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

基本信息

批准号：
10269932
负责人：
Carlos J Orihuela
金额：
$ 41.18万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
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/10269932
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 Structure of respiratory epithelium Surface Tertiary Protein Structure Testing Toxin Variant Virulence Virus Virus Diseases Work co-infection experience influenzavirus mortality mouse model mutant neonatal mice novel pathogen pneumococcal surface protein A superinfection synergism transmission process

项目摘要

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.

甲型流感病毒（IAV）显着增强肺上皮细胞对肺炎球菌溶血素介导的敏感性坏死性凋亡。简而言之，肺炎球菌溶血素是由肺炎链球菌 (Spn) 产生的成孔毒素，而坏死性凋亡是一种不依赖半胱天冬酶的程序性细胞死亡形式，会导致细胞裂解。在此处，我们将确定一个新的关键观察的分子基础和完整的生物学结果：Spn 结合通过肺炎球菌表面蛋白 A (PspA) 坏死性凋亡呼吸道上皮细胞。简而言之，我们的初步结果表明，PspA 与垂死细胞上的宿主衍生的 (h)GAPDH 结合，并且这种特性直接结合影响 IAV/Spn 疾病的严重程度。此外，Spn/脱落的上皮细胞聚集体在鼻咽可能促进 Spn 传播至幼稚宿主。在此我们检验以下假设： IAV/Spn 重复感染会发生高水平的上皮细胞坏死性凋亡，从而促进 PspA 介导的与细胞结合。该特性直接增强 Spn 生长并促进 Spn 传播。目标 1：确定 PspA 介导的坏死性肺上皮细胞粘附的分子基础（LEC）。 Spn 与 LEC 的粘附是 PspA 依赖性的，当细胞发生坏死性凋亡时会增强，并由 PspA 与垂死细胞表面的 hGAPDH 结合。我们将确定 PspA 负责的域对于 hGAPDH 结合，该结构域在 Spn 测序菌株中的保守性如何，以及 hGAPDH 的代表性 PspA 变体。我们将在 PspA 中创建并测试同基因突变体的能力 hGAPDH 结合基序结合垂死的 LEC。我们将确定与 PspA 结合的 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 IAV 重复感染期间疾病严重程度增加所必需的。我们将确定是否 hGAPDH 结合改变了 PspA 的典型作用，即抑制乳铁蛋白介导的杀伤作用。我们将确定 PspA 结合如何影响 Spn 在气道内的定位以及这是如何受到影响的与 IAV 合并感染、中和肺炎球菌溶血素或阻断坏死性凋亡。我们将确定是否 PspA- Spn 介导的与濒临死亡的 LEC 的结合促进了它们在营养受限的条件下的生长。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。目标 3：确定 PspA 介导的粘附对定植和传播的要求。斯普恩在定植过程中与垂死的粘膜上皮细胞结合，并一起通过鼻分泌物排出。脱落的 Spn/宿主细胞聚集体具有传染性，并且被认为可以促进 Spn 在污染物上的存活。我们将确定 PspA 对 Spn/宿主细胞聚集体形成的要求，此外，肺炎球菌溶血素如何， IAV 重复感染和坏死性凋亡抑制会影响其分泌物数量。我们将确定 PspA hGAPDH 结合传输至初始宿主的要求。我们将确定抗体是否针对 PspA 和/或肺炎球菌溶血素可降低传播率并缩短定植时间。