Anthrax lethal factor cleaves mouse nlrp1b in both toxin-sensitive and toxin-resistant macrophages

Abstract

Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.

Figure 1. Nlrp1 protein alignments and constructs.

(A). Alignment of amino acid sequences from the N-terminus of mouse Nlrp1b and rat Nlrp1 proteins. Sequences shown are those of 4 mouse and 2 rat strains, including strains having macrophages that are either sensitive (S) or resistant (R) to LT. The previously identified LT cleavage site after residue 44 in rat CDF Nlrp1 is indicated by an arrow. The red box indicates the region of mouse sequence shown in (B). (B) Nlrp1b constructs used in this study with focus on N-terminal regions containing putative LF cleavage sites. The top two constructs represent the full-length HA-tagged Nlrp1b proteins from the LT-sensitive Balb/cJ (BALB) and the LT-resistant NOD/LtJ (NOD) macrophages, which were expressed in HT1080 cells. Full length NOD Nlrp1b is shorter (1172 aa) than BALB Nlrp1b due to a region downstream of the leucine rich repeat domain that is missing in this protein. The next four constructs represent proteins where aa 3-118 of Nlrp1b were expressed and purified from E. coli as N-terminal GST-tagged proteins. These proteins also contain a C-terminal His6 tag (not represented in figure). In the sequence alignments, residues identical to those in the construct listed above are indicated by quotation marks (“). Putative LF cleavage sites based on previously described motifs are drawn as vertical dotted lines below filled arrows. The MEK4 cleavage site is also aligned with both putative Nlrp1b cleavage sites. The last two sequences are those of constructs having two key lysine residues substituted with alanine.

Figure 2. Cleavage of full length rat and mouse Nlrp1b proteins by LF.

(A) IP (anti-HA pulldown) followed by anti-HA Western blotting of lysates from HT1080 cells expressing HA-tagged mouse Nlrp1b (BALB) or rat Nlrp1(CDF) proteins following treatment with LF (1 µg/ml) for 15 min or 2 h. Cleavage of CDF Nlrp1 leads to appearance of a 6-kDa HA-reactive band and cleavage of BALB Nlrp1b leads to a slightly smaller fragment. (B) IP (anti-HA pulldown) followed by anti-HA Western blotting of lysates from HT1080 cells expressing HA-tagged Nlrp1b proteins or control vector following treatment with LF (1 µg/ml, 30 min). Anti-HA cross-reactive bands not marked as HA-Nlrp1 also appear in vector-transfected controls. (C) Comparison of size of cleavage fragments generated after cleavage of BALB and NOD HA-tagged Nlrp1b (using conditions same as 2B), indicating the smaller size of the fragment generated following cleavage of the BALB protein (Western representative of five similar experiments).

Figure 3. Cleavage of mouse BALB118 and NOD118 Nlrp1 fusion proteins by LF.

(A, B) In vitro cleavage of N-terminally 6His-GST-tagged aa 3-118 of Nlrp1b proteins. Purified proteins (0.53 mg/ml or 0.94 mg/ml, in A and B, respectively) were treated with the indicated molar ratios of LF, or with a 1∶10 molar ratio of the mutant LF E687C (LFm), for 4 h prior to SDS gel electrophoresis and Coomasie staining. F1 and F2 refer to two fragments generated following LF treatment. (C) GST-tagged or double alanine mutant variants (0.44-0.66 mg/ml) were treated with 33 µg/ml LF or LFm for 4 h prior to SDS gel electrophoresis and Coomassie staining.

Figure 4. Caspase-1 activation in bone marrow-derived mouse macrophages.

LPS-primed (1 µg/ml, 2 h) bone marrow-derived macrophages from Balb/cJ or NOD/LtJ mice were treated with LT (1 µg/ml) for 60 or 80 min, or with nigericin at indicated doses for 20 min. Cell lysates were analyzed by Western blotting for IL-1β, and the same samples were probed with caspase-1 p10 antibody to detect caspase-1 cleavage.