Consequences and utility of the zinc-dependent metalloprotease activity of anthrax lethal toxin

Abstract

Anthrax is caused by the gram-positive bacterium Bacillus anthracis. The pathogenesis of this disease is dependent on the presence of two binary toxins, edema toxin (EdTx) and lethal toxin (LeTx). LeTx, the major virulence factor contributing to anthrax, contains the effector moiety lethal factor (LF), a zinc-dependent metalloprotease specific for targeting mitogen-activated protein kinase kinases. This review will focus on the protease-specific activity and function of LF, and will include a discussion on the implications and consequences of this activity, both in terms of anthrax disease, and how this activity can be exploited to gain insight into other pathologic conditions.

Keywords: anthrax; lethal factor; metalloprotease; mitogen-activated protein kinase kinase; pathogenesis; retinal neovascularization; tumorigenesis.

Figure 1

Schematic of the kinase cascade and resulting cellular responses of the MAPK signaling pathways. (A) Generic scheme of the MAPK signaling cascade, whereby an extracellular stimulus activates MAPKKK, which phosphorylates and activates MAPKK, which phosphorylates and activates MAPK, leading to an intracellular biological response. (B) Schematic of the specific MAPK factors within each of the three major MAPK pathways. LeTx targets the MAPKK tier in the cascade, cleaving and inactivating all the MAPKK (MEK1-2, MKK3-7) with the exception of MKK5, the pathway for which is not depicted.

Figure 2

Inhibitory effect of LeTx on MEK signaling pathway in cells. Human melanoma SK-MEL-28 cells were treated with LeTx in a LF concentration-dependent manner (1 μg/mL PA plus 0, 1, 10, or 100 ng/mL LF) for 24 h. Whole cell extracts were then harvested and subjected to immunoblotting with antibodies against MEK1 N-terminus (top panel), MEK1 C-terminus (second panel), phospho-ERK1/2 (third panel), total ERK1/2 (fourth panel) and β actin (bottom panel).

Figure 3

Inhibitory effect of LeTx on human melanoma SK-MEL-28 xenograft tumor growth. Human melanoma SK-MEL-28 cells were subcutaneously injected (10⁷ cells in 100 μl HBSS) into the right side of the dorsalateral area of athymic nude mice (10 mice per group). After tumors were established to a volume of 50 mm³, mice were intravenously injected with either LeTx (PA plus LF) or control (PA plus LF_E687C) at one standard dose (SD, 1 SD equals 10 μg PA plus 2 μg of LF or LF_E687C) every other day for a total of six injections. Tumor growth is presented as average tumor volume (mm³) against days following inoculation of tumor cells. (■) average volume of the total 10 tumors before treatment. (□) average volume of the tumors in control-treated mice. (♦) average volume of the tumors in LeTx-treated mice.