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的受体结合，被细胞蛋白酶裂解，形成寡聚体，并将另外两种毒素蛋白，致死因子 (LF) 或水肿因子 (EF) 转运至细胞质。 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 浓度大幅增加。 LF 是一种金属蛋白酶，可裂解多种丝裂原激活蛋白激酶激酶 (MEK) 并使其失活。在某些啮齿类动物中，LF 还会裂解并激活炎症小体传感器 NLRP1。炎性体是细胞内复合物，在防御病原体的先天免疫感应中发挥作用。巨噬细胞和树突状细胞中 NLRP1 的裂解导致 caspase-1 激活和称为细胞焦亡的快速细胞死亡。 Caspase-1 的激活是由许多其他炎性体传感器（包括 NLRP3、NAIP/NLRC4 和 AIM2 传感器）激活引起的，也会导致促炎细胞因子 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，但其激活机制目前尚不清楚。 MEK 途径的抑制和 NLRP1 裂解介导的免疫反应激活对宿主产生广泛的影响。 为了继续开展长期小鼠基因图谱项目，我们于 2017 年开始使用 NIAID 支持的协作杂交重组近交小鼠集合。该小鼠系列是一种独特的资源，是由 8 位独特的亲本创始人开发的大型近交系品系，其中包括 3 种野生品系。该集合代表了比任何其他近交模型更广泛的遗传多样性，每 100-200 bp 就有分离多态性。 我们正在进行杂交和 SNP 分析，以确定独特 LT 诱导表型的遗传基础。 2017 年，我们还继续研究 NLRP1 炎性体激活在 LT 诱导大鼠快速 1 小时死亡中的作用。虽然我们之前将毒素的敏感性映射到 NLRP1 位点，但炎症小体传感器的激活导致动物快速死亡的机制仍然未知。 我们正在评估 NLRP1 主要表达的造血细胞对致死率的影响。 2017 年，我们还继续研究炎症小体激活在小鼠和大鼠对弓形虫的抵抗中的作用。具体来说，我们研究了不同炎症小体传感器（包括 NLRP1）的激活对啮齿类动物保护性细胞因子反应的诱导作用以及这些细胞因子的细胞来源。 2017 年完成的一项合作研究确定了 ET 介导的 cAMP 释放的分子后果，并显示依赖于小 GTPase Rab11 的内吞循环破坏。这种破坏会影响细胞连接，并通过阻止关键的连接蛋白转运至细胞膜而导致毒素引起的水肿。使用果蝇和哺乳动物细胞，我们发现 cAMP 效应器 PKA 和 Epac/Rap1 的过度激活会在两个不同的步骤中干扰 Rab11 介导的运输。我们进一步描述了 Epac 和小 GTPase Arf6 在 ET 介导的囊泡运输破坏中的保守作用，并表明任一途径的化学抑制都可以防止 ET 诱导的小鼠水肿。这些研究进一步加深了我们对细胞水平上 ET 诱导的血管渗漏的理解。 在其他合作研究的延续中，分析了 ET 和 LT 对大鼠主动脉环的心血管影响。虽然我们之前已经证明 ET 而不是 LT 会抑制去氧肾上腺素刺激的健康大鼠主动脉环的收缩，但在此期间，我们检查了经过毒素输注预处理的大鼠的动脉功能。只有 ET 被发现可以降低动脉压和收缩力。阿德福韦是一种 ET 抑制剂，可逆转这些影响并保护动物免受毒素挑战。这项研究表明，在接受 ET 治疗的大鼠中，低血压和致死率与动脉收缩功能降低有关。