-tected both in the mutants and wild type.Futhermore,the virulence alternation of excreted proteins from mutant was detected by FCM.Meantime,the survival capability of mutants in whole blood were determined in vitro.Re-sults： The correct RNaseHⅠ,RNaseHⅡ and RNaseHⅢ mutants were successfully construced in S.aureus 8325-4;the results of mutants phenotypes showed that compared to wild type,the growth rate of three mutants decreased notably,the hemolysin and excreted proteins reduced remarkably.FCM result showed that the virulence of three mutants weakened by contrast with wild type.Whole blood killing assay result demonstrated that the survival of three mutants decreased notably compared to wild type.Conclusion： RNaseHⅠ,RNaseHⅡ and RNaseHⅢ play an important role in the growth of S.aureus and toxin production.Our results also suggested that RNaseHⅠ,RNaseH Ⅱ and RNaseHⅢ might be involved in the pathogenicity of S.aureus.