Direct imaging of anthrax intoxication in animals reveals shared and individual functions of CMG-2 and TEM-8 in cellular toxin entry

The virulence of Bacillus anthracis is linked to the secretion of anthrax lethal toxin and anthrax edema toxin. These binary toxins consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds either of two specific cell surface receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding, endocytosis, and cytoplasmic translocation of LF and EF. The cellular distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals is not fully elucidated. Herein, we describe a novel assay to image anthrax toxin intoxication in live animals, and we use the assay to visualize TEM-8- and CMG-2-dependent intoxication. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were co-administered to transgenic mice that ubiquitously express a red fluorescent protein in the absence of Cre activity and a green fluorescent protein in the presence of Cre activity, anthrax toxin intoxication could be visualized at single-cell resolution by confocal microscopy. By using this assay, we show that CMG-2 is critical for intoxication in the liver and heart, whereas TEM-8 is required for full intoxication in the kidney and spleen. Other tissues examined were largely unaffected by single deficiences in either receptor, suggesting extensive overlap in TEM-8 and CMG-2 expression. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for identifying on- and off-targets for reengineered toxin variants in the clinical development of cancer treatments. BackgroundAssays for imaging of anthrax toxin intoxication in animals are not available. ResultsAnthrax toxin-Cre fusions combined with fluorescent Cre reporter mice enabled imaging of anthrax toxin intoxication in animals. ConclusionShared and distinct functions of toxin receptors in cellular entry were uncovered. Significance. A simple and versatile assay for anthrax toxin intoxication is described.