Defining mechanisms of heterogeneity in cell fate decisions during Yersinia pseudotuberculosis infection

先天免疫系统通过启动高度保守的细胞死亡反应，在检测到病原体介导的扰动后促进病原体清除，从而提供对细菌病原体的保护。执行子caspase- 3和-7 （Casp3/7）被启动子caspase-8 （Casp8）激活，诱导细胞凋亡，这是一种免疫沉默的细胞死亡形式。相反，由caspase-1 （Casp1）介导的焦亡是高度炎症性的，与炎性小体激活、Gasdermin D （GSDMD）裂解质膜孔形成以及IL-1家族细胞因子的分泌有关。假结核耶尔森氏菌（Yp）是耶尔森氏菌属的三种人类病原体之一，与引起鼠疫的小肠结肠炎耶尔森氏菌和鼠疫耶尔森氏菌并列。Yp利用一个保守的Ill型分泌系统将毒力因子，即耶尔森氏菌外蛋白（Yops）注入宿主细胞质，促进感染。虽然Yops对细菌的毒力很重要，但它们也能使宿主检测到细菌的存在并产生免疫反应。特别是，YopJ阻断炎症基因表达，触发由Casp8介导的宿主细胞死亡途径，并涉及独立于所有已知Casp1激活调节因子的Casp1的激活。令人惊讶的是，大量实验表明，响应Yp感染的Casp8激活诱导凋亡Casp3/7和热亡Casp1的激活，这就提出了单个细胞如何同时经历两种不同形式的细胞死亡的问题。令人惊讶的是，我新的初步显微镜分析显示，单个细胞表现出凋亡或焦亡的形态学特征，这表明单个细胞经历了不同的细胞命运选择，这些选择被基于大量群体的分析所掩盖。有趣的是，Casp3负调控GSDMD，提供了一个内在的负调控机制来限制焦亡。有趣的是，单个yp感染细胞的YopJ注射水平和Casp3切割水平不同，这表明YopJ注射的可变性使细胞能够整合Casp3和-1激活水平，从而调节细胞凋亡和焦亡之间的选择。这些数据和我的初步发现提出了Casp8介导一种新的Casp1激活形式的假设，并且在免疫信号阻断的情况下，单个细胞中Casp8依赖性Casp3/Casp1激活的相对平衡决定了凋亡和焦亡的命运。在这项研究中，我的目标是使用强大的单细胞为基础的方法来解剖单细胞命运的调节感染Yp。在Aim 1中，我将定义在耶尔森菌感染期间Caspase-1激活的机制，我们之前已经证明它是通过一个独立于已知炎症小体成分的途径发生的。在Aim 2中，使用创新的单细胞caspase报告者，我将剖析YopJ的作用，并测试YopJ注射水平是否决定单个细胞的细胞死亡途径选择。了解Yp感染期间细胞死亡调控的分子基础将为抗菌宿主防御提供新的见解，并促进宿主定向方法的发展，以对抗感染。

The innate immune system provides protection against bacterial pathogens by initiating a highly conserved cell death response that promotes pathogen clearance following the detection of pathogen-mediated perturbations. The executioner caspases-3 and -7 (Casp3/7) are activated by the initiator caspase-8 (Casp8) to induce apoptosis, an immunologically silent form of cell death. Conversely, pyroptosis, mediated by caspase-1 (Casp1), is highly inflammatory, and is associated with inflammasome activation, lytic plasma membrane pore formation by Gasdermin D (GSDMD), and secretion of IL-1 family cytokines. Yersinia pseudotuberculosis (Yp) is one of the three human pathogens in the Yersinia genus along with Yersinia enterocolitica and Yersinia pestis, the causative agent of plague. Yp utilizes a conserved type Ill secretion system to inject virulence factors, known as Yersinia outer proteins (Yops) into the host cell cytosol, to facilitate infection. While Yops are important for bacterial virulence, they also enable the host to detect the presence of the bacteria and generate an immune response. In particular, YopJ blocks inflammatory gene expression, triggering a pathway of host cell death mediated by Casp8 and involving activation of Casp1 that is independent of all known Casp1 activation regulators. Surprisingly, bulk assays show that Casp8 activation in response to Yp infection induces activation of both apoptotic Casp3/7 and pyroptotic Casp1, posing the question of how an individual cell might be simultaneously undergo two distinct forms of cell death. Surprisingly, my new preliminary microscopy analysis reveals that individual cells display morphological features of either apoptosis or pyroptosis, suggesting that individual cells undergo distinct cell fate choices masked by bulk population-based analyses. Intriguingly, Casp3 negatively regulates GSDMD, providing a built-in negative regulatory mechanism to limit pyroptosis. Intriguingly, individual Yp-infected cells vary in their levels of YopJ injection and Casp3 cleavage, suggesting that variability in YopJ injection enables cells to integrate levels of Casp3 and -1 activation, thereby regulating the choice between apoptosis and pyroptosis. These data and my preliminary findings provoke the hypothesis that Casp8 mediates a novel form of Casp1 activation, and the relative balance of Casp8-dependent Casp3/Casp1 activation in individual cells determines apoptotic and pyroptotic fates in response to immune signaling blockade. In this fellowship, I aim to use powerful single-cell-based approaches to dissect the regulation of single-cell fates during infection with Yp. In Aim 1, I will define the mechanism of Caspase-1 activation during Yersinia infection, which we previously demonstrated occurs through a pathway independent of known inflammasome components. In Aim 2, using innovative single cell-based caspase reporters, I will dissect the role of YopJ and test whether levels of YopJ injection determine the cell death pathway choice in individual cells. Understanding the molecular basis for the regulation of cell death during Yp infection will provide novel insights into anti-bacterial host defense and facilitate the development of host-directed approaches to combat infections.