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

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.