PspA binds necroptotic cells to cause disease and transmit

甲型流感病毒（IAV）显著增强肺上皮细胞对肺炎球菌溶血素介导的 坏死性凋亡简言之，肺炎链球菌溶血素是由肺炎链球菌（Spn）产生的成孔毒素， 而坏死性凋亡是导致细胞溶解的程序性细胞死亡的不依赖半胱天冬酶的形式。在此， 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后果：Spn结合到 坏死性呼吸道上皮细胞通过肺炎球菌表面蛋白A（PspA）。简而言之，我们的 初步结果表明，PspA与垂死细胞上的宿主来源的（h）GAPDH结合， 有助于IAV/Spn疾病的严重程度。此外，Spn/脱落的上皮细胞聚集体形成于 鼻咽可能促进Spn向未感染宿主的传播。在此，我们测试假设，在 IAV/Spn重叠感染时，发生高水平的上皮细胞坏死性凋亡，促进PspA介导的细胞凋亡。 与细胞结合。这一特性直接增强了Spn的生长，促进了Spn的传播。 目的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，粘附增强，并且由 PspA与死亡细胞表面上发现的hGAPDH结合。我们将确定PspA负责的领域 对于hGAPDH结合，该结构域在Spn的测序菌株中的保守程度，以及 代表性PspA变体与hGAPDH的结合。我们将创建并测试PspA中的同基因突变体的能力， hGAPDH结合基序结合垂死的LEC。我们将鉴定与PspA结合的hGAPDH区域。 目的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重叠感染期间发生的增强的疾病严重程度所必需的。我们将确定是否 hGAPDH结合改变PspA的典型作用，其抑制乳铁蛋白介导的杀伤。我们将 确定PspA结合如何影响气道内Spn的定位以及这如何受到以下因素的影响： 与IAV的共感染、肺炎球菌溶血素的中和或坏死性凋亡的阻断。我们将确定PspA是否- Spn介导的与垂死LEC的结合促进了它们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。 目的3：确定PspA介导的粘附对定植和传播的要求。SPN 在定殖过程中与垂死的粘膜上皮细胞结合，它们一起随鼻分泌物排出。 脱落的Spn/宿主细胞聚集体具有感染性，并被认为促进Spn在污染物上的存活。我们将 确定PspA对形成Spn/宿主细胞聚集体的需要，此外，肺炎球菌溶血素， IAV重叠感染和抑制坏死凋亡影响分泌物中它们的数量。康贝特人将以 在一些实施方案中，PspA hGAPDH是PspA hGAPDH结合以传递至幼稚宿主的需要。我们将确定是否有针对 PspA和/或肺炎球菌溶血素降低传播率并缩短定殖长度。

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.