Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo

Abstract

Anthrax toxin, a major virulence factor of Bacillus anthracis, gains entry into target cells by binding to either of 2 von Willebrand factor A domain-containing proteins, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). The wide tissue expression of TEM8 and CMG2 suggest that both receptors could play a role in anthrax pathogenesis. To explore the roles of TEM8 and CMG2 in normal physiology, as well as in anthrax pathogenesis, we generated TEM8-and CMG2-null mice and TEM8/ CMG2 double-null mice by deleting TEM8 and CMG2 transmembrane domains. TEM8 and CMG2 were found to be dispensable for mouse development and life, but both are essential in female reproduction in mice. We found that the lethality of anthrax toxin for mice is mostly mediated by CMG2 and that TEM8 plays only a minor role. This is likely because anthrax toxin has approximately 11-fold higher affinity for CMG2 than for TEM8. Finally, the CMG2-null mice are also shown to be highly resistant to B. anthracis spore infection, attesting to the importance of both anthrax toxin and CMG2 in anthrax infections.edema toxin ͉ lethal toxin ͉ tumor endothelium marker-8 B acillus anthracis is a Gram-positive, rod-shaped, sporeforming bacterium, and the causative agent of anthrax. Anthrax toxin is the major virulence factor for this organism and responsible for its lethal effects in the host. Although treatment with antibiotics to eliminate the bacteria can be life-saving at the earlier stages of anthrax disease, once enough toxin has been produced, the disease is often lethal despite treatment. Thus, countermeasures that block toxin or limit its effects are essential at later stages of disease (1). Therefore, a detailed understanding of the interaction between anthrax toxin and the host is needed as a basis for developing improved interventions.Anthrax toxin is a 3-part toxin consisting of protective antigen (PA, 83 kDa), edema factor (EF, 90 kDa), and lethal factor (LF, 89 kDa) (2-4). These 3 proteins are individually nontoxic, but can assemble on the cell surface to form toxic complexes. To intoxicate host mammalian cells, PA binds to its cellular receptors, tumor endothelium marker-8 [TEM8, also named anthrax toxin receptor 1 (ANTXR1)] and capillary morphogenesis protein-2 [CMG2, also named anthrax toxin receptor 2 (ANTXR2)] (5, 6) with the involvement of a coreceptor LRP6 (7,8) and is then proteolytically processed to the active form, cell-surface bound PA63. PA63 spontaneously oligomerizes to form a heptamer that binds and delivers LF and EF into the cytosol. EF, which combines with PA to form edema toxin (ET), is a calmodulin-dependent adenylate cyclase that elevates intracellular cAMP levels, thereby causing diverse effects including impairment of phagocytosis and death of experimental animals (9, 10). LF, which combines with PA to form lethal toxin (LT), is a zinc-dependent metalloproteinase that cleaves and inactivates the mitogen-activated protein kinase kinases (MEKs) 1-4, 6, and 7 (11-13), blocking the ERK, p38, and Jun...

Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

“…TEM8 was the first toxin receptor to be described (4) and continues to receive attention as a potential tumor marker. CMG2 appears to be expressed more widely in tissues, has a higher affinity for PA (6), and in our work has been found to be more important for both bacterial pathogenesis (8,22) and tumor targeting (16,21).…”

“…Lethality of PA Variants for Receptor-deficient Mice-The CMG2 Ϫ/Ϫ and TEM8 Ϫ/Ϫ C57BL/6 mice used here were described previously (6,8). These mice and littermate control mice (all 8 -10 weeks old) were challenged intraperitoneally with two doses of PA or PA variants along with FP59 (in 0.5 ml of PBS; doses indicated in the tables and figures) with a 2-day interval.…”

“…CHO cells were grown in minimum essential medium ␣ with 5% FBS, 2 mM glutamine, 5 mM HEPES, pH 7.4, and 50 g/ml gentamicin, with or without 300 g/ml hygromycin B. HeLa cells were cultured in Dulbecco's modified Eagle's medium with 10% FBS, 2 mM glutamine, and 50 g/ml gentamicin. MEF cells were isolated from embryonic day 13.5 embryos of the wild type, CMG2 Ϫ/Ϫ , TEM8 Ϫ/Ϫ and CMG2 Ϫ/Ϫ /TEM8 Ϫ/Ϫ mice (described below) as described previously (6) and cultured in Dulbecco's modified Eagle's medium with 10% FBS, 2 mM glutamine, and 50 g/ml gentamicin. MEF cells were passaged at 85-90% confluence by splitting 1:4 and used within the first six passages.…”

“…Measurement of PA Affinity for Receptors-Schild plot analyses were used as described previously (6,29) to determine the affinity (apparent K d ) of PA variant proteins for cellular receptors. Briefly, the nontoxic PA-U7, PA-U7 I656Q, PA-U7 I656V, and PA-U7 TSR proteins were used as competitors to block the toxicity of wild type PA to CHO cells.…”

The potential of toxin-based drug delivery systems for enhanced nucleic acid therapeutic delivery

“…It is not known if the octameric mutants adopt (1) an identical conformation to those previously described [85,87] and (2) display enhanced serum stability relative to the more physiological octamers, though their translocase activity has been documented both in vitro and in vivo [85,87]. The PA63 (7/8) complex interacts with cellular receptors including; capillary morphogenesis protein 2 (CMG2) [88], integrin beta-1 (ITGB1) [89] and tumour endothelial marker 8 (TEM8) [90] at the cell surface (Table 2). TEM8 and CMG2 are almost ubiquitously expressed throughout the body, and drive the capture, tissue distribution and internalisation of PA [91].…”