bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2021.01.22.427304

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

Merritt, C.·Chun, E. M.·Fattah, R. J.·Moayeri, M.·Paliga, D.·Neumann, S.·Heumann, R.·Leppla, S. H.·Bugge, T. H.
View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

pathologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Merritt, C., Chun, E. M., Fattah, R. J., Moayeri, M., Paliga, D., Neumann, S., Heumann, R., Leppla, S. H., Bugge, T. H.. 2021-01-22. Direct imaging of anthrax intoxication in animals reveals shared and individual functions of CMG-2 and TEM-8 in cellular toxin entry. https://doi.org/10.1101/2021.01.22.427304

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Midlife nonlinearities across scale in Biondi body amyloid formation in the human choroid plexus

Human choroid plexus epithelial cells (CPECs), which produce cerebrospinal fluid and form the blood-cerebrospinal fluid barrier, accumulate discrete intracellular amyloid inclusions called Biondi bodies (BBs). Using systematic manual and machine learning approaches on the choroid plexus glomus, we determined the percentages of CPECs containing thioflavin S-stained BBs as well as BB load and spatial distributions in whole slide images from 77 cases across the human lifespan. On average, BB prevalence was near zero until age 40, rose steeply in the 5th and 6th decades, and exceeded 50% after age 70. As prevalence plateaued, BB load continued to rise with age. Accelerated BB accumulation was seen in two cases of diabetic ketoacidosis, including a 16-year-old, while an 85-year-old with the relatively rare APOE2/E2 genotype had fewer BBs than expected. BB prevalence in the choroid plexus glomus was not associated with Alzheimer's disease, but was lower in females than males. Notably, spatial clustering of BBs was universal across cases while nearest-neighbor analyses suggested cell-to-cell, prion-like BB spread that can account for the nonlinearities seen at tissue and population scales. The abrupt, nonlinear BB accumulation during midlife coincides with or precedes other common aging-associated neuropathologies of the human brain, consistent with broad BB and choroid plexus contributions to brain aging and disease.

pathology↗
bioRxiv2026

Elevated precontractile calcium set point is sustained by limited PMCA1 export in dystrophic muscle

Duchenne muscular dystrophy is caused by loss of dystrophin and marked by elevated muscle calcium, usually attributed to contraction-induced membrane damage. Whether calcium rises before muscle can contract, and what sustains it, is unresolved. In Caenorhabditis elegans embryos and patient-derived human myoblasts, dystrophic muscle carries an elevated resting calcium set point before contraction is possible. Embryonic calcium is elevated 400 minutes after fertilization, before movement is detectable or the neuromuscular junction forms. Silencing the plasma membrane calcium pump gene mca-1 raises calcium in wild-type but not dystrophic animals, whereas silencing its paralogue raises it in both: the limitation is specific to that export route. Dystrophic human myoblasts have less pump; pump inhibition or silencing increases calcium preferentially in healthy cells, and raising pump expression lowers calcium most in dystrophic cells. Constrained calcium export is an early, continuing determinant of the dystrophic set point.

pathology↗
bioRxiv2026

Systematic comparison of gliflozins identifies a selective anti-calcifying effect of canagliflozin beyond canonical SGLT2 inhibition

Background: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) provide important cardiorenal benefits in chronic kidney disease (CKD), but their effects on vascular calcification remain conflicting. Whether potential anti-calcifying effects are shared across gliflozins or involve mechanisms beyond canonical SGLT2 inhibition is unknown. Methods: We systematically compared canagliflozin, dapagliflozin and empagliflozin in a high-phosphate diet-induced mouse model of vascular calcification and complementary ex vivo and in vitro models, including rodent and human vascular tissues and vascular smooth muscle cells (VSMCs). Aortic transcriptional response was investigated by RNA sequencing. VSMC calcification, proliferation, migration, viability, glucose uptake and bioenergetics were investigated using complementary cellular assays, including OsteoSense staining, Annexin V/propidium iodide staining, radiolabeled 2-deoxyglucose uptake and Seahorse ATP rate analysis. SGLT2/1 expression was examined by RT-qPCR, Western blotting and immunofluorescence. We also evaluated sotagliflozin, a dual SGLT1/2 inhibitor, and mizagliflozin, a selective SGLT1 inhibitor. Results: Among the three gliflozins, only canagliflozin significantly reduced aortic calcification in vivo. Its anti-calcifying effect was also observed in rodent aortic rings, human vascular explants, cultured VSMCs and aortic rings from diabetic db/db mice. Aortic RNA-seq revealed that canagliflozin partially reversed HPD-induced transcriptional alterations, including changes in pro-calcific signaling and cellular metabolism, while some endothelial and inflammatory responses were shared among gliflozins. In mouse VSMCs, canagliflozin reduced calcification across all tested concentrations, dose-dependently decreased osteogenic marker expression, and reduced proliferation and migration. Increased apoptotic and damaged cell fractions were observed at higher concentrations, but not at lower concentrations. Canagliflozin also reduced glucose uptake and mitochondrial ATP production, without significantly affecting glycolytic ATP production. Neither dapagliflozin nor empagliflozin reproduced these cellular and metabolic effects at any of the concentrations tested. SGLT2 and SGLT1 expression was low or undetectable in vascular tissues and cells, and neither sotagliflozin nor mizagliflozin reproduced the anti-calcifying effect of canagliflozin in vivo or in vitro. Conclusions: Canagliflozin exhibits a selective anti-calcifying profile among the gliflozins tested, associated with changes in VSMC responses and cellular metabolism. These findings suggest that its vascular protective effects may involve mechanisms beyond canonical SGLT2 inhibition.

pathology↗