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

项目摘要 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 在理解与感染相关的病理生理学方面至关重要。然而， 相关蛋白质效应物，特别是在蛋白质水平上，已经证明是技术上具有挑战性的。我们试图 为了克服与通过连接多路复用的蛋白质效应物直接鉴定相关的障碍， 定量蛋白质组学与细胞类型特异性亲和捕获，以产生富集工作流程，称为 仿生病毒组学（BV）[Lapek等人，2017; ACS Nano]。我们公布的和新的初步数据 这表明，仿生学和定量蛋白质组学的配对是一个强大的途径，捕捉关键 参与毒力的蛋白质效应物。根据我们目前的数据，我们假设：1）BV是一种工具，可以 定义细菌蛋白的宿主细胞类型特异性; 2）BV平台适用于体内小鼠模型 3）细胞类型特异性可用作研究的靶蛋白的分级量度。在此我们 将应用BV研究重要的人类病原体金黄色葡萄球菌的蛋白质组。目标1： 将定义S的片段。金黄色葡萄球菌蛋白质组对人类皮肤细胞的特异性。微生物 然后将使用技术在体外对BV捕获的蛋白质的子集进行功能研究。该目的是 重要的是S。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。在目标2中， 将BV接种到体内系统中以捕获S。金黄色葡萄球菌蛋白效应子， 巨噬细胞中。传统上，在体内，在宿主背景中鉴定细菌蛋白质组 已经被证明是困难的。因此，使用BV在体内捕获和鉴定细菌衍生的蛋白效应物 是高度创新的。值得注意的是，该提案将集中于表征未知功能的蛋白质。怎么能 如果金黄色葡萄球菌基因组中编码的蛋白质中有近一半是 未知的功能？这一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 本研究的前提是建立一种直接捕获具有宿主细胞特异性的蛋白效应子的强大工具 在蛋白质水平上，这一概念在BV平台上独立撰写的观点中得到了强调 [Distler等人，2017; ACS Nano]。总之，这项变革性的工作将成为一种强大的资源， 从尚未达到的宿主细胞特异性角度进行宿主-病原体研究的假设生成工具。

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.