Anthrax lethal toxin induces cell death-independent permeability in zebrafish vasculature

doi:10.1073/pnas.0712195105

Anthrax lethal toxin induces cell death-independent permeability in zebrafish vasculature

*Vascular Biology Program, Children's Hospital Boston and Department of Surgery, Harvard Medical School, Boston, MA 02115; and
^†Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115

Contributed by R. John Collier, December 28, 2007 (received for review November 4, 2007)

Abstract

Vascular dysfunction has been reported in human cases of anthrax, in mammalian models of Bacillus anthracis, and in animals injected with anthrax toxin proteins. To examine anthrax lethal toxin effects on intact blood vessels, we developed a zebrafish model that permits in vivo imaging and evaluation of vasculature and cardiovascular function. Vascular defects monitored in hundreds of embryos enabled us to define four stages of phenotypic progression leading to circulatory dysfunction. We demonstrated increased endothelial permeability as an early consequence of toxin action by tracking the extravasation of fluorescent microspheres in toxin-injected embryos. Lethal toxin did not induce a significant amount of cell death in embryonic tissues or blood vessels, as shown by staining with acridine orange, and endothelial cells in lethal toxin-injected embryos continued to divide at the normal rate. Vascular permeability is strongly affected by the VEGF/vascular permeability factor (VPF) signaling pathway, and we were able to attenuate anthrax lethal toxin effects with chemical inhibitors of VEGFR function. Our study demonstrates the importance of vascular permeability in anthrax lethal toxin action and the need for further investigation of the cardiovascular component of human anthrax disease.

Footnotes

^‡To whom correspondence may be addressed at:
Vascular Biology Program, Children's Hospital Boston, Karp Family Research Laboratories, 12th floor, Room 12.217, 300 Longwood Avenue, Boston, MA 02115-5737.
E-mail: joanne.chan{at}childrens.harvard.edu or jcollier{at}hms.harvard.edu
Author contributions: R.E.B., R.J.C., and J.C. designed research; R.E.B., S.E.S., R.Z., and J.C. performed research; A.P.B. and R.J.C. contributed new reagents/analytic tools; R.E.B., S.E.S., R.Z., and J.C. analyzed data; and R.E.B., R.J.C., and J.C. wrote the paper.
Conflict of interest statement: R.J.C. is cofounder of and an equity holder in PharmAthene, Inc., and consults for CombinatoRx, Inc.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. DQ415957 (CMG2A) and EF591979 (CMG2B)].
This article contains supporting information online at www.pnas.org/cgi/content/full/0712195105/DC1.
Freely available online through the PNAS open access option.