Contribution of the peptidome to CA-MRSA virulence

项目摘要 全面了解细菌致病机理不仅需要详细了解细菌的致病机理， 基因组和蛋白质组，以及感染过程中产生的肽组。整体 这项建议的目的是建立在我们令人兴奋的初步观察，描述识别 在社区相关（CA）的无细胞上清液中检测到的内源性微量蛋白和肽 MRSA培养。在这个组中，我们发现了两种源自未注释基因座的新型微蛋白 在CA-MRSA TCH 1516基因组中。我们发现，这些微蛋白，被称为S。金黄色葡萄球菌微蛋白1 （SAM 1）和S.金黄色葡萄球菌微蛋白2（SAM 2）在葡萄球菌中高度保守， 通过经典的辅助基因调控系统。我们已经开始描述这些因素，表明， SAM 1似乎作为一个典型的溶细胞素。有趣的是，SAM 2具有独特的生物活性， 扰动角蛋白网络，促进从局部S.金黄色皮肤感染， 侵袭性扩散到下层组织。这一建议的核心假设是， 显著有助于CA-MRSA在宿主中引起疾病能力。在目标1中，我们将剖析 SAM 1作为功能性溶细胞素在体外和体内的致病贡献。鉴于SAM 1的生物活性是 由于CA-MRSA被血清脂蛋白抑制，我们将关注其在细胞内环境中的作用。我们 强有力的初步数据表明，SAM 1选择性地结合到不同宿主细胞中的白藜芦醇苷（PHB 类型因此，我们将通过解剖CA-MRSA溶细胞素的潜在作用， PHBs作为SAM 1的目标。在目标2中，我们将对相互作用进行结构和功能分析 SAM 2和角蛋白之间的关系我们的数据表明，这种相互作用在体内具有重要的后果， 从局部感染到侵袭性感染的转变通过微生物和多组学的有力结合， 方法，我们将定义致病所需的SAM 2结构接触点，并将详细说明宿主 在侵入性转换期间受SAM 2影响的通路。在目标3中，评估更广泛的意义 我们的发现，我们将确定致病作用SAM发挥在一个不同的图书馆CA-MRSA菌株， 除了表达生物活性SAM同源物的其他葡萄球菌病原体之外。目标3至关重要，因为 SAM在CA-MRSA TCH 1516以外的其它葡萄球菌中的重要性仍然未知。这项建议 是高度创新的，因为它偏离了当前研究宿主-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。这项工作具有重要意义，因为 它可以推动抗葡萄球菌疗法的发展， 分子范例-肽组。

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.