Anthrax Lethal Toxin Downregulates Claudin-5 Expression in Human Endothelial Tight Junctions

Abstract

Vascular leakage pathologies such as pleural effusion and hemorrhage are hallmarks of anthrax pathogenesis. We previously reported that anthrax lethal toxin (LT), the major virulence factor of anthrax, reduces barrier function in cultured primary human microvascular endothelial cells. Here, we show that LT-induced barrier dysfunction is accompanied by the reduced expression of the endothelial tight junction (TJ) protein claudin-5 but no change in the expression of other TJ components occludin, ZO-1, ZO-2, or the adherens junction (AJ) protein VE-cadherin. The downregulation of claudin-5 correlated temporally and dose-dependently with the reduction of transendothelial electrical resistance. LT-induced loss of claudin-5 was independent of cell death and preceded the appearance of actin stress fibers and altered AJ morphology. Pharmacological inhibition of MEK-1/2, two kinases that are proteolytically inactivated by LT, showed a similar reduction in claudin-5 expression. We found that LT reduced claudin-5 mRNA levels but did not accelerate the rate of claudin-5 degradation. Mice challenged with LT also showed significant reduction in claudin-5 expression. Together, these findings support a possible role for LT disruption of endothelial TJs in the vascular leakage pathologies of anthrax.

Anthrax Pathogenesis

“…Although the involvement of the cardiovascular system was expected based on many earlier animal studies, the assumption had been that endothelial cells were the likely target of this toxin. In vitro studies showing endothelial cell cytoskeletal and junction modifications, coupled with changes in barrier function, indicate that LT does affect these cells (54,215,263,264). A zebrafish model of vascular collapse also suggested endothelial cells were targeted (19).…”

Tpl2 Promotes Innate Cell Recruitment and Effector T Cell Differentiation To Limit Citrobacter rodentium Burden and Dissemination

“…Claudins aid in maintenance of the integrity of the epithelial barrier through the formation of tight junctions. Because Tpl2 signals upstream of the MEK/ extracellular signal-regulated kinase (ERK) pathway (41) and the expression of claudins 2 and 5 on epithelial cell lines requires signaling through the MEK/ERK pathway (42,43), we hypothesized that the expression of these claudins may be reduced by Tpl2 deficiency. Tpl2 Ϫ/Ϫ mice showed a significant reduction in claudin 2 expression compared to wild-type mice at 8 dpi (Fig.…”

“…Claudins are known to play a significant role in maintaining intestinal integrity through the formation of tight junctions. Claudin 2 and claudin 5 require signaling through the MEK/ERK pathway for protein expression on epithelial cell lines (42,43), and expression of the genes for claudin 1 and 2 can be upregulated by IL-17A stimulation (42). Because Tpl2 signals upstream of the MEK/ERK pathway (41) and contributes to IL-17A production in vitro (38) and in vivo (this study), we tested the possibility that Tpl2 regulates intestinal barrier integrity by regulating the expression of claudin 2 and/or 5 on the surface of epithelial cells.…”

Bacillus anthracis toxins

“…The many observations in animal models, combined with the extensive hemorrhaging noted in anthrax patients [290,291] led to the hypothesis that endothelial cells are a primary target for the lethal actions of these toxins. Endothelial cells were shown to have extensive protein expression changes in response to the toxins, which resulted in cytoskeletal and stress fiber function alterations, modified endothelial junctions, altered barrier function, and changes in ability of neutrophils to move through the endothelium [276,[292][293][294][295][296][297][298][299]. Although early reports suggested LT induced endothelial cell death [300], later studies did not find any evidence of endothelial cell apoptosis in response to the toxin [292]; instead, those researchers observed cell arrest [255].…”