The lysosomal Rag-Ragulator complex licenses RIPK1– and caspase-8–mediated pyroptosis by Yersinia

INTRODUCTION An inflammatory innate immune response is a first line of host defense against invading pathogens. Inflammation recruits immune cells to the infection site and activates protective adaptive immune responses. Invasive bacteria have evolved multiple ways to interfere with host innate immune signaling to facilitate infection. The Yersinia effector protein YopJ suppresses proinflammatory cytokine production by inhibiting transforming growth factor–β–activated kinase 1 (TAK1) and nuclear factor κB (NF-κB) activation. To counteract this virulence factor, host cells initiate receptor-interacting serine-threonine protein kinase 1 (RIPK1)–dependent caspase-8–directed gasdermin D (GSDMD) cleavage and inflammatory cell death (pyroptosis) when TAK1 is inhibited. However, how the RIPK1–caspase-8–GSDMD axis is instructed during Yersinia infection is unclear. RATIONALE The best-studied mechanism by which pathogens stimulate inflammatory cell death involves triggering cytosolic sensors, called inflammasomes, which activate inflammatory caspases (1/4/5/11) to process proinflammatory cytokines and cause pyroptosis. Inflammatory caspase cleavage of GSDMD causes cell membrane pores that mediate both pyroptosis and proinflammatory cytokine secretion. An alternate pyroptotic pathway, mediated by activation of RIPK1 and caspase-8, is triggered when the YopJ virulence factor secreted during pathogenic Yersinia infection blocks TAK1 activation. To determine the molecular mechanisms underlying Yersinia activation of RIPK1–caspase-8–dependent pyroptosis, we performed a genome-wide CRISPR screen using Cas9-expressing immortalized mouse bone marrow–derived macrophages infected with a genome-wide library of single-guide RNA–encoding lentiviruses. The genomes of cells resistant to caspase-8– or caspase-11–dependent pyroptosis were sequenced to identify the knocked-out genes required for pyroptosis. RESULTS The screen identified multiple genes in the lysosomal membrane–anchored Folliculin (Flcn)–Folliculin-interacting protein 2 (Fnip2)–Rag-Ragulator complex as necessary for caspase-8– but not caspase-11–mediated pyroptosis. Deficiency of Rag-Ragulator complex genes rendered cells highly resistant to TAK1 inhibition–triggered pyroptosis, indicating a critical and unexpected role of the lysosomal membrane–tethered Rag-Ragulator supercomplex in RIPK1-dependent caspase-8–directed pyroptosis. In response to pathogenic Yersinia or its mimic [lipopolysaccharide (LPS) plus TAK1 inhibitor], a Fas-associated death domain (FADD)–RIPK1–caspase-8–containing complex was recruited to lysosomal membrane–tethered Rag-Ragulator. Activation of RIPK1 phosphorylation, caspase-8 activation, and pyroptosis depended on Rag guanosine triphosphatase (GTPase) activity and Rag-Ragulator lysosomal binding but was independent of the mechanistic target of rapamycin complex 1 (mTORC1), a well-known Rag-Ragulator–regulated complex. By contrast, Rag-Ragulator did not regulate canonical or noncanonical inflammasome-triggered pyroptosis. CONCLUSION Our study revealed an instructive role of metabolic signaling in directing TAK1 inhibition–induced pyroptosis during a pathogenic bacterial infection. Rag-Ragulator is a well-known critical regulator of cellular responses to changes in nutrient availability and metabolism. Here, Rag-Ragulator served as a platform for activating a FADD–RIPK1–caspase-8 complex formed in response to Toll-like receptor (TLR) or tumor necrosis factor receptor (TNFR) ligation. Rag GTPase activity was critical for triggering the pathway. The role found here for Rag-Ragulator in pyroptosis expands its known roles in metabolic regulation to include regulation of the response to pathogenic infection. Rag-Ragulator monitors both metabolism and infection to serve as a central hub for helping to decide whether available nutrients are adequate for cell proliferation and if an infected cell should die and send out inflammatory danger signals. Future studies can further explore the conditions that stimulate caspase-8–mediated pyroptosis and provide more mechanistic details of how it is regulated, as well as investigate whether manipulating this pathway could have therapeutic benefit.