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. 2013;9(6):e1003452.
doi: 10.1371/journal.ppat.1003452. Epub 2013 Jun 20.

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

Joseph Chavarría-Smith  1 , Russell E Vance
Affiliations

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

Joseph Chavarría-Smith et al. PLoS Pathog. 2013.

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Murine NLRP1B from 129S1 mice is cleaved directly by LeTx.
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.
Figure 2
Figure 2. Mouse NLRP1B cleavage by LF is required for inflammasome activation.
A) Both WT and CR2A GFP-HA-NLRP1B were transfected into 293T cells and then treated with LeTx for the indicated times, and cleavage was monitored by immunoblotting with indicated antibodies. B) Immortalized macrophages from a C57BL6 mouse were transduced with both forms of GFP-HA-NLRP1B and then treated with LeTx or LFn-Fla+PA (FlaTox). Pyroptosis was assayed by LDH release and normalized to complete detergent lysis. Error bars represent plus and minus one standard deviation from the mean.
Figure 3
Figure 3. Cleavage of NLRP1B is sufficient to promote inflammasome activation.
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 Casp1 and Il1b 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 Casp1 and Il1b and assayed by immunoblotting.
Figure 4
Figure 4. Proteasome inhibition and FIIND-processing do not affect NLRP1B cleavage by LF.
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.
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