Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors

Abstract

The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a β-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin.

Author Summary

Bacillus anthracis is the bacterium responsible for the anthrax disease. Its virulence is mainly due to 2 factors, the anthrax toxin and the anti-phagocytic capsule. This toxin is composed of three independent polypeptide chains. Two of these have enzymatic activity and are responsible for the effects of the toxin. The third has no activity but is absolutely required to bring the 2 enzymatic subunits into the cell where they act. If one blocks entry into the cells, one blocks the effects of these toxins, which is why it is important to understand how the toxin enters into the cell at the molecular level. Here we identified various molecules that are involved in efficiently bringing the toxin into the cell. First, we found that the actin cytoskeleton plays an important role in organizing one of the two anthrax toxin receptors at the cell surface. Second, we found a cytosolic protein, β-arrestin, that is required to modify the intracellular part of the toxin receptor, to allow uptake. Finally, we directly show, for the first time, that anthrax toxin uptake is mediated by the so-called clathrin-dependent pathway, a very modular entry pathway, but that the toxin utilizes this pathway in an unconventional way.