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.

项目总结