Susceptibility to Anthrax Lethal Toxin-Induced Rat Death Is Controlled by a Single Chromosome 10 Locus That Includes rNlrp1

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Abstract
Anthrax lethal toxin (LT) is a bipartite protease-containing toxin and a key virulence determinant of Bacillus anthracis. In mice, LT causes the rapid lysis of macrophages isolated from certain inbred strains, but the correlation between murine macrophage sensitivity and mouse strain susceptibility to toxin challenge is poor. In rats, LT induces a rapid death in as little as 37 minutes through unknown mechanisms. We used a recombinant inbred (RI) rat panel of 19 strains generated from LT-sensitive and LT-resistant progenitors to map LT sensitivity in rats to a locus on chromosome 10 that includes the inflammasome NOD-like receptor (NLR) sensor, Nlrp1. This gene is the closest rat homolog of mouse Nlrp1b, which was previously shown to control murine macrophage sensitivity to LT. An absolute correlation between in vitro macrophage sensitivity to LT-induced lysis and animal susceptibility to the toxin was found for the 19 RI strains and 12 additional rat strains. Sequencing Nlrp1 from these strains identified five polymorphic alleles. Polymorphisms within the N-terminal 100 amino acids of the Nlrp1 protein were perfectly correlated with LT sensitivity. These data suggest that toxin-mediated lethality in rats as well as macrophage sensitivity in this animal model are controlled by a single locus on chromosome 10 that is likely to be the inflammasome NLR sensor, Nlrp1. Inflammasomes are multiprotein cytoplasmic complexes that respond to a variety of danger signals by activating the host innate immune response. The sensor components of these complexes are NLR (NOD-like receptor) proteins. In this report, a recombinant inbred rat strain collection was used to genetically map anthrax lethal toxin (LT) susceptibility to a limited region of chromosome 10 containing one such sensor, Nlrp1. Similar to its mouse ortholog, Nlrp1b, which controls murine macrophage sensitivity to this toxin, the locus containing rat Nlrp1 was shown to control macrophage sensitivity to anthrax LT. However, unlike the situation in mice, where multiple genetic loci influence animal susceptibility to LT, the single chromosome 10 locus alone appears to control the rapid anthrax LT-induced death, which can occur in as little as 37 minutes. Sequencing of Nlrp1 from 12 rat strains identified polymorphisms which correlated perfectly with animal sensitivity to toxin. These polymorphisms were within the N-terminal 100-amino acid portion of Nlrp1, in an area of unknown function, which suggests that the N-terminus of rodent Nlrp1 could be an important functional domain.