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应答来提供针对细菌病原体的保护，所述细胞死亡应答在检测到病原体介导的扰动后促进病原体清除。执行者半胱天冬酶-3和-7（Casp 3/7）被起始者半胱天冬酶-8（Casp 8）激活以诱导细胞凋亡，这是一种免疫沉默形式的细胞死亡。相反，由半胱天冬酶-1（Casp 1）介导的细胞凋亡是高度炎症性的，并且与炎性小体活化、Gasdermin D（GSDMD）引起的溶解性质膜孔形成和IL-1家族细胞因子的分泌相关。假结核耶尔森氏菌（Yersinia pseudotuberculosis，Yp）是与小肠结肠炎耶尔森氏菌（Yersinia enterocolitica）和鼠疫耶尔森氏菌（Yersinia pestis）一起沿着的三种人类病原体，是鼠疫的病原体。Yp利用保守的III型分泌系统将称为耶尔森氏菌外蛋白（Yops）的毒力因子注射到宿主细胞胞质溶胶中，以促进感染。虽然Yops对细菌毒力很重要，但它们也使宿主能够检测细菌的存在并产生免疫应答。特别是，YopJ阻断炎性基因表达，触发由Casp 8介导的宿主细胞死亡途径，并涉及独立于所有已知Casp 1激活调节因子的Casp 1激活。令人惊讶的是，批量测定显示响应于Yp感染的Casp 8活化诱导凋亡Casp 3/7和焦变性Casp 1两者的活化，提出了单个细胞如何可能同时经历两种不同形式的细胞死亡的问题。令人惊讶的是，我的新的初步显微镜分析显示，单个细胞显示凋亡或焦亡的形态特征，这表明单个细胞经历了不同的细胞命运选择，这些选择被大量基于群体的分析所掩盖。有趣的是，Casp 3负调控GSDMD，提供了一个内置的负调控机制，以限制焦亡。有趣的是，单个Yp感染的细胞在其YopJ注射和Casp 3切割的水平上不同，这表明YopJ注射的可变性使细胞能够整合Casp 3和-1活化的水平，从而调节凋亡和焦亡之间的选择。这些数据和我的初步研究结果激发了这样的假设，即Casp 8介导了一种新形式的Casp 1激活，并且在单个细胞中Casp 8依赖性Casp 3/Casp 1激活的相对平衡决定了响应于免疫信号传导阻断的凋亡和pyroptotic命运。在这个奖学金，我的目标是使用强大的单细胞为基础的方法来剖析单细胞的命运在感染Yp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，我们先前已经证明这是通过一种独立于已知炎性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半胱天冬酶报告基因，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感染期间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.