Role of M3 peptidases in Staphylococcus aureus pathogenesis

7.项目总结/摘要 SaeR/S双组分系统是金黄色葡萄球菌毒力的中心调节因子。是 由膜内传感激酶SaeS和应答调节DNA结合蛋白组成 SaeR.在筛选调节SaeR/S活性的因子过程中，我们发现两个基因中的插入， 编码推定的寡肽内肽酶F（PepF）酶（下文称为PepF 1）的基因 和PepF 2，因为与枯草芽孢杆菌和乳酸乳球菌PepF同源）。PepF是 锌金属肽酶M3家族成员。与真核M3肽酶不同，原核M3肽酶 M3肽酶研究不足，自发现以来一直是个谜。B。枯草杆菌PepF是 迄今为止唯一的功能性特征M3肽酶，并且当过度生产时切割短的 肽，抑制该生物体中关键的孢子形成信号级联反应。S.金黄色葡萄球菌pepF 1和pepF 2 突变体单独和相加地降低SaeR/S活性。而S.金黄色葡萄球菌pepF无效突变体是 在实验室培养基中生长不受影响，包括富含肽的培养基，pepF突变体显示缺陷 在Sae依赖性基因的表达和在人全血中的存活中。值得注意的是，pepF 1 pepF 2 双突变体在全身感染模型中的毒力减弱。总的来说，我们初步的 数据表明，它在调节中起作用，而不是在营养或一般蛋白质周转中起作用。尽管 迄今为止，真核M3肽酶的表征和只有少量的研究， 原核M3肽酶-所有与测试底物-有一个主要的知识缺口，这些 重要的肽酶。在这个提议中，我们将揭示PepFs在促进糖尿病发病机制中的作用。 这种重要的人类病原体，更广泛地探索PepF生物学和M3肽酶活性。 我们将通过两个具体目标来实现这一点。首先，我们将使用一个公正和尖端的质量 基于光谱法肽组学方法来鉴定PepF 1和PepF 2的底物， 对确认的底物进行功能研究。其次，我们将揭示导致 PepFs的基因调控。为此，我们将使用RNA测序来描绘S的集合。金黄色 受S.金黄色葡萄球菌PepF，然后使用诱变来鉴定控制PepF的因子 基因表达。最后，通过与质谱领域的专家合作， 蛋白质组学，我们将产生的数据，第一次，将确定切割偏好， M3家族的底物特异性决定簇。与此同时，我们将获得更深层次的 了解S.金黄色葡萄球菌得到控制， 揭示了环境和生理信号是如何整合到毒力调控中的 网络来精确地上调这种人类病原体的致病潜力。

7. Project Summary/Abstract The SaeR/S two component system is a central regulator of virulence in Staphylococcus aureus. It is composed of an intramembrane sensor kinase SaeS and a response regulator DNA binding protein SaeR. During a screen for factors that modulate SaeR/S activity, we discovered insertions in two genes that encode putative oligoendopeptidase F (PepF) enzymes (hereafter referred to as PepF1 and PepF2 due to homology with Bacillus subtilis and Lactococcus lactis PepFs). PepFs are members of the M3 family of zinc metallopeptidases. Unlike eukaryotic M3 peptidases, prokaryotic M3 peptidases are understudied and have been enigmatic since their discovery. B. subtilis PepF is the only functionally characterized M3 peptidase to date, and when overproduced cleaves a short peptide, inhibiting a key sporulation signaling cascade in that organism. S. aureus pepF1 and pepF2 mutants reduced SaeR/S activity individually and additively. While S. aureus pepF null mutants are unaffected for growth in laboratory media, including one rich in peptides, pepF mutants show defects in expression of Sae-dependent genes, and survival in whole human blood. Notably, a pepF1 pepF2 double mutant is attenuated for virulence in a systemic infection model. Altogether, our preliminary data suggest a role in regulation and not nutrition or general protein turnover. Despite the characterization of eukaryotic M3 peptidases to date and only a small number of studies with prokaryotic M3 peptidases – all with test substrates – there is a major knowledge gap for these important peptidases. In this proposal, we will unravel the role of PepFs in promoting pathogenesis in this important human pathogen and, more broadly, explore PepF biology and M3 peptidase activity. We will do this using two specific aims. First, we will use an unbiased and cutting-edge mass spectrometry-based peptidomics approach to identify substrates of PepF1 and PepF2 and perform functional studies on confirmed substrates. Second, we will reveal the regulatory cascade leading to gene regulation by PepFs. To do so, we will use RNA-Sequencing to delineate the set of S. aureus genes affected by S. aureus PepFs, and then use mutagenesis to identify factors that control pepF gene expression. In the end, by collaborating with an expert in the field of mass spectrometry-based proteomics, we will generate data that, for the first time, will identify cleavage preference and substrate specificity determinants for the M3 family. At the same time, we will gain a deeper understanding how one of the most important virulence regulators in S. aureus is controlled and reveal how environmental and physiological signals are integrated into the virulence regulatory network to precisely upregulate pathogenic potential of this human pathogen.