Structural Insights into Escherichia coli Shiga Toxin (Stx) Glycosphingolipid Receptors of Porcine Renal Epithelial Cells and Inhibition of Stx-Mediated Cellular Injury Using Neoglycolipid-Spiked Glycovesicles.

Johanna Detzner,Caroline Gloerfeld,Helge Karch,Alexander Mellmann,Hans-Ulrich Humpf,Johannes Müthing,Nadine Legros,Gottfried Pohlentz

doi:10.3390/microorganisms7110582

Abstract

Shiga toxin (Stx) producing Escherichia coli (STEC) cause the edema disease in pigs by releasing the swine-pathogenic Stx2e subtype as the key virulence factor. Stx2e targets endothelial cells of animal organs including the kidney harboring the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer). Since the involvement of renal epithelial cells in the edema disease is unknown, in this study, we analyzed the porcine kidney epithelial cell lines, LLC-PK1 and PK-15, regarding the presence of Stx-binding GSLs, their sensitivity towards Stx2e, and the inhibitory potential of Gb3- and Gb4-neoglycolipids, carrying phosphatidylethanolamine (PE) as the lipid anchor, towards Stx2e. Immunochemical and mass spectrometric analysis revealed various Gb3Cer and Gb4Cer lipoforms as the dominant Stx-binding GSLs in both LLC-PK1 and PK-15 cells. A dihexosylceramide with proposed Galα1-4Gal-sequence (Gal2Cer) was detected in PK-15 cells, whereas LLC-PK1 cells lacked this compound. Both cell lines were susceptible towards Stx2e with LLC-PK1 representing an extremely Stx2e-sensitive cell line. Gb3-PE and Gb4-PE applied as glycovesicles significantly reduced the cytotoxic activity of Stx2e towards LLC-PK1 cells, whereas only Gb4-PE exhibited some protection against Stx2e for PK-15 cells. This is the first report identifying Stx2e receptors of porcine kidney epithelial cells and providing first data on their Stx2e-mediated damage suggesting possible involvement in the edema disease.

Highlights

Edema disease usually occurs in pigs shortly after weaning and exhibits typical clinical neurologic signs such as ataxia, convulsions, and paralysis, or causes sudden death [1,2]
The edema disease of swine has been used as a model to study the pathogenesis of similar diseases of human beings due to comparative pathology that manifests as edema disease in swine and hemolytic uremic syndrome (HUS) in humans caused by enterohemorrhagic E. coli (EHEC) that represent the human-pathogenic Stx-producing E. coli (STEC) subgroup [8]
The first approach of this study was the demonstration of the occurrence of Gb3Cer and Gb4Cer, the eponymous GSLs of the globo-series, in neutral GSL fractions prepared from lipid extracts of cultured porcine LLC-PK1 and PK-15 kidney epithelial cell lines

Summary

Introduction

Edema disease usually occurs in pigs shortly after weaning and exhibits typical clinical neurologic signs such as ataxia, convulsions, and paralysis, or causes sudden death [1,2]. Porcine edema disease is an enterotoxemia caused by certain pathogenic strains of Escherichia coli that colonize the small intestine and produce Shiga toxin (Stx) of the Stx2e subtype considered the key virulence factor involved in the pathogenesis of the infection [3,4]. F18ab fimbriae mediate bacterial colonization, while Stx2e upon transfer to the circulation injures brain endothelial cells, ranging from acute swelling to necrosis and detachment from basement membrane, as an early event in the pathogenesis of Stx-producing E. coli (STEC) strains [5]. Despite the low frequency of Stx2e-producing STEC among human clinical isolates and their general association with a mild course of infections [9,10,11], Stx2e-producing E. coli strains have been occasionally isolated from humans with HUS [12,13]. The relationship between swine STEC and human disease requires further evaluation [14,15,16,17,18]

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