金黄色葡萄球菌金属蛋白酶Eep促进细菌毒力的分子机制

Bacterial membrane-associated proteases have important functions in the quality control of proteins, which are necessary for bacteria metabolism, stress response and pathogenesis. The metalloprotease Eep is involved in the secretion of Sex pheromones in Staphylococcus aureus, however, the main function is still unknown. In the previous research, we found that Eep is the possible substrate of FtsH protease. It affects bacteria adhension of host cell and neutrophil killing. Furthermore, Eep increases bacteria virulence in mouse model, showing that Eep is important for pathogenesis in S.aureus. Whether eep is regulated by the known virulence regulatory system in S.aureus? What’s the substrates for Eep in S.aureus? How does the protease affect bacteria virulence by interacting with host cells? Based on our previous study, we have constructed the eep deletion mutant and complement strains and expressed the Eep protein successfully, we intend to analyse the mechanism of how eep is regulated in S.aureus. We will identify the substrate of Eep by proteomic analysis and verify the molecular mechanism of how Eep increases bacteria virulence by degrading the substrate. Our contribution is significant because it advances our understanding of function of protein turnover and it enables us to design new molecules blocking the staphylococcal survival strategies.

细菌蛋白酶调控蛋白质降解，影响细菌代谢、应激及致病力。金葡菌金属蛋白酶Eep仅报道调节性激素分泌。申请人前期工作证实：Eep是细菌ATP依赖蛋白酶FtsH的底物蛋白，Eep促进细菌对宿主细胞粘附及中性粒细胞的杀伤，小鼠菌血症模型证实Eep明显促进细菌致病力，提示Eep在金葡菌致病中发挥重要作用。金葡菌重要毒力调控系统对Eep是否有调控作用？Eep是否通过降解某种底物蛋白，通过其底物蛋白与宿主细胞相互作用，从而影响细菌毒力？为详尽阐明Eep促进金葡菌毒力的具体分子机制，本课题拟在成功构建eep基因敲除株和互补株及获得有生物活性蛋白基础上，从转录和蛋白水平详尽分析金葡菌重要调控系统对Eep的调节及机制，蛋白质谱分析Eep的底物蛋白，细胞水平及体内实验进一步验证Eep通过降解其底物蛋白促进细菌毒力。研究成果将从新角度揭示蛋白质降解调控系统在细菌致病中的重要作用，为筛选新的抗感染药物靶标提供新思路

细菌蛋白酶调控蛋白质降解，影响细菌代谢、应激及致病力。本项目的研究目标阐明金黄色葡萄球菌金属蛋白酶Eep的功能，通过软件预测和蛋白定位分析，证实了Eep是定位于细胞膜的金属蛋白酶，Eep蛋白酶结构含有4个跨膜区，分别是Y3-F28，N53-D73，G351-P373，T401-W420，其中金属蛋白酶Zn结合保守功能域HEYGH位于第一个跨膜区；通过诱导肠球菌聚集实验证实Eep参与调控性激素Cam373的处理加工过程；中性粒细胞杀伤实验证实了Eep在细菌抵抗宿主细胞杀伤过程中发挥重要功能；肺泡上皮细胞A549粘附实验证实Eep促进细菌粘附宿主上皮细胞；小鼠菌血症感染模型提示eep突变后细菌毒力明显降低。为了探讨Eep调控毒力的分子机制，申请人借助蛋白组学分析对野生菌株和eep突变菌株的菌体蛋白组学研究结果发现，仅有6个蛋白表达水平发生改变，提示Eep金属蛋白酶主要不是通过影响菌体蛋白表达水平调控细菌毒力；进一步对分泌的蛋白组学研究发现，有30个蛋白表达量上调，28个蛋白表达下调，我们对重要的毒力分泌蛋白SpA进行Western blot验证证实了Eep正调控SpA蛋白的表达，提示Eep可能主要通过影响分泌蛋白的表达影响细菌毒力；另外发现，调控细菌氨基酸转运的两个重要脂蛋白在eep突变菌株中明显下调，提示Eep可能通过影响重要氨基酸D-蛋氨酸的转运影响细菌毒力。 该研究成果将从新角度揭示蛋白质降解调控系统在细菌致病中的重要作用，为筛选新的抗感染药物靶标提供新思路。