Action of Shiga Toxin Type-2 and Subtilase Cytotoxin on Human Microvascular Endothelial Cells

María M Amaral,Carolina Jancic,Adrienne W Paton,Flavia Sacerdoti,Cristina Ibarra,Horacio A Repetto,James C Paton,Shree Ram Singh

doi:10.1371/journal.pone.0070431

Abstract

The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 −a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.

Highlights

Hemolytic uremic syndrome (HUS) is characterized by nonimmune hemolytic anemia, thrombocytopenia and acute renal failure [1]
Identification of von Willebrand factor (VWF) and PECAM- 1 on human glomerular endothelial cells (HGEC) Cells obtained from human renal glomeruli were cultured and characterized for endothelial phenotype
Toxins modified HGEC morphology Cell morphology was analyzed by phase contrast microscopy and additional staining with H&E, so HGEC were incubated under growth-arrested conditions either with Stx2 (10 ng/ml) or SubAB (3 μg/ml) or without toxins for 24 h

Summary

Introduction

Hemolytic uremic syndrome (HUS) is characterized by nonimmune hemolytic anemia, thrombocytopenia and acute renal failure [1]. The classical form of HUS is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection, the most prevalent infectious agent responsible for the development of this pathology [2]. In Argentina, HUS is endemic with a high incidence of more than 500 cases per year, being the most common cause of acute renal failure and the second leading cause of chronic renal failure in children younger than 5 years old [3,4]. Clinical and histological renal damage has been strongly associated with Stx types 1 and 2 (Stx, Stx2) produced by O157:H7 STEC, strains that only express Stx are highly prevalent in Argentina [5]. STEC are present in the intestinal tract of healthy cattle and disease outbreaks are frequently due to ingestion of undercooked ground beef, manure-contaminated water, vegetables, fruit or unpasteurized milk

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