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上的受体结合，被细胞表面蛋白酶呋喃裂解，然后捕获两个其他两种毒素蛋白，致死因子（LF，90 kDa）或Edema因子或Edema因子（EF，89 KDA）。 PA-LF和PA-EF复合物通过内吞作用进入细胞，LF和EF转移到细胞质。 EF是一种钙和钙调蛋白依赖性腺苷酸环化酶，在细胞内cAMP浓度中导致大量和不受监管的增加。 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（MEKS）。 炭疽毒素进入细胞中的进入取决于两个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最初被鉴定为在结肠癌中上调的蛋白质。 CMG2与该候选肿瘤标记具有很大的序列相似性。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。 在2012年期间，与NIH和我们的实验室有长期关联的印度科学家合作，我们确定炭疽芽孢杆菌生产的两种酶是双重特异性蛋白激酶（DSPK），这些酶是在Ser，THR，THR，THR和Tyr Prastues和Tyr rystrues and PrapphoryLate serstrate on Ser和Thr and Thr残留物上自磷酸化的独特酶。对这些激酶的转录分析，BAS2152（PRKD）和BAS2037（PRKG），表明它们在生长的所有阶段都表达。 发现PRKD与真核双重特异性相似，Tyr磷酸化调节的双重特异性激酶类别，该类别在Ser，Thr和Tyr残基上自磷酸化，并磷酸化，并磷酸化ser and THR在底物上。 发现PRKG是一种真正的双重特异性蛋白激酶，可介导SER，THR和TYR残基上的自磷酸化和底物磷酸化。 通过质谱法鉴定出两个激酶中磷酸化的位点。 Tyr残基的磷酸化调节PRKD和PRKG的激酶活性。 PRPC是唯一已知的炭疽芽孢杆菌/THR蛋白磷酸酶，也被发现具有双重特异性。 染料木黄酮是一种已知的Tyr激酶抑制剂，发现抑制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的可能作用。 此外，发现丙二酶丙酮酸激酶被PRKD磷酸化，而在Ser和Thr残基上，但不是由PRKG磷酸化。 因此，这项研究提供了植物中植物中DSPK的第一个证据，该证据属于不同类别，具有不同的调节模式。 在这个报告期间，我们长期以来一直在表征炭疽毒素水肿因子（EF）蛋白质的结构，功能和作用的长期努力中的重要进步。 在目前的努力中，在小鼠免疫后产生了抗FEF单克隆抗体（MAB），其中四种抗体被充分表征。 MAB 3F2的亲和力为388 pm，对于EF检测最有效，并且似乎是通过与催化域结合来中和EF的第一种抗体。 MAB 7F10显示体外和体内水肿毒素活性的有效中和。它针对N末端保护性抗原结合结构域。四个mAb与水肿因子的三个不同结构域反应，并且都能够通过蛋白质印迹分析检测纯化的水肿因子。 与致命因子毒素成分相互反应的四个mab中都没有。 在全身性水肿毒素挑战模型和皮下孢子挑战水肿模型中，四只mAb保护的小鼠中有三只。 三个mAb的组合显着延迟了涉及颈部孢子注射的小鼠感染模型中死亡的时间。 这项工作中观察到的保护似乎是第一个直接证据，表明单克隆抗体介导的EF单独中和足以延迟炭疽疾病的进展。 在2012年，我们还为开发针对炭疽毒素保护性抗原的新型单克隆抗体的研究做出了贡献。 这些抗体有助于对毒素蛋白的结构 - 功能分析，并且可以基于治疗剂。 我们报道了一种实用的策略，用于开发与当前可用治疗相比，具有实质性优势的简单抗毒素。 该策略采用单个重组的“靶向剂”，该重组剂在两个唯一位点结合A毒素，并结合了一个“清除AB”，该抗体结合了每个靶向剂上存在的两个表位。 靶向剂和清除AB的共同给药可导致毒素装饰多达四个ABS，以促进加速清除率。 将治疗策略应用于两种肉毒杆菌神经毒素（BONT）血清型，并在两个不同的中毒模型中受保护小鼠免受致死性的影响，具有等效于常规抗毒素血清的功效。 靶向剂是单个重组蛋白，该蛋白由两个骆驼抗抗块重链仅AB V（H）（VHH）结合域和两个E-TAG表位组成。 清除mAb是一个抗tag mab。 通过比较使用中和与非中和剂的体内效力，或者在不存在清除AB的存在与存在的情况下，允许对抗毒素疗效中中和和清除的作用的前所未有的见解。 令人惊讶的是，当使用后毒理治疗模型时，即使没有清除AB，也完全保护了毒素中和杂化剂完全保护小鼠免于中毒。 因此，在临床上与肉毒杆菌的小鼠模型中，单个易于生产的重组蛋白与多克隆抗血清一样有效。 该策略应在抗毒素开发和其他疗法中广泛应用，在这些疗法中中和和/或加速清除血清生物分子可以提供治疗益处。 在2012年完成的一个协作项目中，使用了知识和试剂在早期研究细胞蛋白酶在细菌蛋白毒素激活中的作用的研究。 这些工具用于鉴定通过腺相关病毒（AAV）控制细胞感染的细胞决定因素，这是某些基因治疗应用中首选的病毒载体的基础。 为了识别这些关键的细胞决定因素，我们利用了AAV的基因转移能力与正向遗传选择的结合，以鉴定该病毒转导至关重要的蛋白质。 编码FURIN基因的AAV血清型5（AAV5）向量被用来转导脂肪蛋白缺陷型CHO FD11细胞，然后选择依赖Furin的细菌蛋白毒素。 分离了少数自发性突变的细胞特异性抗AAV5转导（并因此存活的毒素处理）。 序列分析表明，它们在血小板衍生的生长因子受体α（PDGFRALPHA）基因的铅序列中都有一个单个氨基酸突变，该基因先前已显示为AAV受体。 该突变的表征表明它抑制了PDGFRALPHA运输，从而在质膜上表达有限。 诱变和转染实验证实了该突变对PDGFRALPHA运输的影响，并且可以通过用野生型PDGFRALPHA转染AAV5抗性表型。 因此，脂肪蛋白缺陷型细胞和用细菌毒素进行选择的工具使遗传筛选鉴定了控制病毒敏感性的有趣的细胞基因。