Abstract
Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization.
Highlights
The innate immune system maintains a delicate balance between pathogen defense and inflammatory disease
To circumvent previously identified issues including a lack of pyroptotic cell death with N-terminal tagging of the p30 fragment of GSDMD [11], a FLAG tag was added following residue 248 in this flexible linker region between the end of the fragment required for pyroptosis (AA 1–244) and before the caspase-1/11 cleavage site located N-terminal to residue 277 (Fig. 1C)
Neither WT-GSDMD nor FLAG-GSDMD allowed propidium iodide (PI) uptake in response to LPS alone (Fig. 1F) but demonstrated similar kinetics in pyroptotic pore formation as measured by PI uptake when LPS-primed and stimulated with nigericin or ATP, an additional activator of the NLRP3 inflammasome that acts in a manner distinct from nigericin (Fig. 1, G and H)
Summary
To study GSDMD in macrophages, Gsdmd was genetically deleted in immortalized bone marrow– derived macrophages (iBMDM) through the use of CRISPR-Cas as previously described (Fig. 1A) [16, 18]. The crystal structure of gasdermin A3 further demonstrated that these three residues are likely key for binding to a phenylalaninetryptophan sequence in the p30 fragment [14] If these novel fluorescent GSDMD tools recapitulate wild-type function, mutation of these residues should release autoinhibition and promote aggregation of the fluorescently tagged GSDMD. Consistent with the kinetics of pyroptotic cell death as demonstrated by PI uptake assays, membrane blebbing began 15 min after the addition of nigericin At this time point, mNeon-GSDMD began to redistribute from a diffusely cytoplasmic localization to form multiple aggregates in the cell that appear to localize at the plasma membrane, as well as various internal membranes
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