Lung endothelial cells are sensitive to epsilon toxin from Clostridium perfringens

Jonatan Dorca-Arévalo,Eduard Dorca,Juan Blasi,Mireia Martín-Satué,Marta Blanch,Benjamín Torrejón-Escribano

doi:10.1186/s13567-020-00748-2

Abstract

The pore-forming protein epsilon toxin (Etx) from Clostridium perfringens produces acute perivascular edema affecting several organs, especially the brain and lungs. Despite the toxin evident effect on microvasculature and endothelial cells, the underlying molecular and cellular mechanisms remain obscure. Moreover, no Etx-sensitive endothelial cell model has been identified to date. Here, we characterize the mouse lung endothelial cell line 1G11 as an Etx-sensitive cell line and compare it with the well-characterized Etx-sensitive Madin-Darby canine kidney epithelial cell line. Several experimental approaches, including morphological and cytotoxic assays, clearly demonstrate that the 1G11 cell line is highly sensitive to Etx and show the specific binding, oligomerization, and pore-forming activity of the toxin in these cells. Recently, the myelin and lymphocyte (MAL) protein has been postulated as a putative receptor for Etx. Here, we show the presence of Mal mRNA in the 1G11 cell line and the presence of the MAL protein in the endothelium of some mouse lung vessels, supporting the hypothesis that this protein is a key element in the Etx intoxication pathway. The existence of an Etx-sensitive cell line of endothelial origin would help shed light on the cellular and molecular mechanisms underlying Etx-induced edema and its consequences.

Highlights

Clostridium perfringens types B and D produce severe and rapidly fatal enterotoxemia in ruminants, causing a lethality rate as high as 100% and substantial economic losses [1]
epsilon toxin (Etx) binds to the plasma membrane of 1G11 cells To study the effects of Etx on vascular endothelial cells, especially those related to the histopathological changes described in lung parenchyma of sheep and goats [49] and mice injected with green fluorescent protein (GFP)-Etx, we used the 1G11 cell line, a mouse lung capillary endothelial cell line
To determine Etx binding to 1G11 cells, we incubated the cells with the non-toxic form GFP-epsilon prototoxin (pEtx), which has a similar binding capacity as the toxic form [20], and the cells were analyzed by confocal microscopy

Summary

Introduction

Clostridium perfringens types B and D produce severe and rapidly fatal enterotoxemia in ruminants, causing a lethality rate as high as 100% and substantial economic losses [1]. Both strains produce one of the most lethal clostridial toxins, the epsilon toxin (Etx). The effects of i.v. injection of Etx have been studied in a series of other animal species [27]. There is currently no cellular model for in vitro studies, and previous research using endothelial cell lines from various animal species (including human umbilical endothelial cells) has not shown any effect of Etx [29], suggesting that only a selected set of endothelial cells are sensitive to Etx

Methods

Results

Conclusion