Browsing by Subject "pyroptosis"

Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. Although pyroptosis and apoptosis have distinct signaling pathways, when a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we also now engineer S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. In both engineered strains, apoptosis occurred somewhat slower than pyroptosis. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche, but failed to clear the bacteria from the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. In order to clear an infection, distinct cell types may have specific tasks that they must complete before they die. In some cells, either apoptotic or pyroptotic signaling may initiate the same tasks, whereas in other cell types these modes of cell death may lead to different tasks that may not be identical in defense against infection. We recently suggested that such diverse tasks can be considered as different cellular “bucket lists” to be accomplished before a cell dies. As demonstrated here, engineering pathogens is a useful method for discovering new details of microbial pathogenesis and host defense. However, engineering can result in off-target effects. We engineer S. Typhimurium to overexpress the secretion signal of the type 3 secretion system effector SspH1 fused with domains of other proteins as cargo. Such engineering had no virulence cost to the bacteria for the first 48 hours post infection in mice. However, after 48 hours the engineered bacteria manifest an attenuation that correlates with the quantity of the SspH1 translocation signal expressed. In IFNg-deficient mice this attenuation was weakened. Conversely, the attenuation was accelerated in the context of a pre-existing infection. We speculate that inflammatory signals change aspects of the target cell’s physiology that make host cells less permissive to S. Typhimurium infection. This increased degree of difficulty requires the bacteria to utilize its T3SS at peak efficiency, which can be disrupted by engineered effectors.

Guanylate binding proteins (Gbps) are a family of large GTPases that are highly stimulated by IFNγ and confer resistance to various viral, protozoan, and bacterial pathogens. Following infections of intracellular pathogens, multiple Gbps can localize to pathogen vacuoles and promote the vesiculation and destruction of these structures. While Gbps have also been implicated in pathways independent of vacuolar disruption, their roles in these processes have been less characterized. In this dissertation, I focus on the mechanism of Gbps downstream of vacuolar disruption in order to further elucidate the role of these proteins during immune responses.

Due to the IFNγ stimulation of caspase-11 pyroptosis, I first addressed the ability of Gbps to promote the non-canonical caspase-11 dependent pathway of pyroptosis. I found that Gbpchr3-/- cells had reduced cell death in response to the vacuolar pathogen, L. pneumophila, and various LPS ligands. Using YFP-Gal3 as a marker for damaged membranes, I showed that there were equivalent levels of damaged pathogen vacuoles between WT and Gbpchr3-/- cells suggesting these proteins promoted pyroptosis independently of vacuolar disruption. Instead, it appears that Gbps modulate the activation of caspase-11 following LPS release into the cytosol.

The recruitment of Gbps is mediated by multiple host proteins including the Immunity Related GTPases and the autophagy conjugation system. I found in the second study that at least one Gbp, Gbp2, was also recruited to damaged vacuoles through the aid of Galectin-3, a β-galactoside binding protein, as well as the autophagy adaptor protein, p62. As all three proteins were also recruited to sterile damaged vesicles created by hypotonic shock, calcium phosphate precipitates, and lysosomal damage, it suggests Gbps are recruited through a universal mechanism which is independent of PAMP recognition. Interactions between p62, Gbp2, and Gal3 present a model whereby p62 facilitates the recruitment of Gbp2 to damaged membranes through interactions with Galectin-3. Their localization to these sites may subsequently facilitate autophagic degradation of membranes or promote the recruitment of pyroptotic complexes to modulate immune functions although this remains to be elucidated.

This dissertation examines the less characterized roles of Gbps downstream of vacuolar disruption. By uncovering these alternative pathways, this work provides a foundation to study the variations within the Gbp family and allows for the field to further understand the mechanisms by which they promote cellular immune responses.