Dissecting Yersinia Yop Targets in Neutrophils

摘要 中性粒细胞（PMNs，多形核细胞）是直接介导免疫应答的关键先天性免疫细胞。 通过释放活性氧（ROS）、脱粒、嗜酸性粒细胞 细胞外陷阱（NET）和吞噬作用。假结核耶尔森菌（Yersinia pseudotuberculosis，Yptb） 通过向中性粒细胞注射称为Yops的关键毒力蛋白来拮抗这些抗菌作用。我们 试图了解介导组织感染中细菌病原体清除的机制， 了解被YopH和YopO破坏的过程。我们已经证明SKAP 2是一个 在组织感染中，中性粒细胞中YopH的基本靶标;然而，SKAP 2不是YopH的唯一基本靶标 在组织感染中。SKAP 2的错义突变与自身免疫性疾病的发生相关 疾病和癌症。这表明SKAP 2信号下游的PMN功能障碍具有广泛的 感染控制之外的影响。我们进一步表明，SKAP 2依赖性机制在 嗜中性粒细胞对于限制另一种革兰氏阴性病原体肺炎克雷伯氏菌（Kp）的生长至关重要， 肺使用来自髓系祖细胞（MP）造血干细胞的PMNs，我们发现SKAP 2是 对于细胞外（ROS）产生是必需的，但对于Kp感染后的脱粒不是必需的。令人惊奇的是， SKAP 2是Kp感染后Syk磷酸化所必需的，但不是感染后Syk磷酸化所必需的。 CLRs和整联蛋白受体的刺激，表明中性粒细胞通过不同的受体识别Kp， 可能是G蛋白偶联受体（GPCR）。本申请的目的是利用我们的专业知识， 小鼠感染模型，MP-PMN遗传操作，细菌遗传学和蛋白质的活细胞可视化 复合物，以了解由YopH和YopO靶向的PMN中的途径。我们将进一步调查 这些目标是否在控制Kp中起关键作用。为此，我们的具体目标是（1）解剖 SKAP 2的模块化功能是在感染后中性粒细胞中产生抗微生物应答所需的 Kp. (2)了解YopO对小鼠感染期间PMN中GPCR信号传导的影响。(3)识别 YopH靶向的SKAP 2-非依赖性途径，以抑制中性粒细胞脱粒。后 完成这些研究后，我们将了解SKAP 2和其他Yop靶向蛋白如何在PMN中发挥作用， 控制Kp和Yptb的感染。深入了解分子机制，信号为 在感染过程中，中性粒细胞释放破坏组织的杀菌因子，ROS和颗粒， 新的和有针对性的方法来操纵这些途径，以增强感染期间的这些活动 与多药耐药细菌，并停止过度损害，由于不受控制的中性粒细胞反应， 各种自身免疫性疾病。

ABSTRACT Neutrophils (PMNs, polymorphonuclear cells) are pivotal innate immune cells that directly mediate pathogen clearance through release of reactive oxygen species (ROS), degranulation, neutrophilic extracellular traps (NETs) and phagocytosis. The bacterial pathogen, Yersinia pseudotuberculosis (Yptb) antagonizes these antimicrobial actions by injecting critical virulence proteins, called Yops, into PMNs. We seek to understand mechanisms that mediate clearance of bacterial pathogens in tissue infection by understanding the processes that are disrupted by YopH and YopO. We have shown that SKAP2 is one essential target of YopH in PMNs in tissue infection; however, SKAP2 is not the only essential target of YopH in tissue infection. Missense mutations in SKAP2 are associated with the development of autoimmune disorders and cancers. This suggests that PMN dysfunction downstream of SKAP2 signaling has wide ranging implications beyond infection control. We have further shown that SKAP2-dependent mechanisms in neutrophils are critical for limiting growth of another Gram-negative pathogen, Klebsiella pneumoniae (Kp), in lungs. Using PMNs derived from myeloid progenitor (MP) hematopoietic stem cells, we found that SKAP2 is essential for extracellular (ROS) production but is not required for degranulation after Kp infection. Surprisingly, SKAP2 is required for Syk phosphorylation after infection by Kp, but not for Syk phosphorylation after stimulation of CLRs and integrin receptors, indicating that neutrophil recognition of Kp is via a distinct receptor, possibly a G protein coupled receptor (GPCR). The objective of this application is to use our expertise in murine infection models, MP-PMN genetic manipulation, bacterial genetics and live-cell visualization of protein complexes, to understand pathways in PMNs that are targeted by YopH and YopO. We will further investigate whether these targets play crucial roles in the control Kp. To this end, our specific aims are to (1) Dissect the modular functions of SKAP2 required for generating anti-microbial responses in neutrophils after infection with Kp. (2) Understand the effects of YopO on GPCR signaling in PMNs during murine infection. (3) Identify the SKAP2-independent pathways that are targeted by YopH to inactivate neutrophil degranulation. After completion of these studies, we will understand how SKAP2 and other Yop-targeted proteins work in PMNs to control infection by Kp and Yptb. A thorough understanding of molecular mechanisms that signal for the release of the tissue damaging bactericidal factors, ROS and granules, by PMNs during infection should lead to novel and targeted approaches to manipulate these pathways to enhance these activities during infection with multidrug resistant bacteria and to stop excessive damage due to uncontrolled PMN responses in a variety of auto-immune diseases.