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: 7732681
• 负责人: Stephen Leppla
• 金额: $ 89.94万
• 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/7732681
• 关键词: ADP ribosylation; Adenylate Cyclase; Affinity; Animal Model; Animals; Anthrax disease; Antigens; Area; Bacillus anthracis; Bacterial Proteins; Bacterial Toxins; Bacteriophages; Binding; Biochemical Genetics; Biological Assay; Calcium; Calmodulin; Cell surface; Cells; Cleaved cell; Complex; Cultured Cells; Cyclic AMP; Cytosol; Diphtheria Toxin; Drug or chemical Tissue Distribution; Eating; Endocytosis; Endopeptidases; Evolution; Excision; Factor Xa; Food; Genetic; Infection; Ion Channel; Laboratories; Mammalian Cell; Metalloproteases; Mitogen-Activated Protein Kinase Kinases; Modification; Molecular Target; Monoclonal Antibodies; Mus; Mutation; N-terminal; Peptide Elongation Factor 2; Peptide Hydrolases; Pharmaceutical Preparations; Plants; Post-Translational Protein Processing; Production; Proteinase-Activated Receptors; Proteins; Pseudomonas aeruginosa toxA protein; Rate; Rattus; Reagent; Relative (related person); Resistance; Ribosomes; Ricin; Role; Site; Structure; Surface; System; Tissues; Toxin; Vaccines; Variant; Virulence Factors; Work; anthrax lethal factor; anthrax toxin; cytotoxicity; edema factor; macrophage; mutant; pressure; receptor; therapeutic target

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). Because the tissue distribution and relative importance of the two alternative toxin receptors, CMG2 and TEM8, are not known, we have sought to develop reagents to distinguish the two. Polyclonal and monoclonal antibodies were developed, but these have not yet proven highly effective in detecting the receptors in cell lysates or on intact cells. A previously described system for bacteriophage display of domain 4 of PA has been used in additional selections, and mutations have been identified that discriminate between the two receptors. Variants of PA have been obtained that retain high affinity for CMG2 while retaining very little affinity for TEM8. Evidence from several areas confirms the view that TEM8 expression is more restricted than that of CMG2, and that the latter is probably the more important receptor. Production of many batches of the toxin proteins for our own use and that of collaborators, and comparison of their potencies, made us aware that the specific activity of LF was variable. Close scrutiny related differences in potency to the identity of the N-terminal residue. By creation of variants having defined N-termini produced by removal of an extension containing a Factor Xa cleavage site, we demonstrated that LF potency appears to be controlled by the N-end rule, which states that the identity of the terminal residue determines the rate of ubiquitinylation and proteosomal degradation. The differences in potency were seen both in the macrophage cytotoxicity assay and in mouse and rat challenges with lethal toxin, the combination of PA and LF. This finding has significance because the macrophage cytotoxicity assay is widely used in characterization of drugs, vaccines, and therapeutics targeting the toxin. Work also continued to understand the role of diphthamide, the unique post-translational modification on elongation factor 2 that is the target of ADP-ribosylation by Pseudomonas exotoxin A and diphtheria toxin. Cells lacking diphthamide are viable, making it difficult to understand why a target for a toxin would be maintained. We have now found that cells lacking diphthamide are 3-fold more susceptible to a ribosome inactivating toxin (RIP), ricin. Although it is speculative, we suggest that one role of diphthamide and a possible reason for its selection during evolution may be the added protection it confers to RIPs. The diphthamide modification is located in the region of the ribosome to which both RIPS and elongation factor 2 bind, and so it may sterically block access by ricin to its target in the ribosome. RIPs may exert a considerable selective pressure because they are produced by many plants, including ones widely eaten as foods.

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)。 由于两种替代毒素受体 CMG2 和 TEM8 的组织分布和相对重要性尚不清楚，因此我们寻求开发试剂来区分两者。 人们开发了多克隆和单克隆抗体，但尚未证明这些抗体在检测细胞裂解物或完整细胞中的受体方面非常有效。 先前描述的 PA 结构域 4 的噬菌体展示系统已用于其他选择，并且已鉴定出区分两种受体的突变。 已获得的 PA 变体保留了对 CMG2 的高亲和力，同时保留了对 TEM8 的极低亲和力。 来自多个领域的证据证实了这样的观点：TEM8 的表达比 CMG2 的表达受到更多限制，并且后者可能是更重要的受体。 生产了多批供我们自己和合作者使用的毒素蛋白，并对它们的效力进行了比较，使我们意识到 LF 的比活性是可变的。 仔细检查与 N 末端残基的特性相关的效力差异。 通过删除含有因子 Xa 切割位点的延伸产生具有定义 N 末端的变体，我们证明了 LF 效力似乎受 N 末端规则控制，该规则表明末端残基的身份决定了速率泛素化和蛋白体降解。 在巨噬细胞细胞毒性测定以及小鼠和大鼠用致命毒素（PA 和 LF 的组合）的攻击中都可以看到效力的差异。 这一发现具有重要意义，因为巨噬细胞细胞毒性测定广泛用于表征针对毒素的药物、疫苗和疗法。 研究人员还继续了解二苯乙醇胺的作用，二苯乙醇胺是延伸因子 2 的独特翻译后修饰，是假单胞菌外毒素 A 和白喉毒素 ADP 核糖基化的目标。 缺乏二苯胺的细胞是有活力的，因此很难理解为什么会保留毒素的目标。 我们现在发现，缺乏二苯甲酰胺的细胞对核糖体失活毒素 (RIP) 蓖麻毒素的敏感性要高出 3 倍。 尽管这是推测性的，但我们认为二苯胺的作用之一以及其在进化过程中被选择的一个可能原因可能是它为 RIP 提供了额外的保护。 二苯甲酰胺修饰位于 RIPS 和延伸因子 2 结合的核糖体区域，因此它可能在空间上阻止蓖麻毒素进入核糖体中的靶标。 RIP 可能会产生相当大的选择压力，因为它们是由许多植物产生的，包括广泛作为食物食用的植物。