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。我们必须对先天免疫系统对细菌的反应有更深入的了解病原体促进开发宿主定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带有结构性蛋白鞭毛蛋白的鞭毛，这是通过细胞外收费受体5（TLR5）和细胞内神经元抑制性识别蛋白5（NAIP5）传感器。鞭毛蛋白的NAIP5敏感性导致形成多蛋白炎症体含有NLR caspase募集结构域4（NLRC4）的复合物，该蛋白4（NLRC4）激活 caspase-1，引发炎性细胞死亡，称为凋亡。在感染过程中，许多病原体包括沙门氏菌肠孢子虫（S. tm）将鞭毛蛋白输送到宿主的细胞质中，导致NAIP5-NLRC4炎症体的激活。多项研究表明，在小鼠中，Naip6也感知鞭毛。为什么有两个不同的传感器用于胞质鞭毛蛋白以及生物状况在此下，Naip6可能会感觉弗拉林尚不清楚。有趣的是，来自许多临床相关的细菌鞭毛蛋白不激活NAIP5的细菌在C末端缺乏精氨酸。此外，截短的S. TM 鞭毛蛋白在其D0域中插入保守的三个氨基酸（称为D0STOP）的固定密码子（称为D0STOP）主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敏感性和细菌病原体可能通过改变该结构域而逃脱NAIP5所必需的。我的初步发现表明，鼠骨髓的TLR2启动衍生巨噬细胞（BMDMS）导致使用异源III传递时，NLRC4依赖对D0STOP鞭毛蛋白的响应的激活耶尔森氏菌的分泌系统（YP）。总共，这些发现和我的初步数据引发了概念上的新假设，即TLR2信号传导Naip6依赖性鞭毛蛋白传感以克服NAIP5的细菌进化。在AIM 1中，我计划确定TLR2如何许可Naip6- TLR2中的NLRC4炎症体激活BMDMS，并测试Naip6是否足以识别鞭毛蛋白使用YP作为输送系统和逆转录病毒表达矢量系统来逃避NAIP5的灵敏度以重新构建 293T细胞中的NAIP5/6-NLRC4炎症体。在AIM 2中，我将评估Naip6鞭毛的贡献在Naip5 - / - 和Naip1-6d/d小鼠中，使用YP作为输送系统引发受保护的宿主反应的检测。这该团契的科学目标是发现一种新的机制，用于先天检测鞭毛和理解为什么小鼠携带两个检测鞭毛蛋白的高度相似的胞质传感器。另一个目标是进步我的培训作为一名科学家，推动未来的职业领导我自己的独立研究小组。强大的精神制 Brodsky博士是宿主病原体相互作用的专家，以及以研究和培训为导向的环境宾夕法尼亚大学将确保成功完成这项研究金。