Structure, function and antigenicity of B. pertussis virulence factors

百日咳博德特氏菌毒力因子的结构、功能和抗原性

• 批准号: 10298432
• 负责人: JENNIFER A MAYNARD
• 金额: $ 56.43万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-09 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10298432
• 关键词: Acylation; Address; Adenylate Cyclase; Adenylate Cyclase Toxin; Anti-Bacterial Agents; Antibodies; Antibody Affinity; Bacteria; Bacterial Adhesins; Bacterial Infections; Bacterial Toxins; Binding; Binding Sites; Biochemical; Biological Assay; Biology; Biophysics; Bordetella pertussis; C-terminal; Calcium; Calcium ion; Calmodulin; Cell membrane; Cells; Complex; Consensus; Cryoelectron Microscopy; Cyclic AMP; Cytoplasm; Data; Developed Countries; Development; Disease; Elements; Environment; Epitopes; Extracellular Space; Family; Fc Receptor; Future; Goals; Gram-Negative Bacteria; Hemolysin; Immune; Immune system; In Vitro; Infant; Infection; Integrins; Intoxication; Length; Leukocytes; Lipase; Macrophage-1 Antigen; Mammalian Cell; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Morbidity - disease rate; Mus; Mutagenesis; N-terminal; Organism; Outcome; Pathogenesis; Pathway interactions; Peptide Hydrolases; Pertussis; Pertussis Toxin; Pertussis Vaccine; Phagocytes; Positioning Attribute; Prevalence; Process; Protein Engineering; Proteins; Research; Resolution; Signaling Protein; Site; Species Specificity; Structure; Therapeutic; Toxic effect; Toxin; Vaccine Antigen; Vaccines; Variant; Virulence Factors; Work; X-Ray Crystallography; base; biophysical analysis; design; genomic data; immunogenicity; improved; inhibiting antibody; insight; interest; member; mortality; mouse model; neutralizing antibody; novel; novel vaccines; pathogen; protein protein interaction; receptor; receptor binding; response; vector-induced

Project Summary Despite widespread use of a vaccine, infection with the bacterium Bordetella pertussis continues to claim the lives of ~200,000 infants annually worldwide and cause significant morbidity and mortality in developed countries, including the US. To develop improved vaccines and therapeutics, we need to better understand how this organism causes disease and identify new vaccine antigens. The adenylate cyclase toxin (ACT) is a leading candidate for inclusion in future pertussis vaccines. ACT is a large (1706 residue), bi-functional toxin with a cell- invasive domain fused to a pore-forming repeat-in-toxin (RTX) hemolysin domain. The RTX domain is composed of five blocks of ~8 nonapeptide motifs separated by linkers of different length and sequence. ACT efficiently targets leukocytes by binding αMβ2 integrins via a site localized to the RTX domain. Receptor binding triggers translocation of the 40 kDa N-terminal adenylate cyclase domain across the host cell membrane where it rapidly converts nearly all intracellular ATP to cAMP, thereby compromising phagocytic and other leukocyte anti- bacterial activities. Although the general features of ACT function have been described, there are few data to support a molecular understanding of any step in the intoxication process for ACT specifically or for RTX proteins more generally. The structural features by which the RTX blocks mediate specific protein–protein interactions, such as receptor binding, and the epitopes and mechanisms by which antibodies inhibit ACT function are not well defined. Our panel of high-affinity antibodies that recognize neutralizing and non-neutralizing epitopes on ACT provide a unique opportunity to address these questions. The long-term goal of this research is to understand structural mechanisms of the complex cellular intoxication process used by the Bordetella adenylate cyclase toxin to incapacitate immune cells. The specific objective is to provide a molecular description of the interaction of ACT’s RTX domain with its receptor and with neutralizing and non-neutralizing antibodies. This will provide mechanistic insights into ACT function and define important vaccine targets such as epitopes susceptible to antibody-mediated neutralization, the receptor-binding site, and pre-translocation conformations. Such information is necessary for the implementation of rational design strategies that seek to more effectively present such targets to the immune system. The expected outcomes include the first structures of an RTX protein containing more than two repeat blocks and the first RTX–antibody and RTX–receptor structures. We will also evaluate structural pathways for RTX antibody escape and species specificity and the impact of such changes on cellular toxicity of the intact ACT protein and bacterial infection using a mouse model. Since there are currently no structural data defining antibody or receptor epitopes for any RTX protein, this work will transform our understanding of this class of bacterial toxin and provide insight into a key pertussis virulence factor.

项目摘要 尽管疫苗的广泛使用，感染百日咳波尔德氏菌的人仍然声称 全世界每年约有20万名婴儿死亡，并在发达国家造成严重的发病率和死亡率， 包括美国。为了开发改进的疫苗和疗法，我们需要更好地了解这一点 生物体导致疾病，并识别新的疫苗抗原。腺苷环化酶毒素(ACT)是一种主要的 未来百日咳疫苗的候选疫苗。ACT是一种大的(1706残基)双功能毒素，具有细胞- 侵袭性结构域与形成孔的毒素重复序列(RTX)溶血素结构域融合。RTX域由 由不同长度和序列的连接子分隔的5个区块的~8个非肽基序。高效行动 通过与αMβ2整合素结合，通过定位于rtx结构域的位点来靶向白细胞。受体结合触发物 40 kDa N末端腺苷环化酶结构域在宿主细胞膜上的移位 将几乎所有细胞内的ATP转化为cAMP，从而损害吞噬细胞和其他白细胞的抗 细菌活动。 虽然已经描述了ACT功能的一般特征，但支持分子的数据很少 了解ACT或更一般的RTX蛋白中毒过程中的任何步骤。 RTX阻断的结构特征介导特定的蛋白质-蛋白质相互作用，如受体 结合，抗体抑制ACT功能的表位和机制还没有很好的定义。我们的 识别ACT上中和表位和非中和表位的一组高亲和力抗体提供了一种 解决这些问题的独特机会。这项研究的长期目标是理解结构 波氏杆菌腺苷环化酶毒素复杂的细胞中毒过程的机制 使免疫细胞丧失能力。具体的目标是提供一个相互作用的分子描述 ACT的RTX结构域与其受体以及中和抗体和非中和抗体的结合。这将提供 对ACT功能的机械性洞察并定义重要的疫苗靶点，如易感表位 抗体介导的中和、受体结合部位和预易位构象。是这样的 信息对于寻求更有效地呈现的合理设计策略的实施是必要的 这样的靶子会影响免疫系统。预期的结果包括RTX蛋白的第一个结构 包含两个以上的重复片段和第一RTX抗体和RTX受体结构。我们还将 评估RTX抗体逃逸和物种特异性的结构途径以及这些变化对 用小鼠模型研究完整ACT蛋白的细胞毒性和细菌感染。因为目前有 没有结构数据定义任何RTX蛋白的抗体或受体表位，这项工作将改变我们的 了解这类细菌毒素，并提供对百日咳毒力的关键因素的洞察。