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 蛋白酶在所有生命王国中都是保守的，并且在 5 个生命体中具有 4 个催化基序 保守的 C 端 TMH（跨膜螺旋），以及数量可变的 N 端 TMH。来自枯草芽孢杆菌的 PrsW 是该家族的创始成员，并执行 Site-1 蛋白水解作用 (S1P) 的 σW 抗 Sigma 因子 (ASF) 响应抗菌肽应激。 S。 金黄色葡萄球菌同源物 (prsS) 因 DNA 损伤剂、细胞壁靶向而上调 抗生素以及血清和巨噬细胞的生长过程中。 prsS 突变体表现出对 DNA 损伤剂、H2O2 和细胞壁靶向抗生素。重要的是，prsS也是必需的 金黄色葡萄球菌具有完全毒力。与此相关的是，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 的表达和删除都与 sigS 的表型相同，这可能是 预计 σS ASF 的 S1P 是否需要 PrsS。然而，尽管我们的数据绑定了函数 PrsS 到 σS，连接两者的候选 ASF 尚未确定。完全有可能 金黄色葡萄球菌中不存在这样的因子——有一些 ECF σ 因子的例子，但没有 ASF。 话虽如此，我们的 PrsS 数据与 σS 数据惊人地相似。无论如何，PrsS 显然是 对金黄色葡萄球菌的感染潜力很重要，从而确定其蛋白水解底物 这些将被证明是非常有用的。此外，还编码了数千种 M82 酶 原核基因组，但只有少数研究对其进行表征。据此，在本 应用程序中，我们将按如下方式询问 PrsS 和 M82 肽酶功能：目标 #1 – 降解组学 识别 PrsS 底物的方法：了解蛋白酶如何发挥作用的主要方式 细胞功能的贡献在于揭示其底物。因此，为了这个目标，我们将使用 公正且前沿的蛋白质组学方法可明确探索完整的子集 由 PrsS 在金黄色葡萄球菌中进行的裂解事件。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非常缺乏。因此，为了这个目标，我们将 对 PrsS 进行结构/功能询问，提供对其作用的详细新见解， 以及有关 M82 系列的一般急需信息。