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: 8745476
• 负责人: Stephen Leppla
• 金额: $ 60.54万
• 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/8745476
• 关键词: ADP ribosylation; Adenylate Cyclase; Affinity; Amidohydrolases; Anabolism; Animal Model; Animals; Anthrax disease; Antibodies; Antigens; Arginine; Bacillus anthracis; Bacterial Proteins; Bacterial Toxins; Base Pairing; Basic Science; Binding; Biochemical Genetics; Botulinum Toxins; Buffers; C-terminal; Candidate Disease Gene; Catalytic Domain; Cell surface; Cells; Charge; Chimeric Proteins; Cleaved cell; Clostridium perfringens; Complement; Couples; Cultured Cells; Cyclic AMP; Cyclodextrins; Cytosol; Data; Dependence; Detection; Deubiquitinating Enzyme; Diagnostic Reagent; Diagnostics Research; Diphtheria Toxin; Dissociation; Dose; Drops; Engineering; Enzymes; Epitopes; Exhibits; Failure; Family; Genes; Genetic; Histidine; Hybrids; Infection; Ion Channel; Ionic Strengths; Laboratories; Libraries; Lysine; MAP Kinase Gene; MAPK14 gene; MEKs; Mass Spectrum Analysis; Measurement; Measures; Membrane Lipids; Metalloproteases; Mining; Molecular Target; Monoclonal Antibodies; Mus; Organism; Pathway interactions; Peptide Elongation Factor 2; Peptide Hydrolases; Pharmaceutical Preparations; Phenylalanine; Property; Proteinase-Activated Receptors; Proteins; Pseudomonas aeruginosa toxA protein; Reaction; Relative (related person); Reporting; Resistance; Role; Saccharomyces cerevisiae; Scheme; Site; Sodium Chloride; Source; Structure; Surface Plasmon Resonance; System; Targeted Toxins; Testing; Therapeutic; Time; Tissues; Toxic effect; Toxin; Translations; Ubiquitin; Ubiquitination; Urokinase; Variant; Virulence; Virulence Factors; Work; amidase; amidation; amino group; anthrax lethal factor; anthrax toxin; antigen binding; base; cell growth; channel blockers; cytotoxic; design; edema factor; improved; inhibitor/antagonist; mutant; neoplastic cell; novel vaccines; receptor; residence; response; synthetic nucleic acid; therapeutic vaccine; trait; tumor; tumor growth; voltage

Anthrax toxin, a major virulence factor of Bacillus anthracis, consists of the cellular binding moiety protective antigen (PA) and the enzymatic moieties lethal factor (LF) and edema factor (EF). To intoxicate host organisms, PA binds to the cellular receptors TEM8 and CMG2 and is proteolytically activated by the ubiquitously expressed cell surface furin protease, resulting in the formation of active PA heptamer, which in turn translocates LF and EF into the cytosol of cells. LF cleaves several MEKs, thereby inactivating the ERK, p38, and JUNK MAPK pathways. EF is an adenylate cyclase that generates abnormally high concentrations of cAMP. PA is a key virulence determinant of anthrax disease, and antibodies to PA protect against infection. Thus, PA is the focus of existing and new vaccines, and structure-function studies of the toxin proteins provide a rational basis on which to improve vaccines and therapeutics. Recent work by others provided evidence that the oligomer PA channel can exist in both heptameric and octameric forms. It was therefore important to assess the relative functional activities of these alternative forms. In 2013 we constructed and screened a highly directed library of PA mutants, and identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. This work provided convincing proof that the octameric form of PA is active. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application. Other bacterial protein toxins in the same family as anthrax toxin also form oligomeric channels to internalize catalytic effector domains. We extended prior work on the ability of cationic b-cyclodextrin derivatives to block PA activity and in 2013 showed that these compounds inhibit two other toxin, the C2 toxin of Clostridium botulinum and iota toxin of Clostridium perfringens. Studies were done in artificial lipid membranes, which provide detailed information about the properties of the oligomeric channels. We studied the voltage and salt dependence of the rate constants of binding and dissociation reactions of two structurally different b-cyclodextrins (AmPrbCD and AMBnTbCD) in the PA, C2, and iota toxin channels. With all three channels, the blocker carrying extra hydrophobic aromatic groups on the thio-alkyl linkers of positively charged amino groups, AMBnTbCD, demonstrated significantly stronger binding compared with AmPrbCD. The more-effective AMBnTbCD blocker shows weaker salt dependence of the binding and dissociation rate constants compared with AmPrbCD. In a search for the putative groups in the channel lumen that are responsible for the short-range forces, we performed measurements with the F427A mutant of PA, which lacks the functionally important phenylalanine clamp. We found that the on-rates of the blockage were virtually conserved, but the residence times and, correspondingly, the binding constants dropped by more than an order of magnitude, which also reduced the difference between the efficiencies of the two blockers. These data will aid in design of improved toxin channel blocking drugs. The anthrax toxin proteins constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, exogenous proteins delivered to the cytosol of cells are subject to ubiquitination on lysines and proteasomal degradation, which limit their potency. We created fusion proteins containing modified ubiquitins with their C-terminal regions fused to the Pseudomonas exotoxin A catalytic domain (PEIII) in order to achieve delivery and release of PEIII to the cytosol. In analyses reported in 2013, we showed that fusion proteins in which all seven lysines of wild-type ubiquitin were retained while the site cleaved by cytosolic deubiquitinating enzymes (DUBs) was removed were nontoxic, apparently due to rapid ubiquitination and proteasomal degradation. Fusion proteins in which all lysines of wild-type ubiquitin were substituted by arginine had high potency, exceeding that of a simple fusion lacking ubiquitin. This variant was less toxic to nontumor tissues in mice than the fusion protein lacking ubiquitin and was very efficient for tumor treatment in mice. The potency of these proteins was highly dependent on the number of lysines retained in the ubiquitin domain and on retention of the C-terminal ubiquitin sequence cleaved by DUBs. It appears that rapid cytosolic release of a cytotoxic enzyme (e.g., PEIII) that is itself resistant to ubiquitination is an effective strategy for enhancing the potency of tumor-targeting toxins. Diphthamide is a modified histidine residue in eukaryotic translation elongation factor 2 (eEF2) that is the target for irreversible ADP-ribosylation by diphtheria toxin (DT). In Saccharomyces cerevisiae, the initial steps of diphthamide biosynthesis are well characterized and require the DPH1-DPH5 genes. However, the last pathway step, amidation of the intermediate diphthine to diphthamide, is ill-defined. In 2013, with our collaborators, the genetic interaction landscapes of DPH1-DPH5 were mined to identify a candidate gene for the elusive amidase (YLR143w/DPH6) and confirm involvement of a second gene (YBR246w/DPH7) in the amidation step. Like dph1-dph5, dph6 and dph7 mutants have undermodified eEF2 forms that evade inhibition by DT. Moreover, mass spectrometry shows that dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2, demonstrating failure to complete the final amidation step. Dph6 is a candidate for the elusive amidase, while Dph7 apparently couples diphthine synthase (Dph5) to diphthine amidation. The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7. Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal 21 frameshifting and altered responses to translation inhibitors. Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, this work paves the way for a detailed mechanistic understanding of diphthamide formation. The monoclonal antibody S9.6 binds DNARNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R-loops. Our group is one of the few sources of the antibody, and we fill dozens of requests for it each year. In 2013 we described a single-chain variable fragment (scFv) of S9.6 and reported its affinities for various synthetic nucleic acid hybrids as measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNARNA and, surprisingly, 2.7 nM for RNARNA hybrids that are AU-rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNARNA hybrid. This work provides information that will increase the value of S9.6 in basic research and as a reagent for diagnostics.

炭疽毒素是炭疽杆菌的主要毒力因子，由细胞结合部分保护性抗原（PA）和酶部分致死因子（LF）和水肿因子（EF）组成。为了使宿主生物体中毒，PA 与细胞受体 TEM8 和 CMG2 结合，并且 由普遍表达的细胞表面弗林蛋白酶进行蛋白水解激活，从而产生 形成活性 PA 七聚体，进而将 LF 和 EF 易位到细胞的胞浆中。 LF 裂解多个 MEK，从而使 ERK、p38 和 JUNK MAPK 通路失活。 EF 是一种腺苷酸环化酶，可产生异常高浓度的 cAMP。 PA 是炭疽病的关键毒力决定因素，PA 抗体可防止感染。因此，PA是现有和新疫苗的焦点，毒素蛋白的结构功能研究为改进疫苗和治疗提供了合理的基础。 其他人最近的工作提供了证据表明寡聚物 PA 通道可以以七聚体和八聚体形式存在。因此，评估这些替代形式的相对功能活动非常重要。 2013年，我们构建并筛选了高度定向的PA突变体文库，并鉴定了彼此互补形成八聚体的变体。这些 PA 变体单独无毒，仅在与互补伙伴结合时才表现出毒性。这项工作提供了令人信服的证据，证明八聚体形式的 PA 具有活性。然后，我们将基质金属蛋白酶和尿激酶纤溶酶原激活剂的激活要求设计成其中两个变体。由此产生的治疗性毒素专门针对表达肿瘤相关蛋白酶的细胞，当使用远低于引起毒性的剂量时，可以完全阻止小鼠体内的肿瘤生长。这种获得相互互补亚基的方案可以与其他寡聚蛋白一起使用，并且可能具有广泛的应用。 与炭疽毒素同一家族的其他细菌蛋白毒素也形成寡聚通道以内化催化效应结构域。我们扩展了之前关于阳离子 b-环糊精衍生物阻断 PA 活性能力的研究，并于 2013 年证明这些化合物可抑制另外两种毒素，即肉毒梭菌的 C2 毒素和产气荚膜梭菌的 iota 毒素。对人造脂质膜进行了研究，提供了有关寡聚通道特性的详细信息。我们研究了两种结构不同的 b-环糊精（AmPrbCD 和 AMBnTbCD）在 PA、C2 和 iota 毒素通道中的结合和解离反应速率常数的电压和盐依赖性。对于所有三个通道，在带正电荷的氨基的硫代烷基连接体上携带额外疏水性芳香基团的阻断剂 AMBnTbCD 与 AmPrbCD 相比，表现出明显更强的结合。与 AmPrbCD 相比，更有效的 AMBnTbCD 阻断剂显示出较弱的结合和解离速率常数的盐依赖性。在寻找通道腔中负责短程力的推定基团时，我们使用 PA 的 F427A 突变体进行了测量，该突变体缺乏功能上重要的苯丙氨酸钳。我们发现阻断剂的结合率实际上是保守的，但停留时间和相应的结合常数下降了一个数量级以上，这也减少了两种阻断剂的效率之间的差异。这些数据将有助于设计改进的毒素通道阻断药物。 炭疽毒素蛋白构成了将细胞毒性酶递送至肿瘤细胞胞质的高效系统。然而，递送到细胞胞质的外源蛋白会受到赖氨酸泛素化和蛋白酶体降解的影响，这限制了它们的效力。我们创建了含有修饰泛素的融合蛋白，其 C 端区域与假单胞菌外毒素 A 催化结构域 (PEIII) 融合，以实现 PEIII 向胞质溶胶的递送和释放。在 2013 年报告的分析中，我们表明，保留野生型泛素的所有七个赖氨酸，同时去除胞质去泛素化酶 (DUB) 切割的位点的融合蛋白是无毒的，这显然是由于快速泛素化和蛋白酶体降解。野生型泛素的所有赖氨酸被精氨酸取代的融合蛋白具有很高的效力，超过了缺乏泛素的简单融合蛋白。与缺乏泛素的融合蛋白相比，该变体对小鼠非肿瘤组织的毒性较小，并且对于小鼠肿瘤治疗非常有效。这些蛋白质的效力高度依赖于泛素结构域中保留的赖氨酸数量以及被 DUB 切割的 C 端泛素序列的保留。看来，本身具有泛素化抗性的细胞毒性酶（例如 PEIII）的快速胞质释放是增强肿瘤靶向毒素效力的有效策略。 Diphthamide 是真核翻译延伸因子 2 (eEF2) 中经过修饰的组氨酸残基，是白喉毒素 (DT) 不可逆 ADP-核糖基化的靶标。在酿酒酵母中，二邻苯甲酰胺生物合成的初始步骤已得到充分表征，并且需要 DPH1-DPH5 基因。然而，最后一个途径步骤，即中间体二酞胺酰胺化为二酞酰胺，尚不明确。 2013 年，我们与合作者一起挖掘了 DPH1-DPH5 的遗传相互作用图谱，以确定难以捉摸的酰胺酶 (YLR143w/DPH6) 的候选基因，并确认第二个基因 (YBR246w/DPH7) 参与酰胺化步骤。与 dph1-dph5 一样，dph6 和 dph7 突变体具有未充分修饰的 eEF2 形式，可逃避 DT 的抑制。此外，质谱分析显示 dph6 和 dph7 突变体特异性地积累二phthine 修饰的 eEF2，表明未能完成最终的酰胺化步骤。 Dph6 是难以捉摸的酰胺酶的候选者，而 Dph7 显然将地菲合酶 (Dph5) 与地菲碱酰胺化偶联。后一个结论基于我们的观察，即 dph7 突变体显示 Dph5 和 eEF2 之间的相互作用急剧上调，表明它们的关联受到 Dph7 的控制。从生理学角度来说，完成二苯胺的合成是实现最佳翻译准确性和细胞生长所必需的，dph 突变体之间的共同特征表明，包括核糖体 21 移码增加和对翻译抑制剂的反应改变。通过鉴定 Dph6 和 Dph7 作为二邻苯二甲酰胺途径酰胺化步骤所需的组分，这项工作为详细了解二邻苯二甲酰胺形成的机制铺平了道路。 单克隆抗体 S9.6 以高亲和力结合 DNARNA 杂交体，使其可用于研究和诊断应用，例如微阵列和 R 环检测。我们的团队是为数不多的抗体来源之一，每年我们都会满足数十个抗体请求。 2013 年，我们描述了 S9.6 的单链可变片段 (scFv)，并报告了通过表面等离子共振 (SPR) 测量的其对各种合成核酸杂交体的亲和力。 S9.6 对于 DNARNA 表现出约 0.6 nM 的解离常数，令人惊讶的是，对于富含 AU 的 RNARNA 杂合体，S9.6 的解离常数为 2.7 nM。 S9.6 scFv 的亲和力似乎不受各种缓冲液条件或低于 500 mM NaCl 的离子强度的强烈影响。与 S9.6 scFv 强烈结合的最小表位包含 DNARNA 杂合体的六个碱基对。这项工作提供的信息将增加 S9.6 在基础研究和作为诊断试剂中的价值。