Biomimetic Virulomics for Capture and Identification of Cell-Type Specific EffectorProteins

项目总结 细菌病原体仍然是一个主要的全球健康问题，相关的高发病率和死亡率。 在宿主−病原体的相互作用中，毒力由病原体产生的生物分子控制 在感染过程中针对不同的组织。具有细胞类型特异性的效应蛋白的鉴定将是 在了解与感染相关的病理生理学方面是最重要的。然而，识别出 事实证明，相关的蛋白质效应器，特别是蛋白质水平上的效应器，在技术上是具有挑战性的。我们在寻找 通过连接多路复用器来克服与直接鉴定蛋白质效应器相关的障碍 具有细胞类型特异性亲和力捕获的定量蛋白质组学，以产生被称为 仿生病毒学(BV)[Lapek等人，2017；ACS Nano]。我们已公布的和新的初步数据 证明生物仿生和定量蛋白质组学的结合是获取关键信息的有效途径 与毒力有关的蛋白质效应物。根据我们目前的数据，我们假设：1)BV是一个可以 确定细菌蛋白的宿主细胞类型特异性；2)BV平台适用于活体小鼠模型 3)细胞类型特异性可作为研究靶向蛋白质的排序指标。在此，我们 将BV应用于研究人类重要病原体金黄色葡萄球菌的蛋白质组。在目标1中，我们 将定义针对人类皮肤细胞的金黄色葡萄球菌蛋白质组的片段。微生物学 然后，技术将被用于在体外对BV捕获的蛋白质的子集进行功能研究。这个目标是 鉴于金黄色葡萄球菌仍然是美国皮肤和软组织感染的主要原因，这一点意义重大。在目标2中，我们 将BV种子转入体内系统，以捕获具有特异性的金黄色葡萄球菌蛋白效应物 活体小鼠体内的巨噬细胞。传统上，在活体内鉴定宿主背景中的细菌蛋白质组 已经被证明是困难的。因此，利用BV在体内捕获和鉴定细菌衍生蛋白效应物 具有很强的创新性。值得注意的是，这项提议将集中在鉴定未知功能的蛋白质上。怎么能 如果S基因组中编码的近一半蛋白质是 未知功能的？这项提议将是在填补这一关键的知识空白方面向前迈出的一步。科学的 本研究的前提是基于一种强大的工具，可以直接捕获具有宿主细胞特异性的蛋白效应物 在蛋白质层面，在BV平台上独立编写的观点中突出了这一概念 [Distler等人，2017；ACS Nano]。总而言之，这项变革性的工作将成为一种强大的资源和 假说生成工具，用于从迄今尚未达到的宿主细胞特异性角度研究宿主病原体。

PROJECT SUMMARY Bacterial pathogens remain a major global health concern with associated high morbidity and mortality rates. Within host−pathogen interactions, virulence is governed by biomolecules produced by the pathogen that target different tissues during infection. Identification of effector proteins with cell-type specificity would be paramount in understanding the pathophysiology associated with infection. However, the identification of relevant protein effectors, particularly at the protein-level, has proven to be technically challenging. We sought to overcome the hurdles associated with direct identification of protein effectors by interfacing multiplexed quantitative proteomics with cell-type specific affinity capture to yield an enrichment workflow, termed Biomimetic Virulomics (BV) [Lapek et al., 2017; ACS Nano]. Our published and new preliminary data demonstrate that the pairing of biomimetics and quantitative proteomics is a powerful avenue to capture key protein effectors involved in virulence. Based on our current data we hypothesize that: 1) BV is a tool that can define host cell-type specificity of bacterial proteins; 2) the BV platform is amenable to an in vivo mouse model of infection; 3) cell-type specificity can be used as a ranking measure to target proteins for study. Herein, we will apply BV to study the proteome of the important human pathogen, Staphylococcus aureus. In Aim 1, we will define the segments of the S. aureus proteome with specificity towards human skin cells. Microbiology techniques will then be used to functionally study a subset of the BV-captured proteins in vitro. This aim is significant given S. aureus remains the leading cause of skin and soft tissue infections in the US. In Aim 2, we will seed BV to an in vivo system in order to capture S. aureus protein effector with specificity towards macrophages in a live mouse. Traditionally, in vivo, identification of bacterial proteomes in the host background has proven to be difficult. Thus, the use of BV to capture and identify bacterial-derived proteins effectors in vivo is highly innovative. Notably, this proposal will focus on characterizing proteins of unknown function. How can we expect to fully understand S aureus infection biology if nearly half of the proteins encoded in its genome are of unknown function? This proposal will be a step forward in filling this critical gap in knowledge. The scientific premise of this study is based on a powerful tool for capturing protein effector with host cell specificity directly at the protein level, a notion that was highlighted in a perspective independently written on the BV platform [Distler et al., 2017; ACS Nano]. Together, this transformative work will serve as a powerful resource and hypothesis-generating tool for host-pathogen studies from an as-yet unattained host cell-specific perspective.