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.

Human genetic variation altering anthrax toxin sensitivity

“…1B) is consistent with a polygenic inheritance model, which also is implied from evidence that multiple genes can affect anthrax toxin lethality (26)(27)(28)(29)(30)(31)(32). Among these genes is CMG2, which encodes a type 1 transmembrane glycoprotein that recently has been shown to be the primary anthrax toxin receptor in rodents (36).…”

“…3B). This finding suggests that the effects we have demonstrated for this SNP in cell culture experiments are modulated in genetically complex cell populations by the actions of other genes altering toxin internalization (26)(27)(28)(29)(30)(31)(32), raising the prospect that larger cohorts may be required for detection of the effects of this SNP in such populations.…”

“…2B) and Western blotting experiments (Fig. 2C) showed that both the extent of CMG2 expression and genetic variation affecting amino acid residue 357, which is located in a hCMG2 region known to be functionally important for endocytosis of toxin (29,30), are determinants of PA internalization. The Western blotting experiment also showed that the amount of PA bound to the macrophage cell surface was modulated by the extent of expression of either CMG2 357A or 357P.…”

“…Analysis of HapMap Project release 28 revealed no cis-acting eQTL loci that might account for such variation. Additionally, analysis in this release of genetic variation in the 17 CGEPs reported to alter anthrax toxin entry into cells (26)(27)(28)(29)(30)(31)(32) revealed that a SNP affecting an amino acid within a proline-rich region of the cytosolic tail of CMG2 (P357A; rs12647691) was among the most frequent nonsynonymous SNPs observed in the lymphoblastoid cells tested. Whereas this SNP was not associated with the observed variation in CMG2 expression in the human cell populations we tested [CEU, P = 0.98, n = 62 (excluding 3 CEU outliers P = 0.41, n = 59); YRI, P = 0.62, n = 84], interestingly, its allele frequency varied significantly among different ethnic populations.…”

“…Three host cell proteins [capillary morphogenesis gene 2 (CMG2), tumor endothelial marker 8 (TEM8), and integrin β1 (ITGB1)], which can interact with each other physically or functionally (25,26), can serve as receptors for the bipartite PA/LF and PA/EF toxins (26)(27)(28). Whereas only CMG2, TEM8, and ITGB1 are known ligands of PA, 14 additional CGEPs can assist PA binding and/or internalization (26,(29)(30)(31)(32)(33).…”