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: 10656458
• 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/10656458
• 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; 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; Predisposition; Prevalence; Process; Protein Engineering; Proteins; Research; Resolution; Signal Transduction; Site; Species Specificity; Structure; Therapeutic; Toxic effect; Toxin; Vaccine Antigen; Vaccines; Variant; Virulence Factors; Work; X-Ray Crystallography; biophysical analysis; design; genomic data; immunogenicity; improved; inhibiting antibody; insight; interest; member; mortality; mouse model; neutralizing antibody; novel; novel vaccines; pathogen; protein protein interaction; protein purification; rational design; receptor; receptor binding; response

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.

项目概要 尽管疫苗被广泛使用，但百日咳博德特氏菌感染仍然是最致命的疾病。 每年影响全世界约 200,000 名婴儿的生命，并在发达国家造成严重的发病率和死亡率， 包括美国在内，为了开发改进的疫苗和治疗方法，我们需要更好地了解这一点。 腺苷酸环化酶毒素 (ACT) 是一种主要的病原体，可引起疾病并识别新的疫苗抗原。 ACT 是一种大型（1706 个残基）双功能毒素，具有细胞毒性。 侵入性结构域与成孔毒素重复序列 (RTX) 溶血素结构域融合。 由不同长度和序列的接头有效分隔的五个约 8 个九肽基序的块。 通过定位于 RTX 结构域的位点结合 αMβ2 整合素来靶向白细胞。 40 kDa N 端腺苷酸环化酶结构域易位穿过宿主细胞膜，并迅速 将几乎所有细胞内 ATP 转化为 cAMP，从而损害吞噬细胞和其他白细胞抗- 细菌活动。 尽管已经描述了 ACT 功能的一般特征，但很少有数据支持分子生物学 了解 ACT 中毒过程中的任何步骤，或者更广泛地了解 RTX 蛋白中毒过程中的任何步骤。 RTX 阻断介导特定蛋白质-蛋白质相互作用的结构特征，例如受体 我们还没有明确定义抗体抑制 ACT 功能的表位和机制。 识别 ACT 上中和性和非中和性表位的一组高亲和力抗体提供了 这项研究的长期目标是了解结构。 博德特氏菌腺苷酸环化酶毒素使用的复杂细胞中毒过程的机制 使免疫细胞丧失能力的具体目标是提供相互作用的分子描述。 ACT 的 RTX 结构域及其受体以及中和和非中和抗体将提供。 对 ACT 功能的机制见解并定义重要的疫苗靶标，例如易受感染的表位 抗体介导的中和、受体结合位点和易位前构象。 信息对于实施合理的设计策略是必要的，这些策略旨在更有效地呈现 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和第一个 RTX-抗体和 RTX-受体结构。 评估 RTX 抗体逃逸和物种特异性的结构途径以及此类变化对 目前已有使用小鼠模型研究完整 ACT 蛋白的细胞毒性和细菌感染。 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 了解此类细菌毒素并深入了解关键的百日咳毒力因子。

项目成果

Outsmarting Pathogens with Antibody Engineering.

通过抗体工程智胜病原体。

• 发表时间: 2023-06-08
• 作者: Qerqez, Ahlam N;Silva, Rui P;Maynard, Jennifer A
• 通讯作者: Maynard, Jennifer A

Structural basis for antibody binding to adenylate cyclase toxin reveals RTX linkers as neutralization-sensitive epitopes.

抗体与腺苷酸环化酶毒素结合的结构基础揭示了 RTX 连接体是中和敏感表位。

• 发表时间: 2021
• 影响因子: 6.7
• 作者: Goldsmith, Jory A;DiVenere, Andrea M;Maynard, Jennifer A;McLellan, Jason S
• 通讯作者: McLellan, Jason S

Structural basis for non-canonical integrin engagement by Bordetella adenylate cyclase toxin.

博德特氏菌腺苷酸环化酶毒素与非典型整合素结合的结构基础。

• 发表时间: 2022-08-16
• 影响因子: 8.8
• 作者: Goldsmith, Jory A;DiVenere, Andrea M;Maynard, Jennifer A;McLellan, Jason S
• 通讯作者: McLellan, Jason S

Antibodies binding diverse pertactin epitopes protect mice from Bordetella pertussis infection.

结合多种百日咳杆菌粘附素表位的抗体可保护小鼠免受百日咳博德特氏菌感染。

• 发表时间: 2022-03
• 作者: Silva, Rui P;DiVenere, Andrea M;Amengor, Dzifa;Maynard, Jennifer A
• 通讯作者: Maynard, Jennifer A

Blockade of the Adenylate Cyclase Toxin Synergizes with Opsonizing Antibodies to Protect Mice against Bordetella pertussis.

腺苷酸环化酶毒素的阻断与调理抗体协同作用，保护小鼠免受百日咳博德特氏菌的侵害。

• 发表时间: 2022-08-30
• 影响因子: 6.4
• 作者: DiVenere, Andrea M;Amengor, Dzifa;Silva, Rui P;Goldsmith, Jory A;McLellan, Jason S;Maynard, Jennifer A
• 通讯作者: Maynard, Jennifer A