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

摘要 我们的小组先前鉴定了一种C-末端加工蛋白酶（SaCtpA，ctpASA），作为完整的蛋白酶所必需的。 金黄色葡萄球菌的毒力。我们证明了SaCtpA是膜相关的和组成性的， 在标准生长下产生，但在感染期间暴露于所遇到的条件时升高 （血清、免疫细胞）。当我们将ctpASA突变体暴露于免疫系统的组分（人血清， 血液和巨噬细胞），我们观察到与WT相比增加的敏感性。此外，使用小鼠 脓毒症和播散模型，我们观察到ctpASA突变体感染小鼠的存活率明显更好 与接种WT的小鼠相比，在向脾、心脏、脑的传播中具有多个对数下降， 和肾脏。最近，一项研究表明，SaCtpA可使SOS诱导的细胞分裂抑制剂失活， 类似于在B中对CtpA所见的方式。枯草芽孢杆菌，其切割SOS诱导的细胞分裂抑制剂YneA（no 与SosA同源）。尽管有这些已发表的著作，关于SaCtpA如何 在S.金黄色葡萄球菌细胞，或介导其毒力的关键作用。首先，SaCtpA是组成型表达的， 在高水平下（因此不是SOS响应的一部分），因此具有单一底物是非常不可能的。 事实上，来自CTP 1和CTP-3家族的对应物具有广泛的底物，来自无数细胞的底物，例如， 流程.第二，S.金黄色葡萄球菌酶与其它CTP酶相比具有独特的结构特征， 包括在细胞质中的大的N-末端延伸，非典型PDZ结构域，和保守的PG结合。 域-其相关性尚未确定。最后，尽管SaCtpA和 SosA在DNA损伤后重新启动细胞分裂，SosA失活背后的机制是完全的 未开发的因此，我们认为，这一系列新的特点，以及无数悬而未决的问题， 关于SaCtpA在S.金黄色葡萄球菌细胞，或介导其毒力的作用，使这一重要的 研究课题。因此，我们将：1.剖析SaCtpA的影响：我们将开始执行一个结构 SaCtpA酶的功能研究，以探索其与其他CTP酶相比的许多独特功能。我们 然后将使用尖端质谱技术全面捕获SaCtpA蛋白水解切割事件， 由自己和他人开发。2.探索CtpA对SosA和细胞分裂的影响： 关于SaCtpA与SosA的结合仍然存在许多问题：这两种蛋白质如何相互作用？ SaCtpA绑定在SosA的哪个位置？SaCtpA识别的基序是什么？SaCtpA如何切割SosA (once，两次，竞争降解）？是否有其他蛋白酶参与？因此，探讨这一目标的重点是 这些问题，揭示了SaCtpA-SosA相互作用的机制。同样，虽然莱克萨- 受控细胞分裂抑制剂广泛存在于革兰氏阳性生物体中，它们如何介导其功能， 仍然未知。因此，我们还将鉴定SosA靶向的分裂体蛋白以抑制细胞分裂。

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.