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溶气素的敏感性 坏死性下垂。简而言之，溶气素是由肺炎链球菌(Spn)产生的致孔毒素， 而坏死性下垂是一种不依赖于caspase的程序性细胞死亡形式，导致细胞溶解。在这里， 我们将确定一项新的关键观察的分子基础和全部生物学后果：SPN与 肺炎球菌表面蛋白A(PSPA)诱导的坏死性呼吸道上皮细胞。简单地说，我们的 初步结果表明，PSPA与死亡细胞上宿主衍生的(H)GAPDH结合，并直接具有这一特性 有助于IAV/Spn疾病的严重程度。此外，Spn/塌陷的上皮细胞聚集体形成在 鼻咽可能会将Spn传播给幼稚的宿主。在这里，我们检验了假设，在 IAV/Spn重叠感染促进PSPA介导的高水平上皮细胞坏死性下垂 绑定到细胞。这一特性直接促进了Spn的生长并促进了Spn的传播。 目的1：确定PSPA介导的与坏死性肺上皮细胞黏附的分子基础 (LEC)。SPN与LEC的黏附是依赖PSPA的，当细胞经历坏死性下垂时会增强，并通过 在死亡细胞表面发现PSPA与hGAPDH结合。我们将确定PSPA负责的域 对于hGAPDH结合，这个结构域在Spn的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中创建并测试等基因突变体的能力 HGAPDH结合基序以结合濒临死亡的LEC。我们将确定hGAPDH与PSPA结合的区域。 目的2：确定PSPA介导的黏附对IAV/Spn肺炎严重程度的生物学影响。 在IAV重叠感染期间，需要PSPA才能增加疾病的严重性。我们将确定是否 HGAPDH结合改变了PSPA的典型作用，即抑制乳铁素介导的杀伤。我们会 确定PSPA结合如何影响Spn在呼吸道内的定位，以及 合并IAV感染、中和肺炎溶血素或阻断坏死性下垂。我们将确定PSPA是否- Spn与垂死的LEC的介导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总体疾病进展。 目的3：确定PSPA介导的黏附对定植和传播的要求。SPN 在定植过程中与死亡的粘膜上皮细胞结合，并在鼻分泌物中一起排出。 脱落的Spn/宿主细胞聚合体具有感染性，被认为可以促进Spn在合胞体上的存活。我们会 确定PSPA对Spn/宿主细胞聚集体形成的需求，此外，肺炎溶血素， IAV的重叠感染和坏死性下垂的抑制会影响分泌物中它们的数量。我们将确定 需要PSPA hGAPDH绑定才能传输到原始主机。我们将确定是否有抗体 PSPA和/或溶气素可降低传播率并缩短定植时间。

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.