Pathophysiological Actions of Anthrax Virulence Determinants

炭疽毒力决定因素的病理生理作用

• 批准号: 8946431
• 负责人: Stephen Leppla
• 金额: $ 45.47万
• 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/8946431
• 关键词: Adenylate Cyclase; Amino Acids; Animals; Anthrax disease; Antibiotics; Antigens; Bacillus anthracis; Bacillus anthracis spore; Backcrossings; Bacteria; Binding Proteins; Blood; Blood Vessels; Calmodulin; Cardiac Myocytes; Caspase-1; Cell Culture Techniques; Cell Death; Cell Line; Cell division; Cell model; Cells; Cessation of life; Chromosomes, Human, Pair 10; Cleaved cell; Complement Factor B; Control Locus; Cyclic AMP; Cytosol; Defect; Disease; Edema; Endothelial Cells; Enzymes; Gene Expression; Gene Expression Profile; Genes; Genetic; Goals; Hepatocyte; Host resistance; Immune response; Immunization; In Vitro; Inbred Strain; Inbreeding; Infection; Inflammatory; Interleukin-1; Interleukin-18; Intervention; Investigation; Knock-out; Knockout Mice; Knowledge; Laboratories; Link; Mammalian Cell; Maps; Mediating; Medical; Metalloproteases; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular Genetics; Morphogenesis; Multiprotein Complexes; Mus; Organ; Parasite resistance; Parasites; Pathogenesis; Pathway interactions; Phenotype; Play; Predisposition; Process; Protein Binding; Proteins; Publications; Publishing; Rat Strains; Rattus; Recombinants; Regulation; Reporting; Reproduction spores; Resistance; Rodent; Rodent Model; Role; Signal Pathway; Signal Transduction; Site; Smooth Muscle Myocytes; Staging; Sterols; Surface; System; Targeted Toxins; Testing; Therapeutic; Tissues; Toxin; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Variant; Vascular Diseases; Vascular System; Vesicle; Virulence; Virulence Factors; Work; anthrax lethal factor; anthrax toxin; base; cell type; cytokine; daughter cell; edema factor; embryonic stem cell; in vivo; killings; macrophage; mortality; mouse model; mutant; neutralizing antibody; novel; overexpression; promoter; receptor; research study; response; sensor; therapeutic development

Anthrax toxin protective antigen protein (PA) binds to receptors on the surface of mammalian cells and transports two other toxin proteins, lethal factor (LF) or edema factor (EF) to the cytosol. The primary receptor for PA is colony morphogenesis protein 2 (CMG-2). EF is a potent calmodulin-dependent adenylyl cyclase that causes large increases in intracellular cAMP concentrations and when injected with PA into animals, induces localized edema or systemic vascular collapse. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs) and the N-terminus of the inflammasome sensor NLRP1/NLRP1b. The cleavage of the MEKs inactivates important cellular signaling pathways. The cleavage of NLRP1 leads to caspase-1 activation and the maturation and release of the pro-inflammatory cytokines IL-1β and IL-18. When injected in animals, LT also causes a unique vascular collapse through unknown mechanisms. These toxins are considered the primary virulence factors of B. anthracis, and immunization against PA provides full protection against challenge with anthrax spores. The toxins play roles in different stages of infection. In the early stages of infection, both toxins work together to impair the innate immune response. At later stages, the induction of localized and systemic vascular dysfunction results in host death. Until the publication of our recent work, it was postulated that the vascular collapse induced by LT and ET was likely due to their actions on endothelial cells, although in vivo experimental evidence for this hypothesis was lacking. This year (2014) we published the culmination of years of analyses performed using genetically modified mice developed in our laboratory to investigate the key tissue targets responsible for the lethal effects of the anthrax toxins. Our laboratory created panels of cell-type specific anthrax receptor (CMG2)-deficient mice, as well as tissue-specific CMG2-expressing mice and challenged them with anthrax toxins or spore to assess the role of each tissue in pathogenesis. We found that LT and ET induce vascular collapse and lethality through targeting very different cell types. In a surprising finding, targeting of endothelial cells by either toxin did not play a dominant role in lethality. Instead, targeting of cardiomyocytes and vascular smooth muscles cells (but not hepatocytes) was found to be required for LT-induced lethality. In contrast, ET manifested its effects through direct targeting of hepatocytes, and despite the known impact of cAMP on vasculature, the direct targeting of the primary cells of the vascular system was not required for this toxins effect. These findings show that in anthrax, host lethality is the result of damage to two different vital systems, thereby providing a better understanding on how anthrax disease pathogenesis progresses. The knowledge of the targeted organs will also aid in development of therapeutics and supportive medical interventions for this disease. In other studies we further expanded on our recent discovery of a novel LF substrate, NLRP1. NLRP1 is a NOD-like receptor (NLR) protein which is part of the inflammasome, a multiprotein complex that activates caspase-1 in response to cytoplasmic danger signals. A consequence of NLRP1 inflammasome activation by LT is macrophage death with concurrent IL-1β/IL-18 maturation and release. Until recently, the only known activator of the NLRP1 was LT. We have now discovered that Toxoplasma gondii can also activate this sensor. T. gondii is an intracellular parasite that infects a wide range of warm-blooded species, but rodents can be sensitive or resistant to this parasite. We noted that the Toxo1 locus conferring Toxoplasma resistance in rats mapped to a small region of rat chromosome 10 containing Nlrp1. In a study published in 2014 we performed experiments to show that the parasite also activated NLRP1. In rats the differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation were found to be correlated with the same eight amino acid NLRP1 sequence that determines LT resistance. Thus, rat strain susceptibility was inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP and transcriptome analyses we narrowed candidate loci for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes which included Nlrp1. Knockdown of NLRP1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of resistant macrophages. Our findings reveal T. gondii as a novel activator of the NLRP1 inflammasome and suggest that the inbred strain susceptibility to infection by this parasite is controlled through inflammasome activation. We further extended these Toxoplasma studies to test the effects of both NLRP1 and the related inflammasome sensor, NLRP3, in parasite-infected mice. We found that Toxoplasma activates the NLRP3 inflammasome in mouse macrophages, leading to a pyroptosis-independent activation of IL-1β, but not IL-18. Strikingly, mice infected with the parasite produced large quantities of IL-18 (but not IL-1β) in a manner dependent on NLRP3 and caspase-1/11. Mice deficient in NLRP3 or NLRP1 infected with the parasite also show that both sensors play a role in controlling resistance to the parasite through an IL-18-dependent pathway which limits parasite replication. These findings established T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in mice and revealed a role for these sensors in host resistance to toxoplasmosis. In studies with collaborators we found that the PA channel translocates LF not only into the cytosol of cells but also the lumen of endosomal intraluminal vesicles (ILVs). LF persists in these ILVs (in both in vitro cell cultures and toxin-injected animals) for up to a week, fully protected from degradation by host enzymes and thus capable of maintaining cleavage of its substrates. This persistence results in a prolonged action of the toxin at various organ sites. We found that ILV-localized LF can also be transmitted to daughter cells upon cell division or delivered outside cells as exosomes, which could then potentially deliver LF to other cells in a manner independent of PA. The implications of these findings are significant for anti-LF therapeutics, because the toxin can maintain its action against the host at sites inaccessible to neutralizing antibodies for longer periods than previously anticipated. Furthermore, these studies may provide an explanation for the high mortality associated with anthrax long after bacteria have been killed by antibiotics. Finally, in another collaborative study, we reported on Nlrp1a regulation in a mouse model where expression of this gene was previously linked to sterol regulatory binding protein 1a (Srebp-1a). Srebp-1a knockout mice used for all previous studies carried the Nlrp1 locus from 129/Ola ES cells used to create this knockout, because the proximity of Srebp-1a and Nlrp1 makes it difficult for backcrossing to replace the 129 locus. The 129/Ola mouse Nlrp1a gene has a promoter defect that does not allow expression of NLRP1a. Our collaborators created a Srebp-1a knockout mouse carrying the functional Nlrp1 locus from C57BL/6J, with normal expression of NLRP1a restored. This study emphasized the importance of genetic fidelity in analyses of mouse phenotypes.

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，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。 PA 的主要受体是集落形态发生蛋白 2 (CMG-2)。 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，并且当将 PA 注射到动物体内时，会诱导局部水肿或全身血管塌陷。 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) 和炎性体传感器 NLRP1/NLRP1b 的 N 末端。 MEK 的裂解会使重要的细胞信号传导途径失活。 NLRP1 的裂解导致 caspase-1 激活以及促炎细胞因子 IL-1β 和 IL-18 的成熟和释放。当注射到动物体内时，LT 还会通过未知机制引起独特的血管塌陷。 这些毒素被认为是炭疽芽孢杆菌的主要毒力因子，针对 PA 的免疫可提供针对炭疽芽孢攻击的全面保护。毒素在感染的不同阶段发挥作用。在感染的早期阶段，两种毒素共同作用，损害先天免疫反应。在后期，局部和全身血管功能障碍的诱导导致宿主死亡。 在我们最近的工作发表之前，人们推测 LT 和 ET 引起的血管塌陷可能是由于它们对内皮细胞的作用所致，尽管缺乏支持这一假设的体内实验证据。 今年（2014 年），我们发表了多年来使用我们实验室开发的转基因小鼠进行的分析的成果，以研究导致炭疽毒素致命影响的关键组织目标。我们的实验室创建了细胞类型特异性炭疽受体 (CMG2) 缺陷型小鼠以及表达组织特异性 CMG2 的小鼠，并用炭疽毒素或孢子对它们进行攻击，以评估每种组织在发病机制中的作用。我们发现 LT 和 ET 通过针对不同的细胞类型来诱导血管塌陷和致死。 令人惊讶的发现是，这两种毒素对内皮细胞的靶向作用在致死率方面并未发挥主导作用。相反，LT 诱导的致死需要靶向心肌细胞和血管平滑肌细胞（但不是肝细胞）。相比之下，ET 通过直接靶向肝细胞来发挥其作用，尽管已知 cAMP 对脉管系统的影响，但这种毒素作用不需要直接靶向血管系统的原代细胞。这些发现表明，在炭疽中，宿主致死是两个不同生命系统受损的结果，从而更好地了解炭疽病的发病机制如何进展。 对目标器官的了解也将有助于开发针对这种疾病的治疗方法和支持性医疗干预措施。 在其他研究中，我们进一步扩展了我们最近发现的一种新型 LF 底物 NLRP1。 NLRP1 是一种 NOD 样受体 (NLR) 蛋白，是炎症小体的一部分，炎症小体是一种多蛋白复合物，可响应细胞质危险信号激活 caspase-1。 LT 激活 NLRP1 炎性体的结果是巨噬细胞死亡，同时 IL-1β/IL-18 成熟和释放。直到最近，唯一已知的 NLRP1 激活剂是 LT。我们现在发现弓形虫也可以激活这个传感器。弓形虫是一种细胞内寄生虫，可感染多种温血物种，但啮齿动物可能对此寄生虫敏感或具有抵抗力。我们注意到，Toxo1 基因座赋予大鼠弓形虫抗性，该基因座映射到大鼠 10 号染色体上含有 Nlrp1 的小区域。在 2014 年发表的一项研究中，我们进行的实验表明该寄生虫也激活了 NLRP1。在大鼠中，弓形虫感染的巨噬细胞对细胞焦亡、IL-1β/IL-18 加工和寄生虫增殖抑制的敏感性差异被发现与决定 LT 抗性的相同 8 个氨基酸 NLRP1 序列相关。因此，大鼠品系的敏感性与炭疽LT诱导的细胞死亡的敏感性呈负相关。利用重组近交大鼠、SNP 和转录组分析，我们将控制弓形虫介导的大鼠巨噬细胞焦亡的候选位点缩小到包括 Nlrp1 在内的四个基因。敲低焦亡敏感巨噬细胞中的 NLRP1 会导致寄生虫复制更高，并防止细胞死亡。相反，在焦亡抗性细胞中，来自弓形虫敏感巨噬细胞的 NLRP1 变体的过度表达会导致抗性巨噬细胞的敏化。我们的研究结果表明弓形虫是 NLRP1 炎症小体的新型激活剂，并表明近交株对这种寄生虫感染的易感性是通过炎症小体激活来控制的。 我们进一步扩展了这些弓形虫研究，以测试 NLRP1 和相关炎症小体传感器 NLRP3 对寄生虫感染小鼠的影响。我们发现弓形虫激活小鼠巨噬细胞中的 NLRP3 炎症小体，导致 IL-1β（而非 IL-18）发生细胞焦亡独立的激活。引人注目的是，感染寄生虫的小鼠以依赖于 NLRP3 和 caspase-1/11 的方式产生大量 IL-18（但不是 IL-1β）。感染寄生虫的 NLRP3 或 NLRP1 缺陷小鼠也表明，这两种传感器通过限制寄生虫复制的 IL-18 依赖性途径在控制对寄生虫的抵抗力方面发挥作用。这些发现证实弓形虫是小鼠体内 NLRP1 和 NLRP3 炎症小体的新型激活剂，并揭示了这些传感器在宿主抵抗弓形虫病中的作用。 在与合作者的研究中，我们发现 PA 通道不仅将 LF 易位到细胞的胞浆中，而且还易位到内体腔内囊泡 (ILV) 的管腔中。 LF 在这些 ILV 中（在体外细胞培养物和注射毒素的动物中）持续长达一周，完全免受宿主酶的降解，因此能够维持其底物的裂解。这种持久性导致毒素在各个器官部位的作用延长。我们发现，ILV 定位的 LF 也可以在细胞分裂时传递给子细胞，或者作为外泌体传递到细胞外，然后可能以独立于 PA 的方式将 LF 传递给其他细胞。这些发现对于抗 LF 疗法具有重要意义，因为该毒素可以在中和抗体无法到达的部位维持其针对宿主的作用，时间比之前预期的更长。此外，这些研究可能为细菌被抗生素杀死很久之后与炭疽相关的高死亡率提供了解释。 最后，在另一项合作研究中，我们报告了小鼠模型中 Nlrp1a 的调​​节，其中该基因的表达先前与甾醇调节结合蛋白 1a (Srebp-1a) 相关。 用于之前所有研究的 Srebp-1a 敲除小鼠携带来自用于创建此敲除的 129/Ola ES 细胞的 Nlrp1 基因座，因为 Srebp-1a 和 Nlrp1 的邻近性使得回交难以替换 129 基因座。 129/Ola 小鼠 Nlrp1a 基因具有启动子缺陷，不允许 NLRP1a 表达。我们的合作者创建了一只携带来自 C57BL/6J 的功能性 Nlrp1 基因座的 Srebp-1a 敲除小鼠，NLRP1a 的正常表达得以恢复。这项研究强调了遗传保真度在小鼠表型分析中的重要性。