Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches.
Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches.
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DOI：
10.1111/2049-632x.12179
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发表时间：
2014-08
影响因子：
3.3
通讯作者：
Valdivia RH
中科院分区：
文献类型：
--
作者：
Snavely EA;Kokes M;Dunn JD;Saka HA;Nguyen BD;Bastidas RJ;McCafferty DG;Valdivia RH
分泌的衣原体蛋白酶CPAF在体外切割一组确定的哺乳动物和衣原体蛋白。因此，这种蛋白酶已被提议调节一系列细菌和宿主细胞功能。然而，它最近受到质疑的程度，它的许多确定的底物构成真正的目标蛋白水解在感染的宿主细胞，而不是文物后裂解降解。在这里，我们阐明了CPAF在细胞感染模型中所起的作用，通过分析沙眼衣原体突变体缺乏CPAF活性。利用反向遗传学方法，我们确定了两个C。第三种菌株包含在cpa中具有无义、功能丧失突变的沙眼衣原体菌株（CT 858），第三种菌株包含在II型分泌（T2 S）机制中的突变，其通过阻断酶原分泌和随后的蛋白水解成熟为活性水解酶来抑制CPAF活性。HeLa细胞感染T2 S −或CPAF− C。沙眼衣原体突变体缺乏可检测到的体外CPAF蛋白水解活性，并且在以前归因于CPAF活性的细胞特征上没有缺陷，包括对星形孢菌素诱导的细胞凋亡的抗性、高尔基体片段化、改变的NFκ B依赖性基因表达和对再感染的抗性。然而，CPAF缺陷突变体确实显示出感染性初级体（EBs）的产生受损，表明该蛋白酶在C.沙眼此外，我们在活细胞中提供了令人信服的证据，即CPAF介导的至少两种宿主蛋白质靶点（波形蛋白丝和核包膜蛋白Lamin-associated protein 1（LAP 1））的蛋白质加工在包涵体膜完整性丧失后迅速发生，但在质膜通透性丧失和细胞溶解之前。CPAF依赖的处理宿主蛋白质与包涵体膜完整性的损失相关，因此我们建议CPAF在感染后期发挥作用，可能在细胞裂解过程中释放EB之前导致感染细胞解体的阶段。
The secreted Chlamydia protease CPAF cleaves a defined set of mammalian and Chlamydia proteins in vitro. As a result, this protease has been proposed to modulate a range of bacterial and host cellular functions. However, it has recently come into question the extent to which many of its identified substrates constitute bona fide targets of proteolysis in infected host cell rather than artifacts of post lysis degradation. Here we clarify the role played by CPAF in cellular models of infection by analyzing Chlamydia trachomatis mutants deficient for CPAF activity. Using reverse genetic approaches, we identified two C. trachomatis strains possessing nonsense, loss-of-function mutations in cpa (CT858), and a third strain containing a mutation in Type II secretion (T2S) machinery that inhibited CPAF activity by blocking zymogen secretion and subsequent proteolytic maturation into the active hydrolase. HeLa cells infected with T2S− or CPAF− C. trachomatis mutants lacked detectable in vitro CPAF proteolytic activity, and were not defective for cellular traits that have been previously attributed to CPAF activity, including resistance to staurosporine-induced apoptosis, Golgi fragmentation, altered NFκB-dependent gene expression, and resistance to reinfection. However, CPAF-deficient mutants did display impaired generation of infectious elementary bodies (EBs), indicating an important role for this protease in the full replicative potential of C. trachomatis. In addition, we provide compelling evidence in live cells that CPAF-mediated protein processing of at least two host protein targets, vimentin filaments and the nuclear envelope protein Lamin-associated protein 1 (LAP1), occurs rapidly after the loss of the inclusion membrane integrity, but before loss of plasma membrane permeability and cell lysis. CPAF-dependent processing of host proteins correlates with a loss of inclusion membrane integrity, and so we propose that CPAF plays a role late in infection, possibly during the stages leading to the dismantling of the infected cell prior to the release of EBs during cell lysis.