Contribution of the peptidome to CA-MRSA virulence

项目总结 要全面了解细菌的发病机制，不仅需要详细了解 基因组和蛋白质组，还有在感染过程中详细阐述的多肽组。整体而言 这项建议的目标是建立在我们令人兴奋的初步观察的基础上，描述了 群落相关(CA)无细胞培养上清液中检测到的内源性微蛋白和多肽 MRSA培养。在这个组中，我们鉴定了两个新的微蛋白，它们起源于一个未注释的基因座 在CA-MRSA TCH1516基因组中。我们发现这些被称为金黄色葡萄球菌微蛋白1的微蛋白 (Sam1)和金黄色葡萄球菌微蛋白2(SAM2)在葡萄球菌中高度保守，并受到调控 由经典的辅助基因调控系统控制。我们已经开始描述这些因素，表明 Sam1似乎是一种典型的溶细胞素。有趣的是，SAM2具有独特的生物活性， 角蛋白网络的扰动促进了体内从局部金黄色葡萄球菌皮肤感染到 侵袭性扩散到下面的组织。这一提议的中心假设是SAM 显著提高了CA-MRSA在宿主中致病的能力。在目标1中，我们将剖析 Sam1作为功能性细胞溶血素在体内外的致病作用。鉴于Sam1的生物活性是 一旦CA-MRSA进入细胞内环境，我们将重点研究其在细胞内的作用。我们的 强有力的初步数据表明，Sam1选择性地与不同宿主细胞中的禁忌素(PHBs)结合 类型。因此，我们将通过剖析CA-MRSA细胞溶血素的潜在作用，将其研究带入一个新的方向 PHBs作为Sam1的靶标。在目标2中，我们将对交互的结构和功能进行分析 在SAM2和角蛋白之间。我们的数据表明，这种相互作用在体内具有重要的影响 从局部感染类型转变为侵袭性感染类型。通过微生物学和多组学的强大结合 方法，我们将定义致病所需的SAM2结构接触点，并详细说明宿主 侵入性转换过程中受SAM2影响的通路。在目标3中，评估更广泛的意义 根据我们的发现，我们将确定SAMS在CA-MRSA菌株的不同文库中所起的致病作用， 除了表达生物活性SAM同源物的其他葡萄球菌病原体。目标3是至关重要的，因为 除了CA-MRSA TCH1516之外，SAM在其他葡萄球菌中的重要性仍不清楚。这项建议 具有很高的创新性，因为它偏离了目前研究宿主-病原体相互作用的重点 通过更成熟的基因组和蛋白质组工作流程。鉴于以下情况，拟议的工作具有重要意义 它可以推动抗葡萄球菌疗法的发展，基于一种相对较新且在很大程度上神秘的 分子范式--多肽。

PROJECT SUMMARY A comprehensive understanding of bacterial pathogenesis not only requires a detailed knowledge of the genome and proteome, but also the peptidome elaborated during the progression of infection. The overall objective of this proposal is to build upon our exciting preliminary observations describing the identification of endogenous microproteins and peptides detected in cell-free supernatants of community-associated (CA) MRSA cultures. Within this group we identified two novel microproteins originating from an unannotated locus in the CA-MRSA TCH1516 genome. We found that these microproteins, termed S. aureus microprotein 1 (SAM1) and S. aureus microprotein 2 (SAM2), are highly conserved among Staphylococci and are regulated by the classical accessory gene regulatory system. We have started to characterize these factors, showing that SAM1 appears to act as a canonical cytolysin. Intriguingly, SAM2 possesses unique bioactivity, the perturbation of keratin networks that promotes an in vivo switch from a localized S. aureus skin infection to an invasive dissemination to the underlying tissues. The central hypothesis of this proposal is that SAMs significantly contribute to CA-MRSA’s ability to cause disease in a host. In Aim 1, we will dissect the pathogenic contributions of SAM1 as a functional cytolysin in vitro and in vivo. Given the bioactivity of SAM1 is inhibited by serum lipoproteins, we will focus on its role once CA-MRSA is in an intracellular environment. Our strong preliminary data suggests SAM1 selectively binds to prohibitins (PHBs) across differential host cell types. Thus, we will take the study of CA-MRSA cytolysins in a new direction by dissecting the potential role of PHBs as targets of SAM1. In Aim 2, we will perform a structure and function analysis of the interaction between SAM2 and keratin. Our data shows that this interaction has an important in vivo consequence during the shift from a local to invasive infection type. By a powerful combination of microbiology and multi-omic approaches, we will define the SAM2 structural contact points required for pathogenesis and will detail host pathways that are impacted by SAM2 during the invasive switch. In Aim 3, to assess the broader significance of our discovery, we will determine the pathogenic roles SAMs play in a diverse library of CA-MRSA strains, in addition to other Staphylococcal pathogens that express bioactive SAM homologs. Aim 3 is critical given that the importance of SAMs in other Staphylococci beyond CA-MRSA TCH1516 remains unknown. This proposal is highly innovative because it departs from the current focus of investigating host-pathogen interactions through the more established genomic and proteomic workflows. The proposed work is highly significant given it can drive the development of anti-Staphylococcal therapies based on a relatively new and largely mysterious molecular paradigm - the peptidome.