Trapping and Proteomic Identification of Cellular Substrates of the ClpP Protease in Staphylococcus aureus

In the important human pathogen Staphylococcus aureus the cytoplasmic ClpP protease is essential for mounting cellular stress responses and for virulence. To directly identify substrates of the ClpP protease, we expressed in vivo a proteolytic inactive form of ClpP (ClpPtrap) that will retain but not degrade substrates translocated into its proteolytic chamber. Substrates captured inside the proteolytic barrel were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry. In total, approximately 70 proteins were trapped in both of the two S. aureus strains NCTC8325-4 and Newman. About one-third of the trapped proteins are previously shown to be unstable or to be substrates of ClpP in other bacteria, supporting the validity of the ClpP-TRAP. This group of proteins encompassed the transcriptional regulators CtsR and Spx, the ClpC adaptor proteins McsB and MecA, and the cell division protein FtsZ. Newly identified ClpP substrates include the global transcriptional regulators PerR and HrcA, proteins involved in DNA damage repair (RecA, UvrA, UvrB), and proteins essential for protein synthesis (RpoB and Tuf). Our study hence underscores the central role of Clp-proteolysis in a number of pathways that contribute to the success of S. aureus as a human pathogen.

作为重要人类致病菌的金黄色葡萄球菌（Staphylococcus aureus），其胞质ClpP蛋白酶（ClpP protease）在介导细胞应激反应与致病力形成过程中发挥不可或缺的作用。为直接鉴定ClpP蛋白酶的底物，我们在体内表达了一种蛋白水解失活型ClpP（ClpPtrap），该变体可结合但不会降解易位进入其蛋白水解腔室的底物。被捕获于蛋白水解桶状结构内的底物会与带有His标签的ClpP复合物共纯化，并通过质谱法完成鉴定。在两种金黄色葡萄球菌菌株NCTC8325-4与Newman中，总计约70种蛋白质被捕获。其中约三分之一的捕获蛋白此前已被证实为不稳定蛋白，或是其他细菌中的ClpP底物，这验证了ClpP捕获法（ClpP-TRAP）的有效性。该类蛋白涵盖转录调节因子CtsR与Spx、ClpC适配蛋白McsB与MecA，以及细胞分裂蛋白FtsZ。新鉴定出的ClpP底物则包括全局转录调节因子PerR与HrcA、参与DNA损伤修复的蛋白质（RecA、UvrA、UvrB），以及蛋白质合成必需的蛋白质（RpoB与Tuf）。因此本研究凸显了Clp蛋白水解在多条通路中的核心作用，这些通路共同促成了金黄色葡萄球菌作为人类致病菌的致病优势。