Research Article

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

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Science  25 Jun 2021:
Vol. 372, Issue 6549, eabg0269
DOI: 10.1126/science.abg0269

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Cell death limits pathogens

During infection, Yersinia inhibition of the protein kinase TAK1 triggers cleavage of the pore-forming protein gasdermin D (GSDMD), which leads to a kind of inflammatory cell death called pyroptosis. In a genome-wide screen, Zheng et al. identified a lysosome-tethered regulatory supercomplex as being a critical driver of Yersinia-induced pyroptosis. The activity of the GTPase Rag and lysosomal tethering of Rag-Ragulator were required to recruit and activate the kinase RIPK1 and protease caspase-8 to cleave GSDMD, which causes cell death and limits infection. By contrast, Rag-Ragulator was not required for inflammasome-mediated pyroptosis. Thus, metabolic signaling on lysosomes can regulate cell death during pathogenic bacterial infection.

Science, abg0269, this issue p. eabg0269

Structured Abstract

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.

Rag-Ragulator serves as a platform for activating a FADD–RIPK1–caspase-8 complex to trigger pyroptosis in response to TLR and TNFR ligation.

When TAK1 is inhibited by the Yersinia effector YopJ or its mimic 5z-7-oxozeaenol (5z7), a FADD–RIPK1–caspase-8–containing complex is recruited to lysosome-tethered Rag-Ragulator, which activates caspase-8– and GSDMD-dependent pyroptosis. TNFRSF, TNFR superfamily; L1-5, Lamtor1-5.

Abstract

Host cells initiate cell death programs to limit pathogen infection. Inhibition of transforming growth factor–β–activated kinase 1 (TAK1) by pathogenic Yersinia in macrophages triggers receptor-interacting serine-threonine protein kinase 1 (RIPK1)–dependent caspase-8 cleavage of gasdermin D (GSDMD) and inflammatory cell death (pyroptosis). A genome-wide CRISPR screen to uncover mediators of caspase-8–dependent pyroptosis identified an unexpected role of the lysosomal folliculin (FLCN)–folliculin-interacting protein 2 (FNIP2)–Rag-Ragulator supercomplex, which regulates metabolic signaling and the mechanistic target of rapamycin complex 1 (mTORC1). In response to Yersinia infection, Fas-associated death domain (FADD), RIPK1, and caspase-8 were recruited to Rag-Ragulator, causing RIPK1 phosphorylation and caspase-8 activation. Pyroptosis activation depended on Rag guanosine triphosphatase activity and lysosomal tethering of Rag-Ragulator but not mTORC1. Thus, the lysosomal metabolic regulator Rag-Ragulator instructs the inflammatory response to Yersinia.

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