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: 10448307
• 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/10448307
• 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; rational design; 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 尽管使用了疫苗的宽度，但bordetella bordetella buttussis的感染仍在声称 全球每年约有200,000名婴儿的生命，并在发达国家引起明显的发病率和死亡率， 包括美国。要开发改进的疫苗和疗法，我们需要更好地了解如何 有机体会引起疾病并鉴定新的疫苗抗原。腺苷酸环化酶毒素（ACT）是领先 候选未来百日咳疫苗的候选人。 ACT是一个大型（1706居住），具有细胞的双功能毒素 浸润结构域融合到形成孔的重复中毒素（RTX）溶血素结构域。 RTX域组成 五个块的五个块的〜8个非肽基序，这些基序被不同的长度和序列的接头隔开。有效地采取行动 通过与RTX结构域结合的位点结合αMβ2整合素来靶向白细胞。受体结合触发器 40 kDa N末端腺苷酸环化酶结构域跨宿主细胞膜的易位 将几乎所有细胞内ATP转换为cAMP，从而损害了吞噬细胞和其他白细胞抗 细菌活动。 尽管已经描述了ACT函数的一般特征，但几乎没有数据支持分子 了解针对ACT的任何步骤或更广泛地针对RTX蛋白的任何步骤。 RTX阻止介导特定蛋白质 - 蛋白质相互作用的结构特征，例如接收器 结合，以及抗体抑制ACT功能的表位和机制尚未很好地定义。我们的 识别ACT中和和非中和表位的高亲和力抗体面板提供了 解决这些问题的独特机会。这项研究的长期目标是了解结构 Bordetella腺苷酸环化酶毒素使用的复杂细胞中毒过程的机制 丧失能力的免疫细胞。具体目标是提供有关相互作用的分子描述 ACT的RTX域具有其接收器，并具有中和和非中和抗体。这将提供 对ACT功能的机械洞察并定义了重要的疫苗靶标，例如易感的表位 抗体介导的谈判，接收器结合位点和预先定位构象。这样的 信息对于实施合理设计策略是必需的，该策略试图更有效地呈现 这样的目标是免疫系统。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块以及第一个RTX - 抗体和RTX-受体结构。我们也会 评估RTX抗体逃逸和规格特异性的结构途径以及此类变化对 使用小鼠模型，完整ACT蛋白和细菌感染的细胞毒性。由于目前有 没有定义任何RTX蛋白的抗体或受体表位的结构数据，这项工作将改变我们的 了解这类细菌毒素，并洞悉关键的百日咳病毒因子。