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

探索新型 M82 蛋白酶在金黄色葡萄球菌毒力中的作用

• 批准号: 10462851
• 负责人: Lindsey Neil Shaw
• 金额: $ 22.43万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-17 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462851
• 关键词: Antibiotics; Archaea; Bacillus subtilis; Binding; Biology; Blood; C-terminal; Cell Wall; Cell physiology; Clostridium difficile; Complement; DNA Damage; Data; Defect; Disease; Disease Progression; Enzymes; Eukaryota; Event; Family; Family member; Firmicutes; Genes; Genome; Growth; Hamsters; Homologous Gene; Human; Hydrogen Peroxide; Infection; Knowledge; Life; Membrane Proteins; Modeling; Multi-Drug Resistance; Mus; Mutagenesis; Mutation; N-terminal; Organism; Outcome; Pathogenicity; Peptide Hydrolases; Phenocopy; Phenotype; Proteins; Proteolysis; Proteomics; Regulation of Proteolysis; Role; Serum; Sigma Factor; Site; Staphylococcus aureus; Stress; Structure; Ursidae Family; Virulence; Virulent; Work; antimicrobial; antimicrobial peptide; experimental study; in vivo Model; insight; macrophage; methicillin resistant Staphylococcus aureus; mutant; novel; pathogen; response; transcription factor

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.

抽象的 M82蛋白酶在生活的所有王国中都是保守的，并且在5个蛋白酶中都有4个催化序列 保守的C末端TMH（跨膜螺旋），以及可变数量的N末端 TMH。枯草芽孢杆菌的PRSW是家族的创始成员，并执行Site-1蛋白解析 （S1P）响应抗菌胡椒应激的σW抗sigma因子（ASF）。 S. 金黄色同源物（PRS）是针对DNA损害剂的响应，细胞壁目标的更新 抗生素，以及血清和巨噬细胞的生长。 PRSS突变体对 DNA损害剂H2O2和细胞壁靶向抗生素。重要的是，也需要PRS 用于金黄色葡萄球菌中的全病毒。与此相关的是，金黄色葡萄球菌中存在一个孤独的ECF-SIGMA因子σs。 在每种方面，对SIGS的PRSS表达和删除 如果需要σSASF的S1P，则期望是否需要PRS。但是，要求我们的数据将 尚未确定连接这两者的候选ASF的PRSS到σs。完全有可能 金黄色葡萄球菌中不存在这样的因素 - 有一些没有ASF的ECFσ因子的例子。 话虽如此，我们的PRS数据与σs的数据非常相似。无论如何，PRS显然是 对金黄色葡萄球菌的传染性潜力很重要，从而确定其蛋白水解底物 被证明是有益的。此外，在内部编码了数千种M82酶 原核基因组，但只有少数研究来表征它们。据此 应用程序我们将询问PRS和M82 Pepperidase的功能如下：AIM＃1 - 降解 识别PRS底物的方法：人们了解蛋白酶的中心方式 有助于细胞功能的是发现其底物。因此，在此目标中，我们将使用 无偏和最前沿的蛋白质组学方法，以探索完整的子集 PRSS在金黄色葡萄球菌中执行的切割事件。目标＃2 - 探索PRS（和M82）蛋白酶 生物学：尽管PRS和M82辣椒酶的重要性很重要，但我们对 驱动其功能的结构特征非常缺乏。根据这个目的，我们将 对PRS进行结构/功能询问，为其作用提供详细的新见解， 以及一般有关M82家族的急需信息。