Pathophysiological Actions of Anthrax Virulence Determinants

炭疽毒力决定因素的病理生理作用

• 批准号: 9161609
• 负责人: Stephen Leppla
• 金额: $ 44.43万
• 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/9161609
• 关键词: Adenylate Cyclase; Affinity; Agonist; Alpaca; Animals; Anthrax disease; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Antigens; Antioxidants; Autoimmune Diseases; Bacillus anthracis; Bacillus anthracis spore; Binding; Biological Assay; Biology; Blood Vessels; Broccoli - dietary; Calmodulin; Canis familiaris; Caspase-1; Cell Death; Cell model; Cell surface; Cells; Cessation of life; Chemicals; Cleaved cell; Clinical Trials; Complement Factor B; Complex; Cyclic AMP; Cytosol; Dendritic Cells; Disease; Edema; Eicosanoids; Endocytosis; Enzymes; Epitopes; Evolution; Genetic Transcription; Goals; Gout; Heating; Immune; Immune response; Immunology; Infection; Inflammation; Inflammatory; Interleukin-1; Interleukin-18; Investigation; Isothiocyanates; Journals; Lead; Leukocytes; Ligands; Link; Liquid substance; Mammalian Cell; Mediating; Metalloproteases; Mitogen-Activated Protein Kinase Kinases; Modeling; Modification; Monoclonal Antibodies; Mus; NF-kappa B; Norepinephrine; Pathogenesis; Pathology; Pathway interactions; Peptide Hydrolases; Peroxisome Proliferators; Pharmaceutical Preparations; Play; Protein Binding; Protein Biosynthesis; Proteins; Proteolysis; Publishing; Recombinants; Reporting; Reproduction spores; Resistance; Response Elements; Rodent Model; Role; Signal Transduction; Staging; Sulforaphane; Surface; Targeted Toxins; Testing; Therapeutic; Toxic effect; Toxin; Urate; Vaccination; Vascular Diseases; Virulence; Virulence Factors; Work; anthrax lethal factor; anthrax toxin; base; cyclopentenone; cytokine; dietary supplements; edema factor; improved; in vivo; inhibitor/antagonist; interest; interleukin-1beta-converting enzyme inhibitor; macrophage; microbial; mouse model; novel; novel therapeutics; pathogen; prevent; protein oligomer; receptor; screening; sensor; therapeutic target; transcription factor

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 several mitogen-activated protein kinase kinases (MEKs) and the N-terminus of the inflammasome sensor NLRP1. The 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 is the resultant effect following 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-1β and IL-18. The screening for and characterization of inhibitors of LT-induced pyroptosis allows for identification of novel anti-inflammatory therapeutics that target multiple other inflammasomes responsible for detecting a variety of intracellular microbial ligands and endogenous danger signals. Inflammasome activation is an important part of the innate immune response, but is also linked to pathology of many inflammatory and autoimmune disorders. In the 2015 reporting period, we identified the natural isothiocyanate, sulforaphane, present in broccoli sprouts and available as a dietary supplement, as an inhibitor of the NLRP1, NLRP3, NAIP/NLRC4 and AIM2 inflammasomes. We found that sulforaphane inhibits autoproteolytic cleavage of caspase-1 in a manner independent of the actions it normally has on transcription factor Nrf2 and the antioxidant response-element pathway, to which many of the anti-inflammatory effects of sulforaphane have been previously attributed. In a separate study, we discovered that the eicosanoid 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2) and related cyclopentenone PGs which have been studied as NF-κB inhibitors can also inhibit caspase-1 activation by multiple inflammasome pathways. This inhibition is independent of the well-characterized role of 15d-PGJ2 as a peroxisome proliferator receptor-γ agonist, its activation of Nrf2, or any anti-inflammatory functions as an inhibitor of NF-κB. Instead, 15d-PGJ2 prevents the autoproteolytic activation of caspase-1 and the maturation of IL-1β through induction of a cellular state inhibitory to caspase-1 proteolytic function. Similar to sulforaphane, the drug effect is not manifested through direct modification or inactivation of the caspase-1 enzyme. But in a manner different from sulforaphane, the effects of 15d-PGJ2 depend on de novo protein synthesis of an unknown inhibitor or protective protein, through actions of the eicosanoid on a yet to be identified transcription pathway. Testing of both sulforaphane and 15d-PGJ2 in vivo, in a mouse model of gout where monosodium urate crystals are used to activate the NLRP3 inflammasome, showed that both chemicals inhibit inflammatory cell recruitment and IL-1β release. Furthermore, in our murine anthrax infection model, both drugs could reverse NLRP1-mediated murine resistance to Bacillus anthracis spore infection. The above two studies report on novel mechanisms of anti-inflammatory action for two NF-kB inhibitors which have been included in clinical trials on the basis of their impact on inflammation. The findings were published this year in the Journal of Leukocyte Biology and the Journal of Immunology. When injected in animals, both ET and LT induce vascular collapse and host death. These toxins are considered the primary virulence factors of B. anthracis, and 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. The targeting of the common component of both toxins, PA, is the basis for the current vaccination against anthrax and most developed monoclonal antibody-based therapeutics. In collaborative work published in 2015 using the approved anti-PA monoclonal raxibacumab, the effects of the antibody on 24 h edema toxin and lethal toxin challenges in a canine model were studied. Raxibacumab was found to augment fluid and norepinephrine therapy to improve survival in toxin challenged animals. In a separate collaborative work, we identified novel antibodies for targeting PA. Variable domains of camelid heavy chain-only antibodies (VHHs) are small single chain, heat- and pH- stable entities that can access epitopes that large standard antibodies cannot. Furthermore, they can be produced in recombinant form and purified rapidly once their sequences are known. VHHs with high affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified, with one similar to most identified anti-PA monoclonal antibodies, in that it inhibited the toxin binding to its cellular receptor. A second novel neutralizing VHH was found to inhibit endocytosis of the PA oligomer while not preventing PA cleavage by cell surface proteases. Both VHHs, as well as a heterodimer of the two displayed neutralizing potency in cell assays and protected mice from anthrax toxin challenge and Bacillus anthracis spore infection. These studies showed the usefulness of VHHs as novel anti-PA agents, and introduced a novel mechanism for neutralization of anthrax 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，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) 和炎症小体传感器 NLRP1 的 N 末端。炎症小体是细胞内复合物，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 激活是许多其他炎性体传感器（包括 NLRP3、NAIP/NLRC4 和 AIM2 传感器）激活后产生的结果，也会导致促炎细胞因子 IL-1β 和 IL-18 的成熟和释放。 LT 诱导的细胞焦亡抑制剂的筛选和表征可以鉴定针对负责检测各种细胞内微生物配体和内源性危险信号的多种其他炎症体的新型抗炎疗法。炎症小体激活是先天免疫反应的重要组成部分，但也与许多炎症和自身免疫性疾病的病理有关。在 2015 年报告期间，我们发现西兰花芽中存在的天然异硫氰酸盐萝卜硫素可作为膳食补充剂，作为 NLRP1、NLRP3、NAIP/NLRC4 和 AIM2 炎症小体的抑制剂。我们发现萝卜硫素抑制 caspase-1 的自蛋白水解裂解，其方式独立于其通常对转录因子 Nrf2 和抗氧化反应元件途径的作用，萝卜硫素的许多抗炎作用先前已归因于该途径。 在另一项研究中，我们发现类花生酸 15-脱氧-Δ(12,14)-PGJ2 (15d-PGJ2) 和相关的环戊烯酮 PG 已被研究作为 NF-κB 抑制剂，也可以通过多种炎症小体抑制 caspase-1 激活途径。这种抑制作用与 15d-PGJ2 作为过氧化物酶体增殖剂受体-γ 激动剂的明确作用、其对 Nrf2 的激活或作为 NF-κB 抑制剂的任何抗炎功能无关。相反，15d-PGJ2 通过诱导 caspase-1 蛋白水解功能的细胞状态抑制来阻止 caspase-1 的自蛋白水解激活和 IL-1β 的成熟。与萝卜硫素类似，药效并不是通过直接修饰或灭活caspase-1酶来体现的。但与萝卜硫素不同的是，15d-PGJ2 的作用取决于未知抑制剂或保护性蛋白质的从头蛋白质合成，通过类二十烷酸对尚未确定的转录途径的作用。 在痛风小鼠模型中，使用单钠尿酸盐晶体激活 NLRP3 炎症小体，对萝卜硫素和 15d-PGJ2 进行体内测试，结果表明这两种化学物质都能抑制炎症细胞募集和 IL-1β 释放。此外，在我们的小鼠炭疽感染模型中，这两种药物都可以逆转 NLRP1 介导的小鼠对炭疽芽孢杆菌孢子感染的抵抗力。 上述两项研究报告了两种 NF-kB 抑制剂抗炎作用的新机制，这些抑制剂已根据其对炎症的影响被纳入临床试验。该研究结果发表在今年的《白细胞生物学杂志》和《免疫学杂志》上。 当注射到动物体内时，ET 和 LT 都会引起血管塌陷和宿主死亡。这些毒素被认为是炭疽杆菌的主要毒力因子，并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。针对两种毒素的共同成分 PA 的靶向是当前炭疽疫苗接种和大多数开发的基于单克隆抗体的疗法的基础。在 2015 年发表的合作研究中，使用已批准的抗 PA 单克隆 raxibacumab，研究了该抗体对犬模型中 24 小时水肿毒素和致命毒素挑战的影响。 Raxibacumab被发现可以增强液体和去甲肾上腺素治疗，以提高毒素挑战动物的生存率。 在另一项合作工作中，我们发现了针对 PA 的新型抗体。骆驼科动物纯重链抗体 (VHH) 的可变域是小型单链、热稳定和 pH 稳定的实体，可以访问大型标准抗体无法访问的表位。此外，一旦知道它们的序列，它们就可以重组形式生产并快速纯化。从免疫羊驼中获得对 PA 有高亲和力的 VHH，并在巨噬细胞毒性测定中筛选炭疽中和活性。鉴定出两类中和性 VHH，其中一类与大多数鉴定的抗 PA 单克隆抗体相似，因为它抑制毒素与其细胞受体的结合。另一种新型中和性 VHH 被发现可抑制 PA 寡聚物的内吞作用，同时不会阻止细胞表面蛋白酶对 PA 的裂解。两种 VHH 以及两者的异二聚体在细胞测定中都显示出中和效力，并保护小鼠免受炭疽毒素攻击和炭疽芽孢杆菌孢子感染。这些研究表明了 VHH 作为新型抗 PA 剂的有用性，并引入了中和炭疽毒素的新机制。