Cardiac microlesion formation during invasive pneumococcal disease

摘要： 四分之一的因社区获得性肺炎（CAP）住院的成年人经历了不良的心脏事件。 活动临床流行病学研究，以及在小鼠、非人灵长类动物和 人类尸检样本表明，肺炎链球菌（Spn），CAP的主要原因，可以侵入 从血液中排出并导致直接的心脏毒性。在心肌内Spn引起病灶区域 我们称之为微损伤，这些损伤破坏了收缩力。我们最近的一项突破 对SPN发病机制的理解是观察到肺炎球菌被心肌细胞摄取 Spn从内部杀死这些细胞此外，致孔毒素肺炎链球菌溶血素和链球菌 丙酮酸氧化酶（SpxB）衍生的H2 O2都是心脏毒性所必需的。在此，我们的目标是获得 理解在Spn摄取后立即在心肌细胞内发生的事件。 沿着这样的路线，来自体外和体内实验的结果，包括Spn-1的双物种RNA测序。 受感染的心脏，揭示了碳可用性，H2O2 产生、生物膜/心脏微损伤形成和肺炎球菌溶血素产生。因此，我们假设， Spn在心肌细胞内以及在心脏微损伤中遇到的葡萄糖限制，导致 代谢和基因表达的变化，增强细菌的心脏毒性。为了验证这个假设 肺炎链球菌溶血素和H2O2如何协同作用杀死心肌细胞，我们将： 目的1：确定环境葡萄糖，代谢和毒力是如何相互联系的。阐明 这些联系的基础、程度和后果，我们将：1）确定有目的地分流 丙酮酸代谢（通过突变）对乙酸盐、乳酸盐和/或甲酸盐的影响 在高和低葡萄糖条件下的基因表达; 2）确定Spn基因表达如何在高葡萄糖和低葡萄糖条件下变化。 心肌细胞摄取细菌后的纵向方式以及这与Spn变化的关系 代谢; 3）确定代谢相关基因对心肌细胞内Spn存活的重要性， 杀死心肌细胞和整个疾病过程。 目的2：确定细菌来源的H2O2与肺炎球菌溶血素一起如何杀死心肌细胞。SpxB 衍生的H2O2和肺炎球菌溶血素都是Spn杀死心肌细胞所必需的;单独使用每一种都是不够的。 为了确定原因，我们将：1）确定H2O2和肺炎球菌溶血素的不同产生如何共同调节 心肌细胞死亡的形式; 2）确定H2O2是否增强肺炎球菌溶血素的产生，其从 Spn或宿主细胞膜靶向;和，3）确定SpxB衍生的H2O2是否有助于离子 这是一种调节异常，以前与肺炎球菌溶血素诱导的坏死性凋亡有关。这一目标，在其 完成，将推进我们对Spn如何杀死宿主细胞的理解。

ABSTRACT: One-in-four adults hospitalized for community-acquired pneumonia (CAP) experience an adverse cardiac event. Clinical epidemiological studies, as well as those performed in mice, non-human primates, and with human autopsy samples indicate that Streptococcus pneumoniae (Spn), the leading cause of CAP, can invade the heart from the bloodstream and cause direct cardiotoxicity. Within the myocardium Spn cause focal areas of damage we have called microlesions and these disrupt contractility. One recent break-through in our understanding of Spn pathogenesis was the observation that pneumococci are taken up by cardiomyocytes and Spn kill these cells from within. What is more, the pore-forming toxin pneumolysin and Streptococcal pyruvate oxidase (SpxB) derived H2O2 were both requisite for cardiotoxicity. Herein, our goal is to gain an understanding of the events that take place within a cardiomyocyte immediately after Spn uptake. Along such lines, results from in vitro and in vivo experiments, including dual-species RNA sequencing of Spn- infected hearts, have revealed highly compelling connections between changes in carbon availability, H2O2 production, biofilm / cardiac microlesion formation, and pneumolysin production. Thus, we hypothesize that glucose restriction encountered by Spn within a cardiomyocyte, and again in cardiac microlesions, results in metabolic and gene expression changes that enhance bacterial cardiotoxicity. To test this hypothesis and learn how pneumolysin and H2O2 work together to kill cardiomyocytes we will: AIM 1: Determine how environmental glucose, metabolism, and virulence are interlinked. To elucidate the basis, extent, and consequences of these connections we will: 1) determine how purposeful shunting of pyruvate metabolism (by means of mutation) towards the production of acetate, lactate, and/or formate impacts gene expression under high and low glucose conditions; 2) identify how Spn gene expression changes in longitudinal fashion after bacterial uptake by a cardiomyocyte and how this is linked to changes in Spn metabolism; 3) determine the importance of metabolism-linked genes to Spn survival within a cardiomyocyte, killing of the cardiomyocyte, and the overall disease process. AIM 2: Determine how bacterial derived H2O2, together with pneumolysin, kills cardiomyocytes. SpxB derived H2O2 and pneumolysin are both required for Spn killing of cardiomyocytes; each alone is insufficient. To determine why we will: 1) determine how varying production of H2O2 and pneumolysin together modulate the form of cardiomyocyte death; 2) determine if H2O2 potentiates pneumolysin production, its release from Spn, or host cell membrane targeting; and, 3) determine if SpxB-derived H2O2 contributes to the ion dysregulation that has previously been implicated in pneumolysin-induced necroptosis. This aim, at its completion, will advance our understanding of how Spn kills host cells.