Defining Mechanisms of NAIP5-independent Flagellin Sensing during Bacterial Infection

细菌感染期间不依赖 NAIP5 的鞭毛蛋白传感的定义机制

• 批准号: 10313353
• 负责人: James Grayczyk
• 金额: $ 6.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313353
• 关键词: Address; Amino Acids; Arginine; Bacteria; Bacterial Antibiotic Resistance; Bacterial Infections; Binding; Biological; Bone Marrow; C-terminal; CASP1 gene; Caspase; Cell Death; Cells; Cessation of life; Chimera organism; Cleaved cell; Complex; Comprehension; Cytosol; Data; Detection; Development; Ectopic Expression; Ensure; Environment; Family; Fellowship; Flagella; Flagellin; Future; Genetic; Goals; Healthcare Systems; Hematopoietic; Immune response; In Vitro; Infection; Infection Control; Inflammasome; Inflammatory; Inflammatory Response; Innate Immune Response; Innate Immune System; Interleukin-1; Legionella pneumophila; Leucine-Rich Repeat; Licensing; Malignant Neoplasms; Mediating; Membrane; Mentorship; Molecular; Mus; Mutation Detection; Nucleotides; Organelles; Outcome; Pasteurella pseudotuberculosis; Pathogen detection; Pattern; Pattern recognition receptor; Pennsylvania; Proteins; Public Health; Research; Research Training; Salmonella typhimurium; Scientist; Signal Transduction; Structural Protein; Structure; System; TLR2 gene; TLR5 gene; Terminator Codon; Tertiary Protein Structure; Testing; Therapeutic; Time; Training; Transcriptional Activation; Type III Secretion System Pathway; United States; Universities; VDAC1 gene; base; career; cell motility; clinically relevant; combat; cytokine; expression vector; extracellular; in vivo; innate immune mechanisms; macrophage; neuronal apoptosis inhibitory protein; novel; pathogen; pathogenic bacteria; receptor; reconstitution; recruit; response; sensor

PROJECT SUMMARY/ABSTRACT: Antibiotic resistant bacterial infections are an immediate public threat to the United States and global healthcare systems. We must gain a deeper understanding of the innate immune system’s response to bacterial pathogens to facilitate development of host-directed therapeutic approaches to combat bacterial infection. Many clinically relevant bacterial pathogens harbor a flagellum comprised of the structural protein flagellin, which is recognized by the extracellular Toll-like receptor 5 (TLR5) and the intracellular neuronal apoptosis inhibitory protein 5 (NAIP5) sensor. NAIP5 sensing of flagellin results in the formation of a multiprotein inflammasome complex containing the NLR caspase recruitment domain-containing protein 4 (NLRC4) which activates caspase-1, triggering an inflammatory cell death called pyroptosis. In the course of infection, a number of pathogens including Salmonella enterica serovar Typhimurium (S. Tm) deliver flagellin into the host cytosol, leading to activation of the NAIP5-NLRC4 inflammasome. Multiple studies suggest that in mice, NAIP6 also senses flagellin. Why there are two distinct sensors for cytosolic flagellin, as well as the biological circumstances under which NAIP6 might sense flagellin is unknown. Intriguingly, bacterial flagellins from many clinically relevant bacteria that do not activate NAIP5, lack a conserved arginine at the C terminus. Moreover, truncated S. Tm flagellin with a stop codon inserted in the place of conserved three amino acids in its D0 domain (termed D0STOP) abrogates recognition of flagellin by the host cell. These observations suggest that the C terminus of flagellin is essential for NAIP5 sensing and that bacterial pathogens may escape NAIP5 by altering this domain. My preliminary findings reveal that TLR2 priming of murine bone marrow derived macrophages (BMDMs) leads to activation of a NLRC4-dependent response to D0STOP flagellin when delivered using the heterologous Type III secretion system of Yersinia pseudotuberculosis (Yp). Altogether, these findings and my preliminary data provoke the conceptually novel hypothesis that TLR2 signaling licenses NAIP6-dependent flagellin sensing to overcome bacterial evasion of NAIP5. In Aim 1, I plan to determine how TLR2 licenses NAIP6- NLRC4 inflammasome activation in TLR2 primed BMDMs and test if NAIP6 is sufficient to recognize flagellins that evade NAIP5 sensing using Yp as a delivery system and a retroviral expression vector system to reconstitute the NAIP5/6-NLRC4 inflammasome in 293T cells. In Aim 2, I will assess the contribution of NAIP6 flagellin detection in eliciting protective host responses using Yp as a delivery system in Naip5-/- and Naip1-6D/D mice. The scientific goal of this fellowship is to uncover a new mechanism for innate detection of flagellin and understand why mice harbor two highly similar cytosolic sensors that detect flagellin. Another goal is to advance my training as a scientist to propel a future career in leading my own independent research group. The strong mentorship by Dr. Brodsky, an expert in host-pathogen interactions, and the research and training-oriented environment at the University of Pennsylvania will ensure successful completion of this fellowship.

项目总结/摘要： 抗生素耐药性细菌感染是对美国和全球的直接公共威胁。 医疗保健系统。我们必须更深入地了解先天免疫系统对细菌的反应。 病原体，以促进开发宿主导向的治疗方法来对抗细菌感染。许多 临床相关的细菌病原体具有由结构蛋白鞭毛蛋白组成的鞭毛， 由细胞外Toll样受体5（TLR5）和细胞内神经元凋亡抑制因子识别， 蛋白5（NAIP 5）传感器。NAIP 5传感鞭毛蛋白导致多蛋白炎性体的形成 含有NLR半胱天冬酶募集结构域蛋白4（NLRC 4）的复合物，其激活 半胱天冬酶-1，触发称为焦亡的炎性细胞死亡。在感染过程中，一些 病原体包括肠道沙门氏菌鼠伤寒血清型（S. Tm）将鞭毛蛋白递送到宿主胞质溶胶中， 导致NAIP 5-NLRC 4炎性体的活化。多项研究表明，在小鼠中，NAIP 6也 感觉鞭毛蛋白。为什么有两种不同的细胞质鞭毛蛋白传感器，以及生物学环境 NAIP6在何种条件下可能感知鞭毛蛋白尚不清楚。有趣的是，来自许多临床相关的细菌鞭毛蛋白 不激活NAIP 5的细菌在C末端缺乏保守的精氨酸。此外，截短S. TM 在其D0结构域中保守的三个氨基酸位置插入终止密码子的鞭毛蛋白（称为D0STOP） 消除宿主细胞对鞭毛蛋白的识别。这些观察结果表明鞭毛蛋白的C末端是 这是NAIP 5传感所必需的，并且细菌病原体可以通过改变该结构域来逃避NAIP 5。我 初步发现表明，小鼠骨髓衍生的巨噬细胞（BMDM）的TLR2引发导致 当使用异源III型递送时，激活对D0STOP鞭毛蛋白的NLRC 4依赖性应答 假结核耶尔森氏菌（Yp.）总之，这些发现和我的初步数据 引发概念新颖的假设，即TLR 2信号传导许可NAIP 6依赖性鞭毛蛋白 感测以克服NAIP 5的细菌逃避。在目标1中，我计划确定TLR2如何许可NAIP6- TLR2致敏的BMDM中的NLRC 4炎性体活化并测试NAIP 6是否足以识别鞭毛蛋白 其使用Yp作为递送系统和逆转录病毒表达载体系统来重建 NAIP 5/6-NLRC 4炎性小体在293T细胞中的表达。在目标2中，我将评估NAIP 6鞭毛蛋白的贡献， 在Naip5-/-和Naip1 - 6D/D小鼠中使用Yp作为递送系统引发保护性宿主应答的检测。的 这项研究的科学目标是揭示鞭毛蛋白的先天检测的新机制，并了解 为什么小鼠具有两个高度相似的细胞质传感器来检测鞭毛蛋白。另一个目标是推进我的训练 作为一名科学家，我希望能在未来的职业生涯中领导自己的独立研究小组。强大的指导 由宿主-病原体相互作用专家Brodsky博士和研究和培训环境在 宾夕法尼亚大学将确保成功完成这项奖学金。