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

天然免疫系统通过启动高度保守的细胞死亡反应来提供对细菌病原体的保护，该反应在检测到病原体介导的扰动后促进病原体的清除。执行者Caspase-3和-7(Caspase-3/7)由启动子Caspase-8(Caspase-8)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，caspase-1(CASP1)介导的上睑下垂是高度炎症的，并与炎症小体激活、Gasdermin D(GSDMD)溶解的质膜孔形成和IL-1家族细胞因子的分泌有关。假结核耶尔森氏菌是耶尔森氏菌属中与小肠结肠炎耶尔森氏菌和鼠疫耶尔森氏菌并列的人类三大病原体之一。Yp利用一种保守的Ill分泌系统将毒力因子，即耶尔森氏菌外部蛋白(YOPs)注入宿主细胞胞浆，以促进感染。虽然YOP对细菌的毒力很重要，但它们也使宿主能够检测到细菌的存在并产生免疫反应。特别是，YopJ阻断了炎症基因的表达，触发了由Casp8介导的宿主细胞死亡途径，并涉及CASP1的激活，该激活不依赖于所有已知的CASP1激活调节因子。令人惊讶的是，批量分析表明，对YP感染做出反应的Casp8激活诱导了凋亡的Casp3/7和焦链CASP1的激活，这提出了一个问题，即单个细胞如何同时经历两种不同形式的细胞死亡。令人惊讶的是，我的新的初步显微镜分析显示，单个细胞显示出凋亡或下垂的形态特征，这表明单个细胞经历了不同的细胞命运选择，而大量基于群体的分析掩盖了这一选择。有趣的是，Casp3负调控GSDMD，提供了一种内置的负调控机制来限制下垂。有趣的是，不同YopJ感染细胞的YopJ注射水平和Casp3裂解水平不同，这表明YopJ注射的差异使细胞能够整合Casp3和-1的激活水平，从而调节细胞在凋亡和下垂之间的选择。这些数据和我的初步发现提出了一种假设，即Casp8介导了一种新的CASP1激活形式，单个细胞中依赖于Casp8的Casp3/CASP1激活的相对平衡决定了免疫信号阻断后的凋亡和焦链命运。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剖析感染yp期间单细胞命运的调节。在目标1中，我将定义在耶尔森氏菌感染过程中Caspase-1激活的机制，我们先前证明这种激活是通过一条独立于已知的炎症体成分的途径发生的。在目标2中，使用创新的基于单细胞的caspase记者，我将剖析YopJ的作用，并测试YopJ注射水平是否决定单个细胞中细胞死亡途径的选择。了解Yp感染过程中细胞死亡调控的分子基础将为抗细菌宿主防御提供新的见解，并有助于开发宿主导向的方法来对抗感染。

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.