Molecular mechanisms underlying organ penetration in disseminated pneumococcal infection

肺炎链球菌（Spn）器官侵袭事件的分子学理解 尽管其后果是毁灭性的，但在菌血症期间仍然难以捉摸。我们仔细研究了 胶囊和血管内皮细胞（VEC）之间的相互作用，并提出了假设，胶囊 动力学是复杂的并且影响Spn跨VEC易位的所有阶段。这包括依恋， 受体介导的内吞作用（RME），对细胞内杀伤的抵抗，以及向基底外侧的运输 表面器官入侵。我们假设的测试将揭示Spn运输的细胞内机制 导致器官入侵我们的研究结果将影响基于基因型的疫苗设计的风险评估策略。 AIM 1.确定血清型如何影响血小板活化因子受体（PAFr）和层粘连蛋白受体 引发Spn的粘附/摄取。我们的初步结果表明，胶囊具有不同类型的拮抗作用 影响细菌与宿主细胞的相互作用，从而影响RME。我们将使用一个全面的小组， 低和高侵袭性疾病能力的同基因胶囊转换突变体，特别关注血清型 属于同一血清群，即，在它们的胶囊中具有单一的和确定的分子差异 结构，以确定特定的生化特征，如乙酰化，如何影响与两者的相互作用， 外周和大脑VEC。我们将量化Spn粘附素表面暴露的血清型依赖性 磷酸胆碱（PC）和胆碱结合蛋白A（CbpA），其对与其配体PAFr和 LR，分别对负责摄取的受体启动的信号传导，以及在总结中，对侵袭率。 AIM 2.确定血清型如何影响细胞内Spn的易位路径， 回收与移除。RME在非吞噬细胞中摄取的货物的降解是由 新的LC 3相关的内吞再循环途径“LANDO”。因此，LANDO可能是一个关键的途径， 通过肺炎球菌穿过血管内皮细胞使用低和高侵入性胶囊突变互换，我们将确定 PAFr和LR启动LANDO的能力。我们将随后确定胶囊的影响， 血清型对Spn跨野生型与LANDO缺陷型VEC运输的影响。我们将把目标定位为 Spn进入pafr-/-、LANDO缺陷和LR阻断小鼠的心脏和大脑，从而评估胶囊如何 在细菌移位与溶酶体中死亡的十字路口调节重要的内吞过程。 AIM 3.确定胶囊脱落如何影响VEC内的细菌命运。然而， 当血管内皮细胞摄取时，血流被包裹，我们的结果表明， 核内体在RME之后，并提供保护以免受低pH和氧化应激的影响， 细菌。我们将确定胶囊的状态（脱落或未脱落）和脱落的重要性， 当他们穿过血管内皮细胞时。我们将确定血清型是否影响由Spn引起的包膜脱落动力学， 胶囊类型和脱落如何影响内体酸化和溶酶体融合。

A molecular understanding of the events responsible for Streptococcus pneumoniae (Spn) organ invasion during bacteremia remains elusive although its consequences are devastating. We have closely examined the interaction between capsule and vascular endothelial cells (VEC) and developed the hypothesis that capsule dynamics are complex and impact all stages of Spn translocation across VEC. This includes attachment, receptor mediated endocytosis (RME), resistance to intracellular killing, and trafficking to the basolateral surface for organ invasion. Testing of our hypothesis will reveal the intracellular mechanics of Spn trafficking that drive organ invasion. Our results will impact risk assessment strategies for serotype-based vaccine design. AIM 1. Determine how serotype impacts platelet-activating factor receptor (PAFr) and laminin-receptor initiated adhesion/uptake of Spn. Our preliminary results indicate capsule has serotype-variable antagonistic effects on the bacterium's interaction with host cells that affects RME. We will use a comprehensive panel of isogenic capsule switch mutants of low and high invasive disease capability, specifically focusing on serotypes belonging to the same serogroup, i.e., having single and defined molecular differences in their capsule structure, to determine how specific biochemical features, such as acetylation, affect interactions with both peripheral and cerebral VEC. We will quantify serotype dependence of surface exposure of the Spn adhesins phosphorylcholine (PC) and choline binding protein A (CbpA), its effect on adhesion to their ligands PAFr and LR, respectively, on receptor-initiated signaling responsible for uptake, and in summate, on invasion rate. AIM 2. Determine how serotype influences the path taken by intracellular Spn for translocation vs recycling vs removal. The degradation of cargo taken up by RME in non-phagocytic cells is mediated by the novel LC3-associated endocytic recycling pathway `LANDO'. Thus, LANDO is likely a critical pathway co-opted by the pneumococcus to cross VEC. Using low and highly invasive capsule mutant swaps, we will determine the ability of PAFr and LR to initiate LANDO. We will subsequently determine the impact of capsule and serotype on Spn trafficking across wild type vs LANDO deficient VEC in vitro. We will characterize targeting of Spn into heart and brain of pafr-/-, LANDO deficient, and LR-blocked mice, thereby assessing how capsule modulates vital endocytic processes at the crossroads of bacterial translocation vs death in the lysosome. AIM 3. Determine how capsule shedding impacts bacterial fate within VEC. Whereas pneumococci in the bloodstream are encapsulated when taken up by VEC, our results suggest capsule is shed within the endosome following RME and provides protection from low pH and oxidative stress which are deployed to kill the bacterium. We will determine the status of capsule (shed or unshed) and the importance of shedding on Spn as they cross the VEC. We will determine if serotype impacts the kinetics of capsule shedding by Spn and how capsule type and shedding influences endosome acidification and lysosome fusion.