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      Inflammasome Sensor NLRP1 Controls Rat Macrophage Susceptibility to Toxoplasma gondii

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          Abstract

          Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded species. Rats vary in their susceptibility to this parasite. The Toxo1 locus conferring Toxoplasma resistance in rats was previously mapped to a region of chromosome 10 containing Nlrp1. This gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin (LT) induced rapid cell death (pyroptosis). We show here that rat strain differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was Nlrp1. Knockdown of Nlrp1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRP1 inflammasome in rat macrophages.

          Author Summary

          Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain “sensor” proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the pro-inflammatory cytokines, IL-1β and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. Rat strains have been previously shown to vary in their susceptibility to this parasite. We report here that rat macrophages from different inbred strains also vary in sensitivity to Toxoplasma induced lysis. We find that NLRP1, an inflammasome sensor whose only known agonist is anthrax LT, is also activated by Toxoplasma infection. In rats there is a perfect correlation between NLRP1 sequence and macrophage sensitivity to Toxoplasma-induced rapid cell death, inhibition of parasite proliferation, and IL-1β/IL-18 processing. Nlrp1 genes from sensitive rat macrophages can confer sensitivity to this rapid cell death when expressed in Toxoplasma resistant rat macrophages. Our findings suggest Toxoplasma is a new activator of the NLRP1 inflammasome.

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          Most cited references 31

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          The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus.

           Hao Xu,  Liping Liu,  Nan Gong (2011)
          Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death. The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component. How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages, is a universal component of the flagellin-NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5-NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity. © 2011 Macmillan Publishers Limited. All rights reserved
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            Innate immune recognition of bacterial ligands by NAIPs dictates inflammasome specificity

            Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the CASPASE1 (CASP1) protease 1,2 . Although genetic data support a critical role for inflammasomes in immune defense and inflammatory diseases 3 , the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing C4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin 4,5 and PrgJ 6 , a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homolog of NLRC4, NAIP5 (NLR family, Apoptosis Inhibitory Protein 5), has been implicated in activation of NLRC4 7–11 , but is widely assumed to play only an auxiliary role 1,2 , since NAIP5 is often dispensable for NLRC4 activation 7,8 . However, Naip5 is a member of a small multigene family 12 , raising the possibility of redundancy and functional specialization among Naip genes. Indeed, we show here that different NAIP paralogs dictate the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that NAIP2/NLRC4 physically associates with PrgJ but not flagellin, whereas NAIP5/NLRC4 associates with flagellin but not PrgJ. Taken together, our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor-ligand model for activation of the NAIP/NLRC4 inflammasomes.
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              Reconstituted NALP1 inflammasome reveals two-step mechanism of caspase-1 activation.

              Interleukin (IL)-1beta maturation is accomplished by caspase-1-mediated proteolysis, an essential element of innate immunity. NLRs constitute a recently recognized family of caspase-1-activating proteins, which contain a nucleotide-binding oligomerization domain and leucine-rich repeat (LRR) domains and which assemble into multiprotein complexes to create caspase-1-activating platforms called "inflammasomes." Using purified recombinant proteins, we have reconstituted the NALP1 inflammasome and have characterized the requirements for inflammasome assembly and caspase-1 activation. Oligomerization of NALP1 and activation of caspase-1 occur via a two-step mechanism, requiring microbial product, muramyl-dipeptide, a component of peptidoglycan, followed by ribonucleoside triphosphates. Caspase-1 activation by NALP1 does not require but is enhanced by adaptor protein ASC. The findings provide the biochemical basis for understanding how inflammasome assembly and function are regulated, and shed light on NALP1 as a direct sensor of bacterial components in host defense against pathogens.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                March 2014
                13 March 2014
                : 10
                : 3
                Affiliations
                [1 ]Massachusetts Institute of Technology, Department of Biology, Cambridge, Massachusetts, United States of America
                [2 ]Molecular Parasitology Section, Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, Maryland, United States of America
                [3 ]Department of Pharmacology, University of California-San Diego, La Jolla, California, United States of America
                [4 ]Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, Maryland, United States of America
                University of Massachusetts, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: KMC MEG JPJS MM. Performed the experiments: KMC GG MAH DC MEG JPJS MM. Analyzed the data: KMC GG MAH SHL MEG JPJS MM. Contributed reagents/materials/analysis tools: MAH MP SHL MEG JPJS MM. Wrote the paper: KMC SHL MEG JPJS MM.

                Article
                PPATHOGENS-D-13-02273
                10.1371/journal.ppat.1003927
                3953412
                24626226

                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                Page count
                Pages: 11
                Funding
                This research was supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases. KMC was supported by National Institutes of Health (F31-AI104170), MAH by a Wellcome Trust-MIT postdoctoral fellowship, and JPJS by National Institutes of Health (R01-AI080621. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Immunology
                Immunity
                Innate Immunity
                Microbiology
                Immunity
                Innate Immunity
                Protozoology
                Parastic Protozoans
                Toxoplasma Gondii

                Infectious disease & Microbiology

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