Dissecting the Influence of a C-Terminal Processing Protease on S. aureus Pathogenesis

摘要 我们的团队之前发现了一种C-末端处理蛋白水解酶(SaCtpA，ctpASA)，是完整 金黄色葡萄球菌的致病力。我们证明了SaCtpA是膜相关的和结构性的 在标准生长条件下产生，但在感染期间遇到条件时会升高 (血清、免疫细胞)。当我们将ctpASA突变体暴露于免疫系统的组件(人血清， 血液和巨噬细胞)，我们观察到与WT相比，敏感性增加。此外，用一只小鼠 在脓毒症和播散模型中，我们观察到ctpASA突变感染的小鼠存活明显好于 与接种了WT的相比，在扩散到脾、心、脑方面有多个对数下降 还有肾脏。最近，一项研究表明，SaCtpA使SOS诱导的细胞分裂抑制物失活，在一种 方式类似于在枯草杆菌中看到的CTPA，它裂解SOS诱导的细胞分裂抑制物YneA(NO 与SOSA同源)。尽管有这些已发表的作品，但关于SaCtpA如何 在金黄色葡萄球菌细胞中发挥作用，或在毒力中发挥关键作用。首先，SaCtpA是结构性表达的 在高水平下(因此不是SOS响应的一部分)，因此不太可能有单一的底物。 事实上，CTP1和CTP-3家族的对应分子有各种各样的底物，来自无数的细胞 流程。其次，与其他CTP酶相比，金黄色葡萄球菌酶具有独特的结构特征， 包括细胞质中一个大的N-末端延伸，一个非典型的PDZ结构域和一个保守的PG结合 领域--其相关性尚待确定。最后，尽管SaCtpA和SACtpA之间存在明显的联系 SOSA在DNA损伤后重新启动细胞分裂，SOSA失活背后的机制完全是 未被开发的。因此，我们认为，这一系列的新奇特征，以及无数悬而未决的问题 关于SaCtpA如何在金黄色葡萄球菌细胞中发挥作用，或调节其在毒力中的作用，这使这一点成为一个重要的 研究课题。因此，我们将：1.剖析SaCtpA的影响：我们将从执行结构开始 对SaCtpA酶的功能进行研究，探索其与其他CTP酶相比的许多独特功能。我们 然后将使用尖端的质谱学技术全球捕获SaCtpA蛋白水解性切割事件， 是我们自己和其他人发展起来的。2.探索CTPA对SOSA和细胞分裂的影响：有 关于SaCtpA与SOSA的结合仍有许多问题：这两种蛋白是如何相互作用的？ SaCtpA在SOSA上绑定在哪里？协会认可的主题(S)是什么？SaCtpA如何切割SOSA (一次，两次，竞争降级)？是否还有其他的蛋白酶参与其中？因此，它是这一目标的重点探索 这些问题，揭示了SaCtpA-SOSA相互作用的机制。同样，尽管Lexa- 受控细胞分裂抑制物在革兰氏阳性生物中广泛存在，它们如何调节其功能是 仍然未知。因此，我们还将确定SOSA靶向的抑制细胞分裂的分裂体蛋白(S)。

Abstract Our group previously identified a C-terminally processing protease (SaCtpA, ctpASA), as being required for full virulence in Staphylococcus aureus. We demonstrated that SaCtpA is membrane associated and constitutive produced under standard growth, but is elevated upon exposure to conditions encountered during infection (serum, immune cells). When we exposed ctpASA mutants to components of the immune system (human serum, blood and macrophages), we observed increased sensitivity in comparison to the WT. Moreover, using a murine model of sepsis and dissemination, we observed that ctpASA mutant infected mice survived significantly better than those inoculated with the WT, and had multiple log decreases in dissemination to the spleen, heart, brain and kidneys. Recently, a study implicated SaCtpA as inactivating an SOS-induced inhibitor of cell division, in a manner akin to that seen for CtpA in B. subtilis, which cleaves the SOS induced cell division inhibitor, YneA (no homology to SosA). Despite these published works, myriad questions remain regarding how SaCtpA functions in the S. aureus cell, or mediates its key role in virulence. Firstly, SaCtpA is constitutively expressed at high levels (and thus is not part of the SOS response), therefore having a single substrate is very unlikely. Indeed, counterparts from CTP1 and CTP-3 families have a wide array of substrates, from myriad cellular processes. Secondly, the S. aureus enzyme has unique structural features compared to other CTP-enzymes, including a large N-terminal extension in the cytoplasm, an atypical PDZ domain, and a conserved PG binding domain - the relevance of which has yet to be determined. Finally, despite a clear link between SaCtpA and SosA to reboot cell division following DNA damage, the mechanism behind SosA inactivation is completely unexplored. Therefore, we suggest that this array of novel features, alongside myriad unanswered questions regarding how SaCtpA functions in the S. aureus cell, or mediates its role in virulence, make this an important topic for study. Accordingly, we will: 1. Dissect the Influence of SaCtpA: We will begin by performing a structure function study of the SaCtpA enzyme, to explore its many unique features compared to other CTP enzymes. We will then globally capture SaCtpA proteolytic cleavage events using cutting edge mass-spectrometric techniques, developed by ourselves and others. 2. Explore the Impact of CtpA on SosA and Cell Division: There are many questions that remain regarding engagement of SaCtpA with SosA: How do these two proteins interact? Where on SosA does SaCtpA bind? What is the motif(s) recognized by SaCtpA? How does SaCtpA cleave SosA (once, twice, compete degradation)? Are other proteases involved? Thus, it is the focus of this aim to explore these questions, and shine light on the mechanism of SaCtpA-SosA interaction. Similarly, although LexA- controlled cell division inhibitors are widespread in Gram-positive organisms, how they mediate their function is still unknown. Therefore, we will also identify the divisome protein(s) targeted by SosA to inhibit cell division.