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.

抽象的 中性粒细胞（PMN，多形核细胞）是直接介导的关键先天免疫细胞 通过释放活性氧 (ROS)、脱颗粒、中性粒细胞清除病原体 细胞外陷阱（NET）和吞噬作用。细菌病原体，假结核耶尔森菌 (Yptb) 通过将称为 Yops 的关键毒力蛋白注入 PMN 来拮抗这些抗菌作用。我们 寻求了解介导组织感染中细菌病原体清除的机制 了解 YopH 和 YopO 扰乱的过程。我们已经证明 SKAP2 是其中之一 组织感染中 PMN 中 YopH 的重要靶标；然而，SKAP2并不是YopH的唯一重要靶点 在组织感染中。 SKAP2 的错义突变与自身免疫性疾病的发生有关 疾病和癌症。这表明 SKAP2 信号下游的 PMN 功能障碍具有广泛的影响 其影响超出了感染控制范围。我们进一步表明 SKAP2 依赖性机制 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菌 (Kp) 的生长至关重要。 肺。使用源自骨髓祖细胞 (MP) 造血干细胞的 PMN，我们发现 SKAP2 对于细胞外 (ROS) 的产生至关重要，但对于 Kp 感染后的脱颗粒来说不是必需的。出奇， SKAP2 是 Kp 感染后 Syk 磷酸化所必需的，但 Kp 感染后 Syk 磷酸化则不需要 刺激 CLR 和整合素受体，表明中性粒细胞对 Kp 的识别是通过不同的受体， 可能是 G 蛋白偶联受体 (GPCR)。该应用程序的目的是利用我们的专业知识 小鼠感染模型、MP-PMN 基因操作、细菌遗传学和蛋白质的活细胞可视化 复合物，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 感染后中性粒细胞中产生抗微生物反应所需的 SKAP2 模块化功能 Kp。 (2)了解YopO在小鼠感染过程中对PMNs中GPCR信号传导的影响。 (3) 识别 YopH 靶向 SKAP2 独立途径来灭活中性粒细胞脱颗粒。后 完成这些研究后，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。全面了解信号的分子机制 感染期间 PMN 释放组织损伤性杀菌因子、ROS 和颗粒，应导致 寻找新的、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 与多重耐药细菌，并阻止由于不受控制的中性粒细胞反应造成的过度损害 多种自身免疫性疾病。