Pathophysiological Actions of Anthrax Virulence Determinants

炭疽毒力决定因素的病理生理作用

• 批准号: 9566673
• 负责人: Stephen Leppla
• 金额: $ 41.45万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9566673
• 关键词: Adenylate Cyclase; Animal Model; Animals; Anthrax disease; Anti-Inflammatory Agents; Anti-inflammatory; Antigens; Binding Proteins; Blood Vessels; CASP1 gene; Calmodulin; Cardiovascular system; Cell Death; Cell membrane; Cell model; Cells; Cellular biology; Cessation of life; Chemicals; Chromosome Mapping; Cleaved cell; Collection; Complex; Concentration Camps; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Dendritic Cells; Drosophila genus; Edema; Extravasation; Genetic; Genetic Polymorphism; Genetic Variation; Goals; Hematopoietic; Hour; Hypotension; Immune; Immune response; Inbred Mouse; Inbred Strain; Inbreeding; Inflammasome; Inflammatory; Infusion procedures; Interleukin-1; Interleukin-18; Mammalian Cell; Maps; Mediating; Metalloproteases; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mus; National Institute of Allergy and Infectious Disease; Pathway interactions; Peptide Hydrolases; Phenotype; Phenylephrine; Phosphotransferases; Play; Process; Protein Kinase; Proteins; Rattus; Recombinants; Recycling; Resistance; Resources; Rodent; Role; Signal Pathway; Signal Transduction; Source; Surface; Toxin; Toxoplasma gondii; Virulence; adefovir; anthrax lethal factor; anthrax toxin; cytokine; edema factor; immune activation; inhibitor/antagonist; macrophage; novel; pathogen; pressure; prevent; receptor; response; sensor; tool; trafficking; tumor

Anthrax toxin protective antigen protein (PA) binds to receptors on the surface of mammalian cells, is cleaved by cellular proteases, forms an oligomer, and transports two other toxin proteins, lethal factor (LF) or edema factor (EF) to the cytosol. EF is a potent calmodulin-dependent adenylyl cyclase that causes large increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves and inactivates several mitogen-activated protein kinase kinases (MEKs). In certain strains of rodents LF also cleaves and activates the inflammasome sensor NLRP1. Inflammasomes are intracellular complexes that play a role in innate immune sensing for defense against pathogens. The cleavage of NLRP1 in macrophages and dendritic cells leads to caspase-1 activation and a rapid cell death termed pyroptosis. Caspase-1 activation, which follows from activation of many other inflammasome sensors, including the NLRP3, NAIP/NLRC4 and AIM2 sensors, also leads to maturation and release of the pro-inflammatory cytokines IL-1 and IL-18. Interestingly, the only other known activator of NLRP1 is Toxoplasma gondii, but the mechanism for its activation is currently unknown. The inhibition of the MEK pathways and NLRP1 cleavage-mediated activation of the immune response have a wide range of consequences for the host. Continuing a long-term mouse gene mapping project, in 2017 we initiated use of the NIAID-supported Collaborative Cross recombinant inbred mouse collection. This mouse collection is a unique resource, a large panel of inbred strains developed from 8 unique parental founders which include three wild strains. The collection represents a wider genetic diversity than in any other inbred model, with segregating polymorphisms at every 100-200 bp. We are in the process of doing crosses and SNP analyses to identify the genetic basis for unique LT-induced phenotypes. In 2017 we also continued our studies into the role of NLRP1 inflammasome activation in the rapid, 1-hour death induced by LT in rats. While we previously mapped sensitivity to the toxin to the NLRP1 locus, the mechanism by which activation of an inflammasome sensor leads to rapid animal death remains unknown. We are in the process of assessing the contribution to lethality of the hematopoietic cells in which NLRP1 is primarily expressed. During 2017 we also continued our studies on the role of inflammasome activation in both murine and rat resistance to Toxoplasma gondii. Specifically, we investigated the contribution of activation of different inflammasome sensors, including NLRP1, to the induction of protective cytokine responses in rodents and the cellular sources of these cytokines. A collaborative study completed during 2017 identified molecular consequences of ET-mediated cAMP release, and showed disruption of endocytic recycling dependent on the small GTPase Rab11. This disruption impacts cellular junctions and contributes to edema induced by the toxin by preventing transport of crucial junction proteins to cell membranes. Using both Drosophila and mammalian cells we showed that over-activation of the cAMP effectors PKA and Epac/Rap1 interferes with Rab11-mediated trafficking at two distinct steps. We further described conserved roles of Epac and the small GTPase Arf6 in ET-mediated disruption of vesicular trafficking and showed that chemical inhibition of either pathway prevents ET-induced edema in mice. These studies further our understanding of ET-induced vascular leakage at the cellular level. In a continuation of other collaborative studies the cardiovascular effects of ET and LT on rat aortic rings were analyzed. While we had previously demonstrated that ET but not LT inhibits phenylephrine stimulated contraction of aortic rings prepared from healthy rats, in this period we examined arterial function in rats pretreated with toxin infusions. Only ET was found to reduce arterial pressure and contractile force. Adefovir, an ET inhibitor, reversed these effects and protected animals from toxin challenge. This study showed that in ET-treated rats, hypotension and lethality are associated with reduced arterial contractile function.

炭疽毒素保护性抗原蛋白（PA）与哺乳动物细胞表面上的受体结合，被细胞蛋白酶裂解，形成低聚物，并将另外两个毒素蛋白传递，即致死因子（LF）或水肿因子（EF）（EF）。 EF是一种有效的钙调蛋白依赖性腺苷酸环化酶，可导致细胞内cAMP浓度大大增加。 LF是一种金属蛋白酶，可裂解并灭活几种有丝分裂原激活的蛋白激酶激酶（MEKS）。在某些啮齿动物菌株中，LF还裂解并激活炎症体传感器NLRP1。炎性症是细胞内复合物，在对病原体防御的先天免疫感中发挥作用。 NLRP1在巨噬细胞和树突状细胞中的裂解导致caspase-1激活，快速细胞死亡称为凋亡。 CASPASE-1激活之后是由于许多其他炎性体传感器的激活，包括NLRP3，NAIP/NLRC4和AIM2传感器，也导致促炎细胞因子IL-1和IL-18的成熟和释放。有趣的是，唯一已知的NLRP1激活剂是弓形虫弓形虫，但其激活的机制目前尚不清楚。 MEK途径和NLRP1裂解介导的免疫反应激活的抑制作用对宿主具有广泛的后果。 继续进行一个长期的小鼠基因映射项目，2017年，我们开始使用NIAID支持的协作交叉重组近交小鼠收集。该鼠标收集是一种独特的资源，是由8个独特的父母创始人开发的大型近交菌株，其中包括三个野生菌株。该集合比在任何其他近交模型中都代表更广泛的遗传多样性，每100-200 bp的多态性隔离。 我们正在进行交叉和SNP分析，以确定独特的LT诱导表型的遗传基础。 2017年，我们还继续研究NLRP1炎症体激活在大鼠中LT诱导的1小时死亡中的作用。虽然我们以前对毒素对NLRP1基因座的敏感性绘制了敏感性，但炎性体传感器激活导致动物死亡的机制仍然未知。 我们正在评估主要表达NLRP1的造血细胞致死性的贡献。 在2017年期间，我们还继续研究炎症体激活在鼠类对弓形虫弓形虫的耐药性中的作用。具体而言，我们研究了包括NLRP1在内的不同炎性体传感器的激活对啮齿动物保护性细胞因子反应的诱导和这些细胞因子的细胞来源的贡献。 一项在2017年完成的协作研究确定了ET介导的cAMP释放的分子后果，并显示内吞回收的破坏取决于小GTPase RAB11。这种破坏会影响细胞连接，并通过防止关键连接蛋白传输到细胞膜而导致毒素引起的水肿。使用果蝇和哺乳动物细胞，我们表明营地效应子PKA和EPAC/RAP1的过度激活在两个不同的步骤下会干扰Rab11介导的贩运。我们进一步描述了EPAC和小GTPase ARF6在ET介导的囊泡运输中的破坏中的保守作用，并表明化学抑制两条途径可阻止小鼠ET诱导的水肿。这些研究进一步了解了我们对ET诱导的细胞水平血管泄漏的理解。 在其他协作研究的延续中，分析了ET和LT对大鼠主动脉环的心血管影响。尽管我们以前证明ET而不是LT抑制了由健康大鼠制备的主动脉环的苯肾上腺素刺激的收缩，但在此期间，我们检查了用毒素输注预处理的大鼠中的动脉功能。仅发现ET可以降低动脉压和收缩力。 ET抑制剂Adefovir逆转了这些作用，并保护动物免受毒素挑战。这项研究表明，在经过ET处理的大鼠中，低血压和致死性与动脉收缩功能降低有关。