Susceptibility to anthrax lethal toxin-induced rat death is controlled by a single chromosome 10 locus that includes rNlrp1

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.

Figure 1. LT sensitivity in rat strains correlates perfectly with macrophage sensitivity.

(A, B) Top panels show representative cytotoxicity assays for rat macrophages treated with toxin. BMDMs were treated with various concentrations of LT and cell viability was assessed after 3 h by MTT assay as described in Materials and Methods. Lower panels show average rat times to death (TTD) after LT treatment (100 µg, IV, n = 3 or n = 4/strain), and the macrophage LT sensitivity phenotypes (S  = sensitive, R = resistant). (*)s indicate progenitor strains and a related congenic strain.

Figure 2. Mapping of the LT sensitivity locus to chromosome 10.

(A) Strain distribution patterns at several markers on chromosome 10 (a complete collection based on published data for all chromosomes is found in Figure S1). Genotypes are SHR-like (H; gray) or BN-Lx-like (B; white). U indicates unknown genotypes. (*) indicates predicted genotypes. (B) Linkage map for markers on chromosome 10. Markers with known locations in the genomic data for chromosome 10 of the Brown Norway rat (BN/SsNMcw; RGSC 3.4) are mapped to their approximate physical locations on the chromosome (indicated with arrows). Expansion indicates the region from 57.7 M to 58.1 M of chromosome 10 and selected open reading frames. (C) SNP data between markers D10Rat77 and D10Rat80 (flanking rNlrp1) for all RI strains (except BXHO8) and their progenitors. SNP heterozygosity is indicated by dual base designation.

Figure 3. Allelic variations in rNlrp1 correlate with LT sensitivity.

Proposed exon structure of the mature mRNA for rNlrp1 is shown at the top. Exons are numbered above and locations of forward (green arrowhead) and reverse (red arrowhead) primers for the primary sequencing reactions are indicated. Approximate domain locations are shown for NACHT (green), LRR (blue), and CARD (red). Amino acid alignments for the five alleles of rNlrp1 are aligned to the exon structure. White hashes are indicative of amino acid changes relative to protein encoded by allele 1. Expansions of the three regions of interest are also shown with alignments of the five alleles. Red letters identify residues that differ from those in the protein encoded by allele 1.