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的腺苷酸环化酶，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（MEKS）。 由于尚不清楚两种替代毒素受体CMG2和TEM8的组织分布和相对重要性，因此我们试图开发试剂以区分两者。 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有效。 先前描述的用于PA域4的噬菌体显示系统已在其他选择中使用，并且已经确定了区分两种受体的突变。 已经获得了PA的变体，该变体保持对CMG2的高亲和力，同时保持对TEM8的亲和力很少。 来自多个领域的证据证实，TEM8表达比CMG2更受限制，而后者可能是更重要的受体。 生产供我们自身使用的毒素蛋白和合作者的许多批次生产，以及其效力的比较，使我们意识到LF的特定活动是可变的。 与N末端残基的身份的效力相关差异。 通过创建通过去除包含因子XA裂解位点的扩展而产生的N-Termini的变体，我们证明了LF效力似乎是由N端规则控制的，该规则指出末端残基的身份决定了泛素化和蛋白质化降解的速率。 在巨噬细胞的细胞毒性测定中也可以看到效力的差异，以及在致命的毒素（PA和LF的结合）中的小鼠和大鼠挑战中的差异。 这一发现具有重要意义，因为巨噬细胞的细胞毒性测定法被广泛用于靶向毒素的药物，疫苗和疗法的表征。 工作还继续理解二乙酰胺的作用，二乙酰胺是对伸长因子2的独特后翻译后修饰，这是假单胞菌Exotoxin A和Diphtheria Toxin的ADP-核糖基化的靶标。 缺乏双乙酰胺的细胞是可行的，因此很难理解为什么要保持毒素的靶标。 现在，我们发现缺乏双乙酰胺的细胞更容易受到核糖体灭活毒素（RIP），ricin的影响。 尽管它具有推测性，但我们建议双智酰胺的一个作用及其在进化过程中选择的可能原因可能是它赋予撕裂的额外保护。 Diphthamide的修饰位于核糖体的区域中，裂口和伸长因子2结合，因此它可能会在核糖体中通过ricin对其靶标的空间阻断。 撕裂可能会施加相当大的选择压力，因为它们是由许多植物产生的，包括被广泛食用的食物。