Downstream of gasdermin D cleavage, a Ragulator-Rag-mTORC1 pathway promotes pore formation and pyroptosis

Background:

Pyroptosis is an inflammatory form of cell death characterized by membrane disruption leading to lytic cell death and release of inflammatory molecules, including IL-1 family cytokines. This membrane disruption is the result of pores formed by the protein gasdermin D (GSDMD), which is cleaved by inflammatory caspases following infection or inflammasome activation. The N-terminal fragment of cleaved GSDMD (NT-GSDMD) localizes to the membrane and oligomerizes to form pores which permit the transit of relatively small molecules (including IL-1 family cytokines) and eventually lead to full lytic cell death. While the upstream events leading to GSDMD cleavage have been more deeply studied, the extent of regulation of pore formation remains unclear. In this preprint, Evavold et al present a forward genetic screen and identify a novel regulatory mechanism of GSDMD pore formation.

 

Key findings:

• Development of a system to study GSDMD pore formation
  A key feature of this preprint is the development of a biological system that allows the specific study of the pore formation stage of pyroptosis. In order to do this, the authors established a cell line system expressing a doxycycline-inducible NT-GSDMD-BFP fusion protein. Induction of NT-GSDMD-BFP expression is sufficient to cause pore formation independent of the upstream pyroptosis machinery (e.g. inflammasome activation), which is easily monitored by uptake of the dye propidium iodide (PI). BFP fusion allows for tracking of NT-GSDMD expression and localization, which is critical to deconvolute the steps of membrane localization and subsequent pore formation. These cell lines were developed in a background of Cas9 transgenic expression, allowing for CRISPR knockout of regulators of the pore formation process. A schematic overview of this system is presented in Figure 1.
• Ragulator/mTORC1 regulate GSDMD pore formation

The authors used their new system to perform a genome-wide CRISPR screen to identify regulators of pore formation. By examining guide sequences depleted from the highest BFP+/PI- cells, the authors identified genes specifically required for pore formation downstream of GSDMD cleavage. The top hits included multiple components of the Rag-Ragulator complex, which is responsible for recruiting mTORC1 to lysosomes for amino acid sensing. Follow-up experiments using targeted knockouts revealed that Ragulator and mTORC1, but not mTORC2, are required for proper pore formation and pyroptosis, but not for membrane localization of NT-GSDMD., In natural pyroptotic settings, such as Salmonella infection, mTORC1-deficient cells were resistant to pyroptosis, even while they showed proper GSDMD cleavage, confirming the role of Ragulator/mTORC1 in regulating GSDMD pore formation.

 

 

Figure 1: Schematic of system to study GSDMD pore formation

An immortalized BMDM cell line expressing Cas9 and a doxycycline-inducible NT-GSDMD-BFP fusion protein was generated. After induction with doxycycline, several outcomes are possible. Failure to properly express the NT-GSDMD fusion protein will result in a BFP and PI-negative cell (propidium iodide, taken up through pores in the membrane). Expression of the NT-GSDMD-BFP fusion protein without pore formation will result in a BFP single-positive cell. Proper pore formation as in normal pyroptosis will result in PI uptake and a BFP/PI double-positive cell. Figure prepared with biorender.

 

Importance:

This study decouples GSDMD cleavage and membrane recruitment from pore formation and downstream cytokine release and cell death. This adds a new layer of regulatory potential to the process of pyroptosis, and promises to reshape how we understand this process of regulated cell death. The potential integration of cellular metabolic state or activity with GSDMD pore formation, through Ragulator/mTORC1, also presents an intriguing new area of study in the field of cell death.

 

Moving forward / Questions for authors:

• The mechanistic process by which Ragulator/mTORC1 regulates pore assembly will be exciting to unravel in future studies. One straightforward question is to ask whether Ragulator/mTORC1 activity is required *during* the process of pyroptosis. Pharmacologic inhibitors could help answer the question of whether Ragulator/mTORC1 activity is required during/after membrane recruitment of NT-GSDMD to facilitate pore formation, or if their deficiency “primes” the cell in advance to be susceptible to pore formation. Either result would be interesting, suggesting either that certain metabolic activities are a key part of active pyroptosis or that certain cells are particularly susceptible/resistant to pyroptosis based on their metabolic state prior to infection.
• What do the depleted sgRNAs from the screen (i.e. the opposite end of the screen from the Ragulator components, shown in Fig 3A) represent? Does the screen distinguish between potential inability to express/maintain the NT-GSDMD fragment (BFP- cells) and increased cell death (BFP+/PI+ cells)?

 

doi: https://doi.org/10.1242/prelights.26212

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