Role of M3 peptidases in Staphylococcus aureus pathogenesis

M3肽酶在金黄色葡萄球菌发病机制中的作用

基本信息

批准号：
10575030
负责人：
Shaun R Brinsmade
金额：
$ 24.17万
依托单位：
GEORGETOWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10575030
关键词：
Affect Animals Antibiotics Attenuated Bacillus Bacillus subtilis Biochemical Biology Blood Cell division Collaborations Cytoplasm DNA-Binding Proteins Data Defect Disease Endocarditis Enzymes Exhibits Family Family member Gene Expression Gene Expression Regulation Genes Genetic Screening Growth Heart Homologous Gene Hospitals Housekeeping Human Individual Infection Knowledge Laboratories Lactococcus lactis Learning Life Mass Spectrum Analysis Membrane Proteins Metalloproteases Modeling Mucous Membrane Mutagenesis Names Order Spirochaetales Organism Osteomyelitis Pathogenesis Pathogenicity Peptide Hydrolases Peptide Signal Sequences Peptides Phosphotransferases Physiological Process Production Proteins Proteobacteria Proteomics Regulation Regulon Resistance Role Sepsis Signal Transduction Site Skin Skin Tissue Soft Tissue Infections Staphylococcus aureus Substrate Specificity Surface System Systemic infection Testing Time Virulence Virulence Factors Visit Zinc antimicrobial combat commensal bacteria human pathogen insight member mouse model mutant neurotransmission neutrophil next generation sequencing novel overexpression preference promoter protein degradation protein-histidine kinase response sensor transcription factor transcriptome sequencing

项目摘要

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.

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。由膜内传感激酶 SaeS 和反应调节 DNA 结合蛋白组成 SaeR。在筛选调节 SaeR/S 活性的因子时，我们发现了两个插入物。编码推定寡肽内肽酶 F (PepF) 的基因（以下简称 PepF1）和 PepF2（由于与枯草芽孢杆菌和乳酸乳球菌 PepFs 同源）。锌金属肽酶 M3 家族的成员，与真核 M3 肽酶不同。 M3 肽酶的研究尚未充分，并且自从枯草芽孢杆菌 PepF 被发现以来一直是个谜。迄今为止唯一具有功能特征的 M3 肽酶，当过量产生时会裂解一个短片段肽，抑制该生物体中关键的孢子形成信号级联反应。突变体单独和相加地降低了 SaeR/S 活性，而金黄色葡萄球菌 pepF 无效突变体则降低了 SaeR/S 活性。 pepF 突变体在实验室培养基（包括富含肽的培养基）中生长不受影响，显示出缺陷值得注意的是，pepF1 pepF2 影响了 Sae 依赖性基因的表达和在全人血液中的存活。总而言之，我们的初步结果是，双突变体在全身感染模型中的毒力减弱。数据表明，它在调节方面发挥作用，而不是在营养或一般蛋白质周转方面发挥作用。迄今为止，真核 M3 肽酶的表征以及只有少数研究原核 M3 肽酶 – 均带有测试底物 – 这些存在重大知识差距在本提案中，我们将揭示 PepF 在促进疾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做到这一点：首先，我们将使用公正且前沿的质量。基于光谱测定的肽组学方法来识别 PepF1 和 PepF2 的底物并执行其次，我们将揭示导致的调控级联。为此，我们将使用 RNA 测序来描述金黄色葡萄球菌组。受金黄色葡萄球菌 PepF 影响的基因，然后使用诱变来识别控制 pepF 的因素最后，通过与基于质谱领域的专家合作。蛋白质组学，我们将生成数据，首次识别切割偏好和同时，我们将对M3家族的底物特异性决定因素有更深入的了解。了解金黄色葡萄球菌最重要的毒力调节因子之一是如何被控制的揭示环境和生理信号如何整合到毒力调节中网络来精确上调这种人类病原体的致病潜力。