Structure and Function of Virulence Factors of Bacillus anthracis

炭疽杆菌毒力因子的结构和功能

• 批准号: 8555954
• 负责人: Stephen Leppla
• 金额: $ 54.12万
• 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/8555954
• 关键词: Adenylate Cyclase; Affect; Affinity; Amino Acids; Animal Model; Animals; Anthrax disease; Antibodies; Antigens; Antitoxins; Bacillus anthracis; Bacterial Proteins; Bacterial Toxins; Binding; Biochemical Genetics; Blood capillaries; Bontoxilysin; Botulism; Calcium; Calmodulin; Catalytic Domain; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Cessation of life; Cleaved cell; Collaborations; Colon Carcinoma; Complex; Cultured Cells; Cyclic AMP; Cytosol; Dependovirus; Detection; Development; Disease Progression; Drug or chemical Tissue Distribution; Edema; Endocytosis; Endothelium; Enzymes; Epitopes; Gene Transfer; Genes; Genetic; Genetic Screening; Genistein; Growth; Growth and Development function; Immune Sera; Immunization; Infection; Infection Control; Injection of therapeutic agent; Intoxication; Ion Channel; Knowledge; Laboratories; Mammalian Cell; Mass Spectrum Analysis; Mediating; Metalloproteases; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular Target; Monoclonal Antibodies; Morphogenesis; Mus; Mutagenesis; Mutate; Mutation; N-terminal; Neck; Pathogenesis; Peptide Hydrolases; Phase; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Platelet-Derived Growth Factor alpha Receptor; Protein Kinase; Protein phosphatase; Proteinase-Activated Receptors; Proteins; Pyruvate Kinase; Reagent; Recombinant Proteins; Recombinants; Regulation; Relative (related person); Reporting; Reproduction spores; Resistance; Role; Scientist; Sequence Analysis; Serotyping; Serum; Site; Specificity; Structure; Surface; System; Testing; Therapeutic; Time; Tissues; Toxin; Transfection; Treatment Efficacy; Tumor Markers; United States National Institutes of Health; Viral; Viral Vector; Virulence Factors; Virus; Virus Receptors; Western Blotting; Work; anthrax lethal factor; anthrax toxin; antigen binding; base; capillary; cell growth; clinically relevant; edema factor; gene therapy; genetic selection; in vitro activity; in vivo; insight; interest; kinase inhibitor; mouse model; mutant; novel; receptor; research study; subcutaneous; tool; trafficking; tumor; vector

Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then captures either of the two other toxin proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis and LF and EF translocate to the cytosol. EF is a calcium- and calmodulin-dependent adenylyl cyclase that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). Entry of anthrax toxin into cells depends on two related cell surface receptors, tumor endothelium marker 8 (TEM8) and capillary morphogenesis gene product 2 (CMG2). TEM8 was initially identified as a protein upregulated in colon cancers. CMG2 has substantial sequence similarity to this candidate tumor marker. The tissue distribution and the relative importance of the two toxin receptors in toxin action are not well understood. During 2012, in collaboration with an Indian scientist having a long association with NIH and our laboratory, we identified two enzymes produced by Bacillus anthracis as being dual specificity protein kinases (DSPKs), unique enzymes that autophosphorylate on Ser, Thr, and Tyr residues and phosphorylate substrates on Ser and Thr residues. Transcriptional analyses of these kinases, Bas2152 (PrkD) and Bas2037 (PrkG), showed that they are expressed in all phases of growth. PrkD was found to be similar to the eukaryotic dual specificity Tyr phosphorylation- regulated kinase class of dual specificity kinases, which autophosphorylates on Ser, Thr, and Tyr residues and phosphorylates Ser and Thr residues on substrates. PrkG was found to be a bona fide dual specificity protein kinase that mediates autophosphorylation and substrate phosphorylation on Ser, Thr, and Tyr residues. The sites of phosphorylation in both of the kinases were identified through mass spectrometry. Phosphorylation on Tyr residues regulates the kinase activity of PrkD and PrkG. PrpC, the only known B. anthracis Ser/Thr protein phosphatase, was also found to possess dual specificity. Genistein, a known Tyr kinase inhibitor, was found to inhibit the activities of PrkD and PrkG and affect the growth of B. anthracis cells, indicating a possible role of these kinases in cell growth and development. In addition, the glycolytic enzyme pyruvate kinase was found to be phosphorylated by PrkD on Ser and Thr residues but not by PrkG. Thus, this study provides the first evidence of DSPKs in B. anthracis that belong to different classes and have different modes of regulation. This reporting period also saw an important advance in our long-standing efforts to characterize the structure, function, and role in pathogenesis of the edema factor (EF) protein of anthrax toxin. In the current effort, anti-EF monoclonal antibodies (MAb) were produced following immunization of mice, and four of the antibodies were fully characterized. MAb 3F2 has an affinity of 388 pM, was most effective for EF detection, and appears to be the first antibody reported to neutralize EF by binding to the catalytic domain. MAb 7F10 shows potent neutralization of edema toxin activity in vitro and in vivo; it targets the N-terminal protective antigen-binding domain. The four MAb react with three different domains of edema factor, and all were able to detect purified edema factor by western blot analysis. None of the four MAb cross-reacted with the lethal factor toxin component. Three of the four MAb protected mice in both a systemic edema toxin challenge model and a subcutaneous spore challenge edema model. A combination of three of the MAb significantly delayed the time to death in a mouse infection model involving spore injection in the neck. The protection observed in this work appears to be the first direct evidence that monoclonal antibody-mediated neutralization of EF alone is sufficient to delay anthrax disease progression. In the year of 2012, we have also contributed in studies developing novel monoclonal antibodies to anthrax toxin protective antigen. These antibodies aid in structure-function analysis of the toxin proteins and can for the basis of therapeutics. We reported a practical strategy for development of simple antitoxins having substantial advantages over currently-available treatments. The strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab V(H) (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacies of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of the clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antisera in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit. A collaborative project completed during 2012 used knowledge and reagents developed in earlier studies on the role of cellular proteases in activation of bacterial protein toxins. These tools were used to identify cellular determinants controlling the infection of cells by adeno-associated viruses (AAV), which are the basis of viral vectors preferred in certain gene therapy applications. To identify these critical cellular determinants, we took advantage of the gene transfer abilities of AAV in combination with a forward genetic selection to identify proteins critical for transduction by this virus. AAV serotype 5 (AAV5) vectors encoding the furin gene were used to transduce furin-deficient CHO FD11 cells, followed by selection with furin-dependent bacterial protein toxins. A small number of spontaneously mutated cells specifically resistant to AAV5 transduction (and therefore surviving the toxin treatment) was isolated. Sequence analysis showed that they all had a single amino acid mutation in the leader sequence of the platelet-derived growth factor receptor alpha (PDGFRalpha) gene, which was previously shown to the be AAV receptor. Characterization of this mutation showed that it inhibited PDGFRalpha trafficking, resulting in limited expression on the plasma membrane. Mutagenesis and transfection experiments confirmed the effect of this mutation on PDGFRalpha trafficking, and the AAV5 resistant phenotype could be rescued by transfection with wild type PDGFRalpha. Thus, the tools of furin-deficient cells and selection with bacterial toxins enabled a genetic screen that identified interesting cellular genes controlling viral sensitivity.

炭疽毒素保护性抗原蛋白（PA，83 kDa）与哺乳动物细胞表面的受体结合，被细胞表面蛋白酶弗林蛋白酶切割，然后捕获其他两种毒素蛋白，致死因子（LF，90 kDa）或水肿因子（EF，89 kDa）。 PA-LF 和 PA-EF 复合物通过内吞作用进入细胞，并且 LF 和 EF 易位至胞质溶胶。 EF 是一种钙和钙调蛋白依赖性腺苷酸环化酶，可导致细胞内 cAMP 浓度大幅且不受调节地增加。 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)。 炭疽毒素进入细胞取决于两种相关的细胞表面受体：肿瘤内皮标记物 8 (TEM8) 和毛细血管形态发生基因产物 2 (CMG2)。 TEM8 最初被鉴定为结肠癌中表达上调的蛋白质。 CMG2 与该候选肿瘤标志物具有显着的序列相似性。两种毒素受体在毒素作用中的组织分布和相对重要性尚不清楚。 2012 年，我们与一位与 NIH 和我们实验室有长期合作的印度科学家合作，鉴定了炭疽杆菌产生的两种酶，即双特异性蛋白激酶 (DSPK)，这是一种在 Ser、Thr 和 Tyr 残基上自磷酸化的独特酶，磷酸化 Ser 和 Thr 残基上的底物。对这些激酶 Bas2152 (PrkD) 和 Bas2037 (PrkG) 的转录分析表明，它们在生长的所有阶段都有表达。 发现 PrkD 与双特异性激酶的真核双特异性 Tyr 磷酸化调节激酶类相似，其对 Ser、Thr 和 Tyr 残基进行自磷酸化，并对底物上的 Ser 和 Thr 残基进行磷酸化。 PrkG 被发现是一种真正的双特异性蛋白激酶，可介导 Ser、Thr 和 Tyr 残基的自身磷酸化和底物磷酸化。 通过质谱法鉴定了两种激酶的磷酸化位点。 Tyr 残基的磷酸化调节 PrkD 和 PrkG 的激酶活性。 PrpC 是唯一已知的炭疽杆菌 Ser/Thr 蛋白磷酸酶，也被发现具有双重特异性。 Genistein 是一种已知的 Tyr 激酶抑制剂，被发现可以抑制 PrkD 和 PrkG 的活性并影响炭疽芽孢杆菌细胞的生长，表明这些激酶在细胞生长和发育中可能发挥作用。 此外，还发现糖酵解酶丙酮酸激酶被 PrkD 在 Ser 和 Thr 残基上磷酸化，但不被 PrkG 磷酸化。 因此，本研究首次证明炭疽芽孢杆菌中的 DSPKs 属于不同纲并具有不同的调节模式。 在本报告期内，我们在表征炭疽毒素水肿因子（EF）蛋白的结构、功能和发病机制中的作用方面的长期努力也取得了重要进展。 在目前的工作中，在对小鼠进行免疫接种后产生了抗 EF 单克隆抗体 (MAb)，并且其中四种抗体得到了充分表征。 MAb 3F2 的亲和力为 388 pM，对于 EF 检测最有效，并且似乎是第一个报道的通过与催化结构域结合来中和 EF 的抗体。 MAb 7F10 在体外和体内均显示出有效的中和水肿毒素活性；它的目标是 N 端保护性抗原结合结构域。四种 MAb 与水肿因子的三个不同结构域发生反应，并且全部能够通过蛋白质印迹分析检测纯化的水肿因子。 四种 MAb 均未与致死因子毒素成分发生交叉反应。 四种单克隆抗体中的三种在全身水肿毒素激发模型和皮下孢子激发水肿模型中均保护小鼠。 在涉及颈部注射孢子的小鼠感染模型中，三种单克隆抗体的组合显着延迟了死亡时间。 这项工作中观察到的保护作用似乎是第一个直接证据，证明单克隆抗体介导的 EF 中和作用足以延缓炭疽病的进展。 2012年，我们还参与了开发针对炭疽毒素保护性抗原的新型单克隆抗体的研究。 这些抗体有助于毒素蛋白的结构功能分析，并可作为治疗的基础。 我们报告了一种开发简单抗毒素的实用策略，与目前可用的治疗方法相比具有显着优势。 该策略采用单一重组“靶向剂”，在两个独特位点结合毒素，以及“清除抗体”，结合每个靶向剂上存在的两个表位。 靶向剂和清除抗体的共同施用会导致毒素被多达四种抗体修饰，以促进加速清除。 该治疗策略适用于两种肉毒杆菌神经毒素（BoNT）血清型，并在两种不同的中毒模型中保护小鼠免于死亡，其功效与传统抗毒素血清相当。 靶向剂是单一重组蛋白，由两个骆驼抗 BoNT 仅重链 Ab V(H) (VHH) 结合域和两个 E-tag 表位的异二聚体组成。 清除单克隆抗体是抗 E-tag 单克隆抗体。 通过比较使用中和剂与非中和剂的治疗的体内功效或清除抗体的存在与不存在，可以前所未有地深入了解毒素中和和清除在抗毒素功效中的作用。 令人惊讶的是，当使用中毒后治疗模型时，即使在没有清除抗体的情况下，毒素中和异二聚体剂也能完全保护小鼠免受中毒。 因此，在临床相关的肉毒中毒小鼠模型中，单一的、易于生产的重组蛋白与多克隆抗血清一样有效。 该策略应该广泛应用于抗毒素开发和其他疗法，其中血清生物分子的中和和/或加速清除可以提供治疗益处。 2012 年完成的一个合作项目使用了早期研究中开发的知识和试剂，这些研究涉及细胞蛋白酶在细菌蛋白毒素激活中的作用。 这些工具用于识别控制腺相关病毒（AAV）感染细胞的细胞决定因素，这是某些基因治疗应用中首选病毒载体的基础。 为了识别这些关键的细胞决定因素，我们利用 AAV 的基因转移能力与正向遗传选择相结合来识别对该病毒转导至关重要的蛋白质。 使用编码弗林蛋白酶基因的 AAV 血清型 5 (AAV5) 载体转导弗林蛋白酶缺陷的 CHO FD11 细胞，然后用弗林蛋白酶依赖性细菌蛋白毒素进行选择。 分离出少量对 AAV5 转导具有特异性抗性的自发突变细胞（因此在毒素处理后仍能存活）。 序列分析显示，它们的血小板源性生长因子受体α（PDGFRα）基因的前导序列中均存在单个氨基酸突变，该突变此前已被证明是AAV受体。 该突变的特征表明它抑制 PDGFRalpha 运输，导致质膜上的表达有限。 诱变和转染实验证实了该突变对PDGFRα运输的影响，并且可以通过用野生型PDGFRα转染来挽救AAV5抗性表型。 因此，弗林蛋白酶缺陷细胞的工具和细菌毒素的选择使得遗传筛选成为可能，从而识别出控制病毒敏感性的有趣的细胞基因。