Dissecting Yersinia Yop Targets in Neutrophils

解析中性粒细胞中的耶尔森氏菌 Yop 靶标

• 批准号: 10570181
• 负责人: Joan C Mecsas
• 金额: $ 71.77万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-11 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10570181
• 关键词: Agonist; Antibacterial Response; Atherosclerosis; Autoimmune Diseases; Bacteria; Biological Models; Cells; Chemotaxis; Communicable Diseases; Critical Pathways; Cytoplasmic Granules; Dependence; Development; Diabetes Mellitus; Disease; Event; Functional disorder; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Genetic; Goals; Gram-Negative Bacterial Infections; Growth; Hematopoietic stem cells; Immune; Infection; Infection Control; Integrins; Klebsiella Infections; Klebsiella pneumoniae; Learning; Location; Lung; Lung infections; Lupus; MAPK3 gene; Malignant Neoplasms; Measures; Mediating; Membrane; Missense Mutation; Modeling; Molecular; Multi-Drug Resistance; Multiple Bacterial Drug Resistance; Mus; Mutation; Myelogenous; PLCgamma2; Pasteurella pseudotuberculosis; Pathway interactions; Phagocytosis; Phosphorylation; Phosphorylation Site; Play; Predisposition; Process; Production; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Protein-Serine-Threonine Kinases; Proteins; Reactive Oxygen Species; Role; Signal Pathway; Signal Transduction; Syndrome; Testing; Therapeutic; Time; Tissues; Virulence; Visualization; Work; Yersinia; antimicrobial; bacterial genetics; bactericide; extracellular; genetic manipulation; in vitro Assay; in vivo; insight; migration; mutant; neutrophil; novel; pathogen; pathogenic bacteria; phosphatidylinositol 3-kinase gamma; prevent; progenitor; protein complex; receptor; response; rhoB p20 GDI; scaffold; skills

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.

摘要 中性粒细胞(PMN，多形核细胞)是关键的先天免疫细胞，直接介导 通过释放活性氧(ROS)、脱颗粒、中性粒细胞清除病原体 胞外陷阱(Net)和吞噬作用。细菌病原体假结核耶尔森氏菌(Yptb) 通过将称为YOPs的关键毒力蛋白注入PMN来对抗这些抗菌作用。我们 寻求了解在组织感染中介导细菌病原体清除的机制 了解YopH和YOPO中断的流程。我们已经证明了SKAP2是一个 YopH在组织感染中中性粒细胞中的重要靶点；然而，SKAP2不是YopH唯一的基本靶点 在组织感染中。SKAP2错义突变与自身免疫的发生 疾病和癌症。这表明，SKAP2信号下游的PMN功能障碍具有广泛的范围 感染控制之外的影响。我们进一步证明了SKAP2依赖的机制在 中性粒细胞对限制另一种革兰氏阴性病原体肺炎克雷伯菌(Klebsiella Penoniae，KP)的生长至关重要 肺部。使用来自髓系祖细胞(MP)造血干细胞的PMN，我们发现SKAP2是 对细胞外(ROS)的产生是必需的，但在KP感染后脱颗粒不是必需的。令人惊讶的是， Kp感染后Syk的磷酸化需要SKAP2，而感染后Syk的磷酸化不需要SKAP2 刺激CLRs和整合素受体，表明中性粒细胞对KP的识别是通过一个不同的受体， 可能是G蛋白偶联受体(GPCR)。此应用程序的目标是将我们的专业知识用于 小鼠感染模型、MP-PMN基因操作、细菌遗传学和蛋白质活细胞可视化 复合体，以了解YopH和YOPO靶向的PMN中的通路。我们将进一步调查 这些目标是否在控制Kp中发挥关键作用。为此，我们的具体目标是(1)剖析 肺炎衣原体感染后中性粒细胞产生抗微生物反应所需的SKAP2的模块化功能 Kp.(2)了解YOPO在小鼠感染过程中对中性粒细胞GPCR信号的影响。(3)确定 YopH靶向灭活中性粒细胞脱颗粒的SKAP2非依赖途径。之后 完成这些研究，我们将了解SKAP2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。对信号的分子机制的透彻理解 中性粒细胞在感染过程中释放的组织破坏性杀菌因子ROS和颗粒应导致 以新的和有针对性的方法来操纵这些途径，以在感染期间增强这些活动 并阻止由于PMN反应不受控制而造成的过度损害 各种自身免疫性疾病。