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.

摘要 M82蛋白酶在生命的所有王国中是保守的，并且在5个王国中具有4个催化基序。 保守的C-末端TMH（跨膜螺旋），以及可变数量的N-末端 TMH。来自B的PrsW。枯草芽孢杆菌是该家族的创始成员，并进行位点1-蛋白水解 (S1P)的σW抗σ因子（ASF）的抗微生物肽应激反应。色葡萄 金黄色葡萄球菌同源物（prsS）在DNA损伤剂、细胞壁靶向 抗生素，以及在血清和巨噬细胞中的生长期间。prsS突变体显示对 DNA损伤剂、H2 O2和细胞壁靶向抗生素。重要的是，还需要prsS 对S.金黄色。与此相关，S中存在一个单独的ECF-sigma因子σS。金黄色。 在每个方面，prsS的表达和缺失都模仿了sigS的表型， 期望σS ASF的S1 P是否需要PrsS。然而，尽管我们的数据将 PrsS到σS，连接两者的候选ASF尚未确定。完全有可能 在S中不存在这样的因子。金黄色葡萄球菌-有没有ASF的ECF σ-因子的例子。 也就是说，我们的PrsS数据与σS数据惊人地相似。无论如何，PrsS显然是 重要的是感染潜力的S。金黄色葡萄球菌，从而确定其蛋白水解底物 会提供很多信息此外，数千种M82酶编码在 原核生物的基因组，但只有少数研究存在表征他们。因此，在此 应用中，我们将如下询问PrsS和M82肽酶功能：目的#1 -降解组 识别PrsS底物的方法：人们理解蛋白酶如何 对细胞功能的作用是揭示其底物。为此，我们将使用 公正和尖端的蛋白质组学方法，以明确探索完整的子集， PrsS在S.金黄色。目标#2 -探索PrsS（和M82）蛋白酶 生物学：尽管PrsS和M82肽酶的重要性很大，但我们对PrsS和M82肽酶的理解是， 驱动其功能的结构特征严重缺乏。为此，我们将 对PrsS进行结构/功能调查，对其作用提供详细的新见解， 以及关于M82家族的一般信息。