Defining SaeR/S-dependent Neutrophil-S. aureus Interactions that Determine the Outcome of Infection

金黄色葡萄球菌(S.金黄色葡萄球菌）在美国是细菌感染的最常见原因之一， 在医院和社区中引起严重程度从轻度到致命的各种类型的感染 设置.在社区中，S.金黄色葡萄球菌通常引起皮肤和软组织感染， 每年1300万次门诊就诊（在美国）但也可引起严重的表现 在没有潜在风险的个体中发生筋膜炎和脓毒症。S. 金黄色葡萄球菌感染的主要原因是耐药率高和S.金黄色葡萄球菌培养 对抗生素的耐药性使得很难开发具有长期潜力的治疗方法 对S.金黄色。本申请中提出的研究将拓宽我们对宿主细胞的认识。 病原体相互作用，为未来研究提供了基础，旨在智能设计新的 疫苗和治疗细菌感染的疗法。为此，本项目将研究S.金黄色葡萄球菌使用 SaeR/S双组分基因调控系统逃避先天免疫。SaeR/S系统对于以下方面至关重要： 逃避中性粒细胞杀伤;然而，确切机制依赖于SaeR/S，导致中性粒细胞 功能障碍未被定义。三个特定的目的将检验SaeR/S介导的上调表达的假设。 S.金黄色葡萄球菌毒力基因破坏了正常情况下 感染在第一个目的中，通过RNA-seq的宿主病原体RNA表达将用于确定直接的 SaeR/S调控的细菌基因对人中性粒细胞基因表达的影响及解决 SaeR/S组分对嗜中性粒细胞和病原体基因同时表达个体贡献。在 第二，我们将确定SaeR/S依赖性变化是如何影响中性粒细胞人髓过氧化物酶（MPO） 活性氧（ROS）的产生改变了中性粒细胞凋亡和中性粒细胞 细胞外陷阱（NET）形成。在第三个目标中，我们将利用一种新的中性粒细胞体外启动模型，通过 刺激的PBMC，以确定初始SaeR/S介导的免疫细胞相互作用对后续 中性粒细胞功能。这些离体原代人细胞研究将用以下鼠模型进行补充 以彻底评估这些免疫应答。本提案中概述的实验结果 将提高我们对宿主-病原体相互作用的全面理解，通过特异性地鉴定新的 SaeR/S介导的S.以克服有效嗜中性白细胞应答。集体 这项研究将致力于我们长期目标，以确定宿主之间的相互反应途径 和病原体，这将突出病原体引起感染以及宿主 解决疾病。研究结果将为疫苗和免疫治疗的开发提供有价值的信息。

Staphylococcus aureus (S. aureus) is one of the most frequent causes of bacterial infections in the U.S. and is responsible for diverse types of infections ranging in severity from mild to fatal in both hospital and community settings. In the community, S. aureus typically causes skin-and-soft-tissue infections with an estimated 12 – 13 million outpatient visits per-year (in the U.S.) but can also cause severe manifestations including necrotizing fasciitis and sepsis in individuals with no underlying risk. One of the most clinically significant aspects of S. aureus infections is the high prevalence of drug resistance and the innate ability for S. aureus to develop resistance to antibiotics making it very difficult to develop therapeutics that will have potential for long-term efficacy on S. aureus. The research proposed in this application will broaden our understanding of host- pathogen interactions, providing the foundation for future studies aimed at the intelligent design of novel vaccines and therapies to treat bacterial infection. To that end, this project will study how S. aureus uses the SaeR/S two-component gene regulatory system to evade innate immunity. The SaeR/S system is essential for evasion of neutrophil killing; however, exact mechanisms dependent on SaeR/S resulting in neutrophil dysfunction are not defined. Three specific Aims will test the hypothesis that the SaeR/S-mediated up- regulation of S. aureus virulence genes undermines an effective neutrophil response that would normally clear infection. In Aim One, host pathogen RNA expression via RNA-seq will be used to determine the direct influence of SaeR/S-regulated bacterial genes on human neutrophil gene expression as well as resolve the individual contribution of SaeR/S components on neutrophil and pathogen gene expression simultaneously. In Aim Two, we will determine how SaeR/S-dependent changes in neutrophil human myeloperoxidase (MPO) activity and the production reactive oxygen species (ROS) alters neutrophil apoptosis and neutrophil extracellular trap (NET) formation. In Aim Three, we will utilize a novel ex vivo model of neutrophil priming by stimulated PBMCs to determine the role of initial SaeR/S-mediated immune cell interactions on subsequent neutrophil function. These ex vivo primary human cell studies will be complemented with murine models of infection to thoroughly evaluate these immune responses. Findings from experiments outlined in this proposal will improve our overall understanding of host-pathogen interactions by specifically characterizing novel SaeR/S-mediated mechanisms used by S. aureus to overcome an effective neutrophil response. Collectively this research will address our long-term objective to characterize reciprocal response pathways between host and pathogen that will highlight key factors needed for both the pathogen to cause infection and for the host to resolve disease. Results will provide valuable information for vaccine and immunotherapeutic development.