Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry

Carly Merritt,Elizabeth M Chun,Rasem J Fattah,Lakmali M Silva,Quinn Q Ma,Mahtab Moayeri,Dennis Paliga,Sebastian Neumann,Rolf Heumann,Stephen H Leppla,Thomas H Bugge

doi:10.1016/j.jbc.2021.101467

Abstract

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. 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 coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.

Highlights

Assays for imaging of anthrax toxin intoxication in animals are not available
It has long been noted that LF residues 1-254 suffice to achieve translocation of a variety of “passenger” polypeptides and other molecules into the cytoplasm of the cells in a PA63-dependent manner [14,15]. These intoxication is initiated by the binding of PA to include other bacterial toxins and bacterial either of two receptors, capillary morphogenesis proteins [14,16,17,18,19,20], fluorescent proteins [21], protein-2 (CMG-2) or tumor endothelial markerviral proteins [22,23,24,25], eukaryotic proteins
Organs from mice injected with PA alone displayed only red fluorescence (Supplemental Figure 3 B, B’, E, E’, H, H’, K, K’, N, N’). When analyzing these organs from mTmG+/0 mice injected with LFn-nuclear localization signal (NLS)-Cre in combination with PA, both red (Supplemental Figure 3 C, F, I, L, O) and green fluorescence were readily detected in the heart, kidney, liver, lungs, and spleen (Supplemental Figure 3 C’, F’, I’, L’, O’), indicating that PA-dependent translocation of LFn-NLS-Cre to the cytoplasm of cells in these organs takes place and that the subsequent nuclear import of the fusion protein leads to the excision of the floxed mTomato gene

Summary

Background

Assays for imaging of anthrax toxin intoxication in animals are not available. Results: Anthrax toxin-Cre fusions combined with fluorescent Cre reporter mice enabled imaging of anthrax toxin intoxication in animals. Conclusion: Shared and distinct functions of toxin receptors in cellular entry were uncovered. Significance: A simple and versatile assay for anthrax toxin intoxication is described

INTRODUCTION

RESULTS

DISCUSSION

EXPERIMENTAL PROCEDURES