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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/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，以促进针对宿主的治疗方法的开发，以对抗细菌感染。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，该鞭毛蛋白是被细胞外 Toll 样受体 5 (TLR5) 和细胞内神经元凋亡抑制因子识别蛋白 5 (NAIP5) 传感器对鞭毛蛋白的 NAIP5 感应导致多蛋白炎症小体的形成。含有 NLR caspase 募集结构域蛋白 4 (NLRC4) 的复合物，该蛋白可激活 caspase-1，引发称为细胞焦亡的炎症细胞死亡。包括肠沙门氏菌鼠伤寒血清型 (S.Tm) 在内的病原体将鞭毛蛋白传递到宿主细胞质中，多项研究表明，NAIP6 也会导致 NAIP5-NLRC4 炎症小体的激活。为什么胞质鞭毛蛋白有两种不同的传感器，以及生物环境有趣的是，许多临床相关的细菌鞭毛蛋白在何种情况下 NAIP6 可能会感知。不激活 NAIP5 的细菌在 C 末端缺乏保守的精氨酸，而且 S.Tm 被截短。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。对于 NAIP5 传感至关重要，并且细菌病原体可以通过改变该结构域来逃避 NAIP5。初步研究结果表明，TLR2 启动小鼠骨髓源性巨噬细胞 (BMDM) 会导致使用异源 III 型递送时，激活对 D0STOP 鞭毛蛋白的 NLRC4 依赖性反应总而言之，这些发现和我的初步数据。激发了概念上新颖的假设，即 TLR2 信号传导许可 NAIP6 依赖性鞭毛蛋白在目标 1 中，我计划确定 TLR2 如何许可 NAIP6-。 TLR2 引发的 BMDM 中 NLRC4 炎性体激活并测试 NAIP6 是否足以识别鞭毛蛋白使用 Yp 作为传递系统和逆转录病毒表达载体系统来重建，从而逃避 NAIP5 传感 293T 细胞中的 NAIP5/6-NLRC4 炎性体在目标 2 中，我将评估 NAIP6 鞭毛蛋白的贡献。使用 Yp 作为 Naip5-/- 和 Naip1-6D/D 小鼠的递送系统来诱导保护性宿主反应的检测。该奖学金的科学目标是发现一种鞭毛蛋白先天检测的新机制并了解为什么小鼠拥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，领导我自己的独立研究小组推动未来的职业生涯。布罗德斯基博士是宿主与病原体相互作用以及以研究和培训为导向的环境方面的专家宾夕法尼亚大学将确保该奖学金的成功完成。