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

剖析 C 末端加工蛋白酶对金黄色葡萄球菌发病机制的影响

• 批准号: 10382392
• 负责人: Lindsey Neil Shaw
• 金额: $ 37.38万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-05 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10382392
• 关键词: Bacillus subtilis; Binding; Blood; Brain; C-terminal; Cell Cycle; Cell division; Cell physiology; Cells; Corynebacterium glutamicum; Cytoplasm; DNA Damage; Disease; Enterococcus faecalis; Enzymes; Event; Exposure to; Family; Genetic Transcription; Growth; Heart; Heat Stress Disorders; Human; Immune; Immune system; Infection; Kidney; Light; Link; Mediating; Membrane; Multi-Drug Resistance; Mus; Mutation; Mycobacterium tuberculosis; N-terminal; Organ; Organism; Pathogenesis; Peptide Hydrolases; Production; Proteins; Publishing; Role; SOS Response; Sepsis; Serine; Serum; Signal Transduction; Son of Sevenless Proteins; Specificity; Spleen; Staphylococcus aureus; Stress; Structure; Techniques; Time; Virulence; Virulent; Visualization; Work; cellular targeting; inhibitor; insight; macrophage; methicillin resistant Staphylococcus aureus; mouse model; mutant; novel; pathogen; protein degradation; repaired

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.

抽象的 我们的小组之前发现了一种 C 端加工蛋白酶（SaCtpA、ctpASA），这是完整的 金黄色葡萄球菌的毒力。我们证明 SaCtpA 是膜相关的和组成型的 在标准生长下产生，但在暴露于感染期间遇到的条件后会升高 （血清、免疫细胞）。当我们将 ctpASA 突变体暴露于免疫系统的成分（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，使用小鼠 在脓毒症和传播模型中，我们观察到 ctpASA 突变体感染的小鼠存活率明显更高 与接种 WT 的患者相比，向脾脏、心脏、大脑的传播有多个对数减少 和肾脏。最近，一项研究表明 SaCtpA 可以使 SOS 诱导的细胞分裂抑制剂失活， 类似于枯草芽孢杆菌中 CtpA 的方式，它裂解 SOS 诱导的细胞分裂抑制剂 YneA（无 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功能，或介导其毒力中的关键作用。首先，SaCtpA组成型表达 处于高水平（因此不是 SOS 响应的一部分），因此不太可能有单一底物。 事实上，CTP1 和 CTP-3 家族的对应物具有广泛的底物，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， 包括细胞质中的大 N 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 失活背后的机制完全是 未经探索。因此，我们建议这一系列新颖的功能以及无数未解答的问题 关于 SaCtpA 如何在金黄色葡萄球菌细胞中发挥作用，或介导其毒力作用，使其成为一个重要的研究领域。 研究主题。因此，我们将： 1. 剖析 SaCtpA 的影响：我们将首先执行一个结构 SaCtpA酶的功能研究，探索其与其他CTP酶相比的许多独特功能。我们 然后将使用尖端质谱技术在全球范围内捕获 SaCtpA 蛋白水解裂解事件， 由我们自己和其他人开发。 2.探讨CtpA对SosA和细胞分裂的影响：有 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 结合在 SosA 上的什么位置？ SaCtpA 识别的基序是什么？ SaCtpA 如何裂解 SosA （一次、两次、竞争降级）？是否涉及其他蛋白酶？因此，本次研究的重点是探索 这些问题，揭示了 SaCtpA-SosA 相互作用的机制。同样，虽然 LexA- 受控细胞分裂抑制剂广泛存在于革兰氏阳性生物中，它们如何介导其功能是 仍然未知。因此，我们还将鉴定 SosA 靶向抑制细胞分裂的分裂体蛋白。