The Receptors that Mediate the Direct Lethality of Anthrax Toxin

Shihui Liu,Yi Zhang,Benjamin Hoover,Stephen Leppla

doi:10.3390/toxins5010001

Abstract

Tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) are the two well-characterized anthrax toxin receptors, each containing a von Willebrand factor A (vWA) domain responsible for anthrax protective antigen (PA) binding. Recently, a cell-based analysis was used to implicate another vWA domain-containing protein, integrin β1 as a third anthrax toxin receptor. To explore whether proteins other than TEM8 and CMG2 function as anthrax toxin receptors in vivo, we challenged mice lacking TEM8 and/or CMG2. Specifically, we used as an effector protein the fusion protein FP59, a fusion between the PA-binding domain of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas aeruginosa exotoxin A. FP59 is at least 50-fold more potent than LF in the presence of PA, with 2 μg PA + 2 μg FP59 being sufficient to kill a mouse. While TEM8−/− and wild type control mice succumbed to a 5 μg PA + 5 μg FP59 challenge, CMG2−/− mice were completely resistant to this dose, confirming that CMG2 is the major anthrax toxin receptor in vivo. To detect whether any toxic effects are mediated by TEM8 or other putative receptors such as integrin β1, CMG2−/−/TEM8−/− mice were challenged with as many as five doses of 50 μg PA + 50 μg FP59. Strikingly, the CMG2−/−/TEM8−/− mice were completely resistant to the 5-dose challenge. These results strongly suggest that TEM8 is the only minor anthrax toxin receptor mediating direct lethality in vivo and that other proteins implicated as receptors do not play this role.

Highlights

Bacillus anthracis is gram-positive, spore-forming, rod-like bacterium that causes anthrax disease through bacteremia as well as by secreting exotoxins—anthrax lethal toxin and edema toxin [1].Anthrax toxins are AB-type toxins, consisting of the cellular receptor binding moiety, protective antigen (PA), and the two enzymatic moieties, lethal factor (LF) and edema factor (EF)
In addition to delivering the native effectors LF and EF, PA has been used to deliver other polypeptides fused with LF amino acids 1–254 (LFn), the PA-binding domain of LF
capillary morphogenesis protein-2 (CMG2)−/−/Tumor endothelium marker-8 (TEM8)−/− mice continued to be successful breeders. These results demonstrate that integrin β1 or other potential PA binding proteins are unable to mediate lethality caused by anthrax toxins in vivo

Summary

Introduction

Bacillus anthracis is gram-positive, spore-forming, rod-like bacterium that causes anthrax disease through bacteremia as well as by secreting exotoxins—anthrax lethal toxin and edema toxin [1]. We have previously generated TEM8- and CMG2-null mice (CMG2−/− and TEM8−/−, respectively) by deleting their TM domains Through analyzing these mice, we have shown previously that CMG2 is the major anthrax toxin receptor in vivo and TEM8 only plays a minor role in anthrax toxin pathogenesis [5]. This is due, at least partially, to the 10-fold higher affinity of PA for CMG2 as compared to TEM8 Another cell surface vWA domain-containing protein, integrin β1, was reported to mediate killing of the mouse macrophage cell line RAW264.7 by LT, suggesting that integrin β1 is another anthrax toxin receptor [6]. Domain-containing proteins functioning as anthrax toxin receptors in vivo remains as an important issue to be addressed

Results and Discussion

Cell Culture and Cytotoxicity Assay

Toxin Challenge Studies

Conclusions