金黄色葡萄球菌ATP依赖蛋白酶ClpP与毒力调控蛋白SaeS之间的调节关系

The proteases system have important functions in the quality control of proteins, which are necessary for bacterial homeostasis in Staphylococcus aureus. ATP-dependent protease ClpP is important for stress response, biofilm formation and pathogenesis, but the exact mechanism is still unknown. In the previous study, we found that ClpP regulates the expression of the important virulence regulator SaeS significantly. The expression of SaeS was increased significantly in clpP mutant strain compared to wild-type. More importantly, the SaeS-controlled target genes were also down-regulated by ClpP. How ClpP protease and ATPase chaperons regulate expression of SaeS? Whether the regulatory network between ClpP and SaeS contributes to virulence? In order to identify regulatory network between ClpP and SaeS systemically, we will analyze the regulation relationship between ClpP and SaeS on transcriptional and translational level in detail. We will identify the role and regulatory mechanism of the unknown proteins connecting ClpP and SaeS. Furthermore, we will verify the contribution to virulence by the regulatory network between ClpP and SaeS using mouse model. Our study is significant because it advances our understanding of regulatory network of ClpP protease, it identifies the mechanism of how protease affects virulence and it enables us to design new molecules blocking the staphylococcal survival strategies.

金葡菌蛋白酶系统调控蛋白质降解，维持细菌稳态平衡。ATP依赖蛋白酶ClpP在细菌应激反应、生物膜形成和毒力等方面发挥重要作用，但具体调节机制有待阐明。申请人前期工作提示ClpP对毒力调控蛋白SaeS有明显调节作用，clpP基因敲除后SaeS表达降低，且SaeS调控的下游靶基因表达明显降低。ClpP蛋白酶及其分子伴侣如何发挥对SaeS的调控作用？ClpP与SaeS之间是否存在相互调节关系？这种调节关系是否最终促进了细菌的毒力？为系统研究ClpP与SaeS之间的调节关系，本课题拟采取转录组和蛋白组学分析技术，从整体探讨ClpP与SaeS在转录或翻译水平调节关系，明确连接ClpP和SaeS中间分子的作用及调节机制。动物体内模型进一步明确ClpP与SaeS之间调节关系最终对细菌毒力的影响。研究结果将为补充完善蛋白酶ClpP的调控网络、阐明蛋白酶调控细菌毒力的机制、筛选新的抗感染药物靶标提供新思路。

金黄色葡萄球菌蛋白酶系统调控蛋白质降解，维持细菌稳态平衡。ATP依赖蛋白酶在细菌应激反应、生物膜形成和毒力等方面发挥重要作用，但具体调节机制有待阐明。SaeRS二元调控系统促进细菌溶血活性和致病力，金黄色葡萄球菌Newman菌株的SaeS蛋白含有一个点突变SaeSL18P，我们前期的研究结果发现，ATP依赖蛋白酶FtsH直接降解调控SaeSL18P，非常有意思的是，ATP依赖蛋白酶ClpP敲除后，SaeSL18P的表达水平显著降低，提示ClpP保护SaeS蛋白稳定性。我们的研究进一步发现，ClpP对SaeS蛋白稳定性的调控依赖于FtsH和钼蝶玲合成蛋白A（MoeA）的共同作用。ClpP并不影响ftsH的转录表达，但是促进FtsH蛋白的表达，尽管如此，体外酶活性检测发现ClpXP并不能直接降解调控FtsH。转座子插入突变文库筛选发现moeA通过抑制SaeS的转录表达抑制金葡菌的溶血活性，并且ClpXP可以直接降解调控MoeA蛋白。最后，通过小鼠菌血症感染模型，发现MoeA促进细菌的毒力。综上所述，我们的结果证实ClpP依赖FtsH和MoeA共同作用正调控SaeS蛋白表达。尽管moeA负调控SaeS蛋白活性，但是MoeA可能通过影响细菌生理过程促进细菌致病力。