Heterodimeric integrin complexes containing β1-integrin promote internalization and lethality of anthrax toxin

Abstract

To kill macrophages, the lethal factor component of Bacillus anthracis toxin binds to a carrier protein (PA), which then interacts with the CMG2 receptor protein on the cell surface and is endocytosed into the cytoplasm. CMG2, as well as TEM8, a second PA receptor not present on macrophages, contain a von Willebrand A domain that is crucial for toxin binding. Here we report that integrin β1, another cell surface von Willebrand A domain protein, can mediate and potentiate anthrax toxin endocytosis. By using microarray-based analysis to globally correlate gene expression profiles with toxin sensitivity, we associated toxin effects with the integrinactivating proteins osteopontin and CD44. Further study showed that PA binds to α4β1-and α5β1-integrin complexes, leading to their conjoint endocytosis, and also interacts-weakly relative to CMG2 but comparably to TEM8-with purified α5β1 complex in vitro. Monoclonal antibody directed against β1-integrin or its α integrin partners reduced PA/integrin endocytosis and anthrax toxin lethality, and hyaluronic acid-which interferes with CD44-mediated integrin activation-had similar effects. Remarkably, whereas deficiency of CMG2 protected macrophages from rapid killing by large toxin doses (>50 ng/mL), by 24 h the toxin-treated cells were dead. Such late killing of CMG2-deficient cells by high dose toxin as well as the late death observed during exposure of CMG2-producing macrophages to low-dose toxin (<1 ng/mL), was dependent on integrin function. Effects of inactivating both CMG2 and integrin were synergistic. Collectively, our findings argue strongly that β1-integrin can both potentiate CMG2-mediated endocytosis and serve independently as a low-affinity PA receptor.CD44 | microarray | osteopontin | CMG2 | lethal factor

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.…”

“…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.…”

“…Entry of each of these proteins into cells is mediated by interaction with a third B. anthracis-generated protein, protective antigen (PA). 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).…”

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].…”