Direct proteolytic cleavage of NLRP1B is necessary and sufficient for inflammasome activation by anthrax lethal factor.

Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1) protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR) or AIM2-like receptor (ALR) protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF) protease that is a key virulence factor secreted by Bacillus anthracis, the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just B. anthracis. By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense

Direct Proteolytic Cleavage of NLRP1B Is Necessary and Sufficient for Inflammasome Activation by Anthrax Lethal Factor

<div><p>Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1) protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR) or AIM2-like receptor (ALR) protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF) protease that is a key virulence factor secreted by <i>Bacillus anthracis</i>, the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just <i>B. anthracis</i>. By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense.</p></div

Murine NLRP1B from 129S1 mice is cleaved directly by LeTx.

<p>A) Protein sequence alignment of the N-terminal region of murine NLRP1B (129S1 allele) and rat NLRP1 (Fischer/CDF allele) was determined by ClutalW with a BLOSUM series matrix. The LF cleavage motif and cleavage site are identified in the rat allele by the bar and arrow above the rat sequence. B) GFP-HA-NLRP1B was transfected into HEK 293T cells and then treated with 1 µg/ml LeTx over the indicate time points followed by immunoblotting (IB) for HA on non-boiled lysates, and boiled lysates when probed with MEK2 and beta-actin antibodies. The arrow-head refers to the LeTx-dependent N-terminal cleavage fragment. C) HA-NLRP1B expressed in 293T cells, immunoprecipitated (IP) with anti-HA beads, and treated with recombinant LF (rLF) for 2 h, followed by immunoblotting for HA. D) Graphic representation of the GFP-HA-NLRP1B construct and annotated functional domains. The different forms of NLRP1B observed are shaded in gray along with their predicted molecular weights, when immunoblotted with an anti-HA antibody.</p

Cleavage of NLRP1B is sufficient to promote inflammasome activation.

<p>A) 293T cells were transfected with WT, TEV-site2 or TEV-site1 GFP-HA-NLRP1B along with empty vector, TEV expression vector, or a LF expression plasmids. In all conditions cells were also co-transfected with <i>Casp1</i> and <i>Il1b</i> expression vectors. Cleavage of GFP-HA-NLRP1B and IL-1β was determined 24 h post transfection. B) Immortalized B6 macrophages were transduced with a retrovirus encoding the indicated GFP-HA-NLRP1B form followed by a sequential transduction with a TEV-expression retrovirus co-expressing THY1.1. Percent transduction was determined by measuring expression of the respective retroviral integration markers (GFP and anti-THY1.1-PE-Cy7) by flow cytometry, and are expressed in relative fluorescent units (RFU). The numbers within each quadrant represent the percentage of live cells within the respective quadrant. C) RAW264.7 macrophages were transduced with GFP-HA-NLRP1B and a Tet-On construct expressing the indicated gene. Cells were treated with 5 µg/ml doxycycline for 20 h and supernatants were assayed for LDH release. D) 293T cells were transfected with empty vector, FL-NLRP1B-HA, the truncated NLRP1B-HA, or ΔLRR HA-NLRP1B, along with <i>Casp1</i> and <i>Il1b</i> and assayed by immunoblotting.</p

Proteasome inhibition and FIIND-processing do not affect NLRP1B cleavage by LF.

<p>A) 293T cells expressing GFP-HA-NLRP1B were co-treated with 1 µg/ml LeTx and 10 µM MG132 (proteasome inhibitor) or the DMSO vehicle and assayed for cleavage. B) Cleavage susceptibility of WT and S984A (FIIND mutant) GFP-HA-NLRP1B was determined in 293T cells at the indicated time points.</p

Functional and Evolutionary Analyses Identify Proteolysis as a General Mechanism for NLRP1 Inflammasome Activation

<div><p>Inflammasomes are cytosolic multi-protein complexes that initiate immune responses to infection by recruiting and activating the Caspase-1 protease. Human NLRP1 was the first protein shown to form an inflammasome, but its physiological mechanism of activation remains unknown. Recently, specific variants of mouse and rat NLRP1 were found to be activated upon N-terminal cleavage by the anthrax lethal factor protease. However, agonists for other NLRP1 variants, including human NLRP1, are not known, and it remains unclear if they are also activated by proteolysis. Here we demonstrate that two mouse NLRP1 paralogs (NLRP1A^B6 and NLRP1B^B6) are also activated by N-terminal proteolytic cleavage. We also demonstrate that proteolysis within a specific N-terminal linker region is sufficient to activate human NLRP1. Evolutionary analysis of primate NLRP1 shows the linker/cleavage region has evolved under positive selection, indicative of pathogen-induced selective pressure. Collectively, these results identify proteolysis as a general mechanism of NLRP1 inflammasome activation that appears to be contributing to the rapid evolution of NLRP1 in rodents and primates.</p></div

NLRP1A is activated by N-terminal proteolysis.

<p>(<b>A</b>) The amino acid sequence of the first 244 residues of NLRP1A^B6 was aligned to NLRP1B¹²⁹ and NLRP1B^B6. The arrow above the alignment indicates the LF-cleavage site in NLRP1B¹²⁹. (<b>B</b>) 293T cells were transfected for 36h with 200ng empty vector (V) or plasmids pcDNA3.1-HA-NLRP1B¹²⁹ or -NLRP1A^B6-MYC (CMV promoter) along with 200ng mCASP1 and 200ng mIL-1β expression vectors. Cells were treated overnight with anthrax lethal toxin (LeTx, 1μg/ml) 24h post-transfection, and then lysates were analyzed by immunoblotting (IB) with the indicated antibodies. (<b>C</b>) 293T cells were transfected for 36h with plasmids encoding 400ng GFP-HA-NLRP1B¹²⁹ or GFP-HA-NLRP1A^B6 (under the control of the LTR promoter in pMSCV) or with mutants engineered to contain an N-terminal TEV protease site. Cells were also co-transfected with 100ng empty vector (V) or plasmids encoding TEV-protease (pTEV) or lethal factor protease (pLF), plus 300ng empty pMSCV. (<b>D</b>) To assess IL-1β cleavage, cells were transfected as in C, but with 8ng of GFP-HA-NLRP1B and co-transfected with vectors encoding 200ng mCASP1 and 200ng mIL-1β. For panels B-D, data shown are representative of at least three similar experiments.</p