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，可裂解几种有丝分裂原激活的蛋白激酶激酶（MEK）和炎性体传感器NLRP1的N端。炎性症是细胞内复合物，在对病原体的防御中具有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激活是许多其他炎性体传感器激活（包括NLRP3，NAIP/NLRC4和AIM2传感器）激活后产生的效果，也导致促炎性细胞因子IL-1β和IL-18的成熟和释放。 对LT诱导的凋亡的抑制剂的筛查和表征，可以鉴定出新型的抗炎治疗剂，这些抗炎疗法针对多种其他负责检测各种细胞内微生物配体和内源性危险信号的其他炎症。炎性体激活是先天免疫反应的重要组成部分，但也与许多炎症性和自身免疫性疾病的病理学有关。在2015年的报告期间，我们确定了在西兰花芽中存在的天然异硫氰酸硫酸硫酸硫磺，并作为饮食补充剂可用，是NLRP1，NLRP3，NAIP/NLRC4和AIM2炎症的抑制剂。我们发现，硫烷抑制caspase-1的自由溶解裂解的方式与通常对转录因子NRF2和抗氧化剂反应元途径的作用无关，以前已归因于磺胺的许多抗炎作用。 在另一项研究中，我们发现eicosanoid 15-脱氧 - δ（12,14）-pGJ2（15d-PGJ2）和相关的环烯酮PGs已研究为NF-κB抑制剂，也可以通过多个炎症组途径抑制CASPASE-1激活。这种抑制与15d-PGJ2作为过氧化物酶体增殖物受体-γ激动剂的特征性作用无关，其激活NRF2或任何抗炎功能是NF-κB的抑制剂。取而代之的是，15d-PGJ2通过诱导细胞态抑制caspase-1蛋白水解功能来阻止caspase-1的自由蛋白解激活和IL-1β的成熟。与磺胺类似，药物作用并非通过直接修饰或caspase-1酶的直接修饰或失活而表现出来。但是，以与硫烷不同的方式，15d-PGJ2的作用取决于通过耶形烷类的作用在尚未鉴定的转录途径上，抑制剂或保护蛋白的从头蛋白合成。 在痛风的小鼠模型中，在体内测试了硫二烷和15d-PGJ2，其中使用单钠晶体晶体激活NLRP3炎性体，表明两种化学物质都抑制了炎性细胞募集和IL-1β释放。此外，在我们的鼠炭疽感染模型中，两种药物都可以逆转NLRP1介导的鼠类对炭疽芽孢杆菌孢子感染的耐药性。 以上两项研究报告了两种NF-KB抑制剂的新型抗炎作用机制，这些机制已根据其对炎症的影响包括在临床试验中。这些发现于今年发表在《白细胞生物学杂志》和《免疫学杂志》上。 当注射动物时，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芽孢杆菌的主要毒力因子，并且在不同感染阶段起着作用。在感染的早期阶段，两种毒素共同起作用，以损害先天的免疫反应。在后来的阶段，局部和全身血管功能障碍的诱导导致宿主死亡。 PA的两种毒素的共同成分的靶向是针对炭疽和最开发的基于单克隆抗体的疗法的当前疫苗接种的基础。在2015年使用经批准的抗PA单克隆raxibacumab发表的合作工作中，研究了抗体对犬类模型中24 h水肿毒素和致命毒素挑战的影响。发现raxibacumab可以增加液体和去甲肾上腺素的疗法，以提高毒素挑战性动物的生存。 在另一项协作工作中，我们确定了针对PA的新型抗体。骆驼型重链抗体（VHHS）的可变结构域是小型的单链，热和ph稳定的实体，可以访问大型标准抗体无法使用的表位。此外，它们可以以重组形式产生，并在其序列已知后迅速纯化。对PA的高亲和力的VHH是从免疫羊驼中获得的，并筛选了炭疽中和巨噬细胞毒性测定中和中和活性。鉴定了两类中和VHHS，一种与大多数鉴定的抗PA单克隆抗体相似，因为它抑制了毒素与其细胞受体的结合。发现第二种新颖的中和VHH可以抑制PA低聚物的内吞作用，同时又不防止细胞表面蛋白酶的PA裂解。 VHHS以及两者的异二聚体都表现出细胞测定中的中和效力，并受到保护的小鼠免受炭疽毒素挑战和炭疽芽孢杆菌孢子感染的影响。这些研究表明，VHHS作为新型抗PA药物的有用性，并引入了一种中和炭疽毒素的新机制。