Proteasome activity is required for anthrax lethal toxin to kill macrophages

Abstract

Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines such as RAW 264.7. LF is translocated by PA into the cytosol of target cells, where it acts as a metalloprotease to cleave mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins. In this study, we show that proteasome inhibitors such as acetyl-Leu-Leu-norleucinal, MG132, and lactacystin efficiently block LeTx cytotoxicity, whereas other protease inhibitors do not. The inhibitor concentrations that block LF cytotoxicity are similar to those that inhibit the proteasome-dependent IkappaB-alpha degradation induced by lipopolysaccharide. The inhibitors did not interfere with the proteolytic cleavage of MEK1 in LeTx-treated cells, indicating that they do not directly block the proteolytic activity of LF. However, the proteasome inhibitors did prevent ATP depletion, an early effect of LeTx. No overall activation of the proteasome by LeTx was detected, as shown by the cleavage of fluorogenic substrates of the proteasome. All of these results suggest that the proteasome mediates a toxic process initiated by LF in the cell cytosol. This process probably involves degradation of unidentified molecules that are essential for macrophage homeostasis. Moreover, this proteasome-dependent process is an early step in LeTx intoxication, but it is downstream of the cleavage by LF of MEK1 or other putative substrates.

FIG. 1

Proteasome inhibitors protect RAW 264.7 cells from LeTx. Cells were exposed for 2 h to 500 ng (each) of PA and LF per ml in the presence of inhibitors at the indicated concentrations. Cytotoxicity was measured by MTT assay, and cell viability was calculated as described in Materials and Methods. Data are averages of five independent experiments.

FIG. 2

Proteasome inhibitors block degradation of IκB-α induced by LPS in RAW 264.7 cells. (A) Cells were incubated with 1 μg of LPS per ml for the indicated times and lysed for Western blotting with antibodies against IκB-α. (B) Proteasome inhibitors were incubated with cells for 30 min before LPS was added for a further 30-min incubation. Cells were then lysed for Western blotting with antibodies against IκB-α. These blots showed a single immunoreactive band of approximately 40 kDa, corresponding to IκB-α, and only this portion of the blot is shown. Results are representative of three independent experiments.

FIG. 3

Proteasome inhibitors do not block intracellular proteolytic cleavage of MEK1 by LF. (A) RAW 264.7 cells were treated with 500 ng (each) of PA and LF per ml for the indicated times and lysed for Western blotting with antibodies against the N terminus or C terminus of MEK1. (B) Cells were incubated with proteasome inhibitors at the indicated concentrations together with PA and LF for 1 h and lysed for Western blotting, as described for panel A. These blots showed a single immunoreactive band of 43 kDa, corresponding to MEK1, and only this part of the blot is shown. Results are representative of three independent experiments.

FIG. 4

Proteasome inhibitors block ATP depletion induced by LeTx. RAW 264.7 cells were treated with 500 ng (each) of PA and LF per ml in the absence or presence of proteasome inhibitors for 1 h. ATP was released from the cells and measured by bioluminescence from luciferin/luciferase, as described in Materials and Methods. Data are averages of three independent experiments.

FIG. 5

LeTx does not induce proteasome activity. Cells were treated with or without 500 ng (each) of PA and LF per ml for 1 h. Cell lysates were prepared and incubated with fluorogenic substrates for 15 min at 37°C. Proteasome specific activity was calculated as the difference between fluorescence in the absence and fluorescence in the presence of lactacystin. VKM, LLE, and LLVY stand for the fluorogenic substrates Z-VKM-AMC, Z-LLE-AMC, and SLLVY-AMC, respectively.