Exploring the Role of a Novel M82 Protease in S. aureus Virulence

摘要 M82蛋白酶在所有生命王国中都是保守的，并在5个物种中携带4个催化基序 保守的C-末端TMH(跨膜螺旋)，伴随着数量可变的N-末端 嗯。来自枯草杆菌的Prsw是该家族的创始成员，执行1位蛋白分解 (S1P)σW抗Sigma因子(ASF)对抗菌肽应激的反应。S。 金黄色葡萄球菌同源基因(PRSS)上调是对DNA损伤剂、细胞壁靶向的反应 抗生素，以及在血清和巨噬细胞生长期间。PRSS突变体对 DNA损伤剂、过氧化氢和靶向细胞壁的抗生素。重要的是，PRSS也是必需的 对金黄色葡萄球菌的完全毒力。与此相关的是，在金黄色葡萄球菌中存在一个单独的ECF-Sigma因子，σS。 在每一方面，PRSS的表达和缺失都与SIGS相似，哪一种可能 预计σS ASF的S1P是否需要PRSS。然而，尽管我们的数据将 PRSS到σS，连接两人的候选人ASF还没有确定。完全有可能 在金黄色葡萄球菌中不存在这样的因子--有ECFσ的例子--没有ASF的因子。 尽管如此，我们的PRSS数据与σS的数据惊人地相似。 对金黄色葡萄球菌的感染潜力很重要，从而决定了它的蛋白分解底物是什么 ARE将被证明是非常有用的。此外，数以千计的M82酶在 原核生物基因组，但目前只有少数几项研究对它们进行了描述。因此，在这方面， 应用我们将询问PRSS和M82肽酶功能如下：目标#1-变性 鉴定PRSS底物的方法：人们理解蛋白酶如何 对细胞功能的贡献是通过揭开它的底物。因此，在这个目标中，我们将使用 无偏见和尖端的蛋白质组方法，以确定探索完整的子集 PRSS在金黄色葡萄球菌中执行的切割事件。目标2-探索PRSS(和M82)蛋白酶 生物学：尽管PRSS和M82肽酶很重要，但我们对 驱动其功能的结构特征严重缺乏。因此，在这一目标中，我们将 对PRSS的结构/功能进行询问，对其作用提供详细的新见解； 以及关于M82系列的一般信息。

ABSTRACT M82 proteases are conserved across all kingdoms of life, and bear 4 catalytic motifs across 5 conserved C-terminal TMH (Transmembrane Helices), alongside a variable number of N-terminal TMH. PrsW from B. subtilis is the founding member of the family and performs Site-1-Proteolysis (S1P) of the σW Anti-Sigma Factor (ASF) in response to antimicrobial peptide stress. The S. aureus homolog (prsS) is upregulated in response to DNA-damaging agents, cell wall-targeting antibiotics, and during growth in serum and macrophages. prsS mutants display sensitivity to DNA-damaging agents, H2O2, and cell wall-targeting antibiotics. Importantly, prsS is also required for full virulence in S. aureus. Connected to this, a lone ECF-sigma factor, σS, exists in S. aureus. In each regard, the expression and deletion of prsS phenocopies that for sigS, which one might expect if PrsS is required for S1P of a σS ASF. However, despite our data tying the function of PrsS to σS, a candidate ASF connecting the two has not been identified. It is entirely possible that such a factor does not exist in S. aureus – there are examples of ECF σ-factors that have no ASF. With that said, our PrsS data is strikingly similar to that for σS. Regardless, PrsS is clearly important to the infectious potential of S. aureus, thus determining what its proteolytic substrates are would prove highly informative. Additionally, thousands of M82 enzymes are encoded within prokaryotic genomes, yet only a handful of studies exist characterizing them. Accordingly, in this application we will interrogate PrsS and M82 peptidase function as follows: Aim #1 – Degradomic Approaches to Identify PrsS Substrates: The central way one understands how a protease contributes to cellular function is by uncovering its substrates. Therefore, in this aim we will use unbiased and cutting edge proteomic approaches to definitively explore the complete subset of cleavage events performed by PrsS in S. aureus. Aim #2 – Exploring PrsS (and M82) Protease Biology: Despite the importance of PrsS, and M82 peptidases at large, our understanding of the structural features that drive their function is sorely lacking. Accordingly, in this aim we will undertake a structure/function interrogation of PrsS, providing detailed new insight into its role, and much needed information on the M82 family in general.