PspA binds necroptotic cells to cause disease and transmit

PspA 结合坏死性凋亡细胞引起疾病并传播

• 批准号: 10470379
• 负责人: Carlos J Orihuela
• 金额: $ 40.77万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-24 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10470379
• 关键词: Adhesions; Affinity; Animals; Antibodies; Apoptosis; Bacteria; Bacterial Adhesins; Binding; Binding Proteins; Biological; Caspase; Cell Death; Cells; Complement; Cytolysis; Data; Desiccation; Development; Disease; Disease Progression; Epithelial Cells; Family; Immune; Inflammation; Influenza; Influenza A virus; Learning; Length; Lung; Mediating; Membrane; Membrane Proteins; Modeling; Molecular; Morbidity - disease rate; Mucous Membrane; Nasopharynx; Nose; Nutrient; Persons; Phosphatidylserines; Pilot Projects; Pneumococcal Infections; Pneumococcal Pneumonia; Pneumonia; Predisposition; Property; Protein Binding Domain; Proteins; Publishing; Research; Resistance; Role; Secondary Prevention; Severities; Severity of illness; Streptococcus pneumoniae; Streptococcus pneumoniae plY protein; Surface; Tertiary Protein Structure; Testing; Toxin; Variant; Virulence; Virus; Virus Diseases; Work; airway epithelium; co-infection; experience; influenzavirus; mortality; mouse model; mutant; neonatal mice; novel; pathogen; pneumococcal surface protein A; superinfection; synergism; transmission process

Influenza A virus (IAV) profoundly enhances the susceptibility of lung epithelial cells for pneumolysin-mediated necroptosis. Briefly, pneumolysin is the pore-forming toxin produced by Streptococcus pneumoniae (Spn), whereas necroptosis is a caspase-independent form of programmed cell death that results in cell lysis. Herein, we will determine the molecular basis and full biological consequence of a new key observation: Spn binds to necroptotic respiratory epithelial cells via Pneumococcal surface protein A (PspA). Briefly, our preliminary results show that PspA binds to host-derived (h)GAPDH on dying cells and this property directly contributes to IAV/Spn disease severity. Furthermore, Spn/sloughed epithelial cell aggregates formed in the nasopharynx likely promote Spn transmission to a naive host. Herein we test the hypothesis that during IAV/Spn superinfection a high level of epithelial cell necroptosis occurs that promotes PspA-mediated binding to cells. This property directly enhances Spn outgrowth and promotes Spn transmission. AIM 1: Determine the molecular basis for PspA-mediated adhesion to necroptotic lung epithelial cells (LEC). Spn adhesion to LEC is PspA-dependent, enhanced when cells undergo necroptosis, and mediated by PspA binding to hGAPDH found on the surface of dying cells. We will identify the domain of PspA responsible for hGAPDH binding, how conserved this domain is across sequenced strains of Spn, and the affinity of representative PspA variants to hGAPDH. We will create and test the ability of isogenic mutants in the PspA hGAPDH binding motif to bind dying LEC. We will identify the region of hGAPDH that is bound by PspA. AIM 2: Determine the biological impact of PspA-mediated adhesion on IAV/Spn pneumonia severity. PspA is required for the enhanced disease severity that occurs during IAV superinfection. We will determine if hGAPDH binding alters the canonical role of PspA, which is inhibiting lactoferricin-mediated killing. We will determine how PspA-binding influences the localization of Spn within the airway and how this is impacted by co-infection with IAV, neutralization of pneumolysin, or blocking of necroptosis. We will determine if PspA- mediated binding of Spn to dying LEC promotes their outgrowth in otherwise nutrient restricted conditions. We will determine how antibody against the hGAPDH-binding motif of PspA alters overall disease progression. AIM 3: Determine the requirement of PspA mediated adhesion to colonization and transmission. Spn binds to dying mucosal epithelial cells during colonization and they are together expelled in nasal secretions. Sloughed Spn/host cell aggregates are infectious and thought to promote Spn survival on fomites. We will determine the requirement for PspA on the formation of Spn/host cell aggregates, moreover, how pneumolysin, IAV superinfection, and necroptosis inhibition influences their number in secretions. We will determine the requirement of PspA hGAPDH binding for transmission to a naive host. We will determine if antibody against PspA and/or pneumolysin reduces transmission rates and shortens the length of colonization.

流感病毒（IAV）深刻增强了肺上皮细胞对肺炎介导的易感性 坏死性。简而言之，肺炎是由肺炎链球菌（SPN）， 而坏死性是一种无依赖性的编程细胞死亡形式，导致细胞裂解。在此处， 我们将确定一个新的关键观察结果的分子基础和完整的生物学结果：SPN与 通过肺炎球菌表面蛋白A（PSPA）的坏死性呼吸性上皮细胞。简而言之，我们的 初步结果表明，PSPA在垂死的细胞上与宿主衍生的（H）GAPDH结合，该特性直接结合 导致IAV/SPN疾病严重程度。此外，在形成 鼻咽可能会促进SPN向天真的宿主传播。在此，我们检验了以下假设 IAV/SPN超级感染高水平的上皮细胞坏死性发生，促进PSPA介导的 与细胞结合。该特性直接增强了SPN的生长并促进SPN传播。 目标1：确定Pspa介导的对坏死性肺上皮细胞的粘附的分子基础 （LEC）。 SPN对LEC的粘附是PSPA依赖性的，当细胞发生坏死时增强，并由 PSPA与垂死细胞表面发现的HGAPDH结合。我们将确定PSPA负责的领域 对于HGAPDH结合，该域在SPN的测序菌株中如何保守，以及 HGAPDH的代表性PSPA变体。我们将创建并测试PSPA中的等生突变体的能力 HGAPDH结合基序以结合垂死的LEC。我们将确定受PSPA约束的HGAPDH区域。 目标2：确定PSPA介导的粘附对IAV/SPN肺炎严重程度的生物学影响。 PSPA是在IAV近距离感染期间发生的增强疾病严重程度所必需的。我们将确定是否 HGAPDH结合改变了PSPA的规范作用，PSPA抑制了乳霉素介导的杀戮。我们将 确定PSPA结合如何影响SPN在气道中的定位以及如何影响。 与IAV共同感染，肺炎的中和或坏死作用。我们将确定PSPA-是否 SPN与死亡LEC的介导的结合促进了其在其他营养限制条件下的生长。我们 将确定针对PSPA的HGAPDH结合基序的抗体如何改变整体疾病进展。 AIM 3：确定PSPA介导的对定殖和传播的粘附的需求。 SPN 在定殖过程中与垂死的粘膜上皮细胞结合，并在鼻分泌物中共同排出。 Sloughed SPN/宿主细胞聚集体具有感染性，并被认为可以促进Fomites上的SPN存活。我们将 确定PSPA对SPN/宿主细胞聚集体形成的需求，此外 IAV超染色和坏死抑制会影响分泌物的数量。我们将确定 PSPA HGAPDH结合以传播到幼稚的宿主的要求。我们将确定抗体是否针对 Pspa和/或肺炎降低了传输速率并缩短定殖长度。