Pathophysiological Actions of Anthrax Virulence Determinants

炭疽毒力决定因素的病理生理作用

• 批准号: 10014140
• 负责人: Stephen Leppla
• 金额: $ 97.13万
• 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/10014140
• 关键词: Adenylate Cyclase; Animal Model; Animals; Anthrax disease; Antibiotics; Antigens; Bacillus anthracis; Bacillus cereus; Bacteria; Binding Proteins; CASP1 gene; Calmodulin; Cancer cell line; Cell Death; Cell model; Cells; Cellular biology; Cellular translocation; Characteristics; Cleaved cell; Collaborations; Complex; Cyclic AMP; Cytoprotection; Cytosol; Dendritic Cells; Development; Disease; Dopamine; Dose; Edema; Enzymes; Food Poisoning; Functional disorder; Goals; Image; Imagery; Immune; Indium-111; Infection; Inflammasome; Inflammatory; Innate Immune Response; Interleukin-1; Interleukin-18; Knockout Mice; Label; Lung; MAPK14 gene; Maintenance; Maleimides; Malignant Neoplasms; Mammalian Cell; Matrix Metalloproteinases; Mediating; Metalloproteases; Methods; Mitogen-Activated Protein Kinase Kinases; Modeling; Modification; Mus; N-terminal; Neurons; Neurotoxins; Nuclear Receptors; Organ; Parkinson Disease; Pathogenesis; Pathology; Pathway interactions; Peptide Hydrolases; Permeability; Pharmacology; Phosphotransferases; Play; Protein Kinase; Proteins; Pulmonary artery structure; Radiolabeled; Rattus; Reporting; Resistance; Rodent; Role; Signal Pathway; Signal Transduction; Site; Surface; Therapeutic; Toxin; Tumor Tissue; Tyrosine 3-Monooxygenase; Virulence; Wild Type Mouse; Work; X-Ray Computed Tomography; anthrax lethal factor; anthrax toxin; cancer cell; cell type; cytokine; dopaminergic neuron; edema factor; imaging agent; in vivo imaging; insight; macrophage; mutant; novel; overexpression; pressure; protective effect; pulmonary vascular permeability; receptor; receptor binding; recruit; sensor; single photon emission computed tomography; tool; tumor; tumor specificity

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. LF is a metalloprotease that cleaves and inactivates several mitogen-activated protein kinase kinases (MEKs). In rodents LF also cleaves and activates inflammasome sensor NLRP1. The inflammasomes are intracellular complexes that play a role in innate immune response. The cleavage of NLRP1 in macrophages and dendritic cells leads to caspase-1 activation, a rapid cell death termed pyroptosis, maturation and release of the pro-inflammatory cytokines IL-1 and IL-18 and recruitment of innate immune cells. The inactivation of the MEK pathways by the toxin, alongside the ability to redirect the toxins to specific cell types through modification of the cleavage site on the receptor binding PA moiety allows the use of these toxins as anti-tumor therapeutics. For example, PA can be modified to be cleaved by specific matrix metalloproteinases (MMPs) which are overexpressed in certain cancers. In FY2019 in a collaboration with UK colleagues, we utilized radiolabeling of modified toxin proteins to image MMP activity in tumor tissues. We first characterized PA mutants in which the furin cleavage site in PA was modified to an MMP-specific sequence. DOTA-GA maleimide was conjugated to LF to allow its labeling by two different methods and functionality of the toxin for cellular translocation was verified post-labeling. After development of efficient 111-Indium-labeling for the purpose of imaging MMP activity in tumors, the labeled LF toxin was used in conjunction with mutant PA proteins which require MMP for activation to assess MMP activity in cancer cell lines. MMP activity of a panel of cancer cells could be correlated to levels of activation of mutant PA. Furthermore, SPECT/CT imaging in vivo allowed tracking of labeled PA and tumor visualization in animals. Only modified PA proteins and not wild type toxin delivered labeled LF molecules to MMP-expressing tumors, showing the specificity of the tumor-targeting by mutant PA molecules. We believe labeled LF and modified PA molecules may be promising imaging agents for proteolytic activity in cancer. In other collaborative studies during this reporting period we utilized the unique characteristics of the anthrax toxins for delivery of cargo to neuronal cells. We created a novel fusion of the N-terminal region of LF to nuclear receptor-related 1 (Nurr1), a protein which is important in maintenance of dopaminergic neurons. Delivery and subsequent cytoplasmic release of Nurr1 was associated with increased levels of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, and with a concentration-dependent protection of cells against a neurotoxin, 6-hyroxydopamine. These protective effects suggest Nurr1 delivery as a potential new treatment option for Parkinsons disease. We continued our work on the multicomponent Bacillus cereus pore-forming toxin HBL, associated with food poisoning induced by this bacterium. We had previously demonstrated stepwise sequential assembly of the tripartite toxin on cells. In these new studies the ability of the HBL toxin to activate the NLRP3 inflammasome to induce pyroptosis and secretion of inflammatory cytokines was investigated. We found the NLRP3 inflammasome played an important role in mediating lethality by B. cereus in mice, as knockout mice were resistant to infection at doses where wild type mice succumbed. Furthermore, pharmacological inhibition of the NLRP3 inflammasome was able to protect against bacterium-induced lethality, suggesting one of the roles the HBL toxin plays is in induction of inflammasome-mediated pathology. Finally, during this year, in a continuation of collaborative studies on anthrax toxin-induced effects on pulmonary vascular permeability in the isolated rat lung model, we found that lethal toxin (LT) increases pulmonary arterial pressure and pulmonary permeability, while edema toxin (ET) decreases pulmonary artery pressure and does not alter permeability in isolated lungs. Both toxins may induce organ dysfunction through these opposing effects during lethal infection. These findings suggest potentially different therapeutic approaches may be needed at different stages of infection depending on levels of each toxin.

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。 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。在啮齿类动物中，LF 还会裂解并激活炎症小体传感器 NLRP1。炎性小体是在先天免疫反应中发挥作用的细胞内复合物。巨噬细胞和树突细胞中 NLRP1 的裂解导致 caspase-1 激活、称为焦亡的快速细胞死亡、促炎细胞因子 IL-1 和 IL-18 的成熟和释放以及先天免疫细胞的募集。 毒素对 MEK 途径的失活，以及通过修饰受体结合 PA 部分上的裂解位点将毒素重定向到特定细胞类型的能力，使得这些毒素可以用作抗肿瘤治疗。例如，PA 可以被修饰以被在某些癌症中过度表达的特定基质金属蛋白酶 (MMP) 裂解。 2019 财年，我们与英国同事合作，利用修饰毒素蛋白的放射性标记对肿瘤组织中的 MMP 活性进行成像。我们首先表征了 PA 突变体，其中 PA 中的弗林蛋白酶切割位点被修改为 MMP 特异性序列。 DOTA-GA 马来酰亚胺与 LF 缀合，允许通过两种不同的方法对其进行标记，并且细胞易位毒素的功能在标记后得到验证。在开发出用于肿瘤 MMP 活性成像的有效 111-Indium 标记后，标记的 LF 毒素与需要 MMP 激活的突变 PA 蛋白结合使用，以评估癌细胞系中的 MMP 活性。一组癌细胞的 MMP 活性可能与突变 PA 的激活水平相关。此外，SPECT/CT 体内成像可以跟踪标记的 PA 和动物体内的肿瘤可视化。只有修饰的 PA 蛋白而非野生型毒素才能将标记的 LF 分子递送至表达 MMP 的肿瘤，这显示了突变型 PA 分子靶向肿瘤的特异性。我们相信标记的 LF 和修饰的 PA 分子可能是有前途的癌症蛋白水解活性成像剂。 在本报告期间的其他合作研究中，我们利用炭疽毒素的独特特性将货物输送到神经元细胞。我们创建了 LF N 末端区域与核受体相关 1 (Nurr1) 的新型融合，这是一种对维持多巴胺能神经元非常重要的蛋白质。 Nurr1 的递送和随后的细胞质释放与酪氨酸羟化酶（多巴胺合成中的限速酶）水平的增加有关，并且与细胞对抗神经毒素 6-羟基多巴胺的浓度依赖性保护有关。这些保护作用表明 Nurr1 递送可以作为帕金森病的潜在新治疗选择。 我们继续研究多组分蜡状芽孢杆菌成孔毒素 HBL，该毒素与该细菌引起的食物中毒有关。我们之前已经证明了三方毒素在细胞上的逐步顺序组装。在这些新研究中，研究了 HBL 毒素激活 NLRP3 炎性体以诱导细胞焦亡和炎性细胞因子分泌的能力。我们发现NLRP3炎性体在介导蜡状芽孢杆菌对小鼠的致死作用中发挥着重要作用，因为敲除小鼠在野生型小鼠死亡的剂量下对感染具有抵抗力。此外，对 NLRP3 炎症小体的药理学抑制能够防止细菌诱导的致死，这表明 HBL 毒素所起的作用之一是诱导炎症小体介导的病理。 最后，今年，在离体大鼠肺模型中炭疽毒素对肺血管通透性影响的继续合作研究中，我们发现致死毒素（LT）会增加肺动脉压和肺通透性，而水肿毒素（ET）会增加肺动脉压和肺通透性。 ）降低肺动脉压力并且不改变离体肺的通透性。这两种毒素都可能在致命感染期间通过这些相反的作用引起器官功能障碍。这些发现表明，根据每种毒素的水平，在感染的不同阶段可能需要不同的治疗方法。