Shiga Toxin-Bearing Microvesicles Exert a Cytotoxic Effect on Recipient Cells Only When the Cells Express the Toxin Receptor

Abstract

Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic Escherichia coli strains capable of causing hemolytic uremic syndrome. Our group has previously shown that the toxin can reach the kidney within microvesicles where it is taken up by renal cells and the vesicles release their cargo intracellularly, leading to toxin-mediated inhibition of protein synthesis and cell death. The aim of this study was to examine if recipient cells must express the globotriaosylceramide (Gb3) toxin receptor for this to occur, or if Gb3-negative cells are also susceptible after uptake of Gb3-positive and toxin-positive microvesicles. To this end we generated Gb3-positive A4GALT-transfected CHO cells, and a vector control lacking Gb3 (CHO-control cells), and decreased Gb3 synthesis in native HeLa cells by exposing them to the glycosylceramide synthase inhibitor PPMP. We used these cells, and human intestinal DLD-1 cells lacking Gb3, and exposed them to Shiga toxin 2-bearing Gb3-positive microvesicles derived from human blood cells. Results showed that only recipient cells that possessed endogenous Gb3 (CHO-Gb3 transfected and native HeLa cells) exhibited cellular injury, reduced cell metabolism and protein synthesis, after uptake of toxin-positive microvesicles. In Gb3positive cells the toxin introduced via vesicles followed the retrograde pathway and was inhibited by the retrograde transport blocker Retro-2.1. CHO-control cells, HeLa cells treated with PPMP and DLD-1 cells remained unaffected by toxin-positive microvesicles. We conclude that Shiga toxin-containing microvesicles can be taken up by Gb3-negative cells but the recipient cell must express endogenous Gb3 for the cell to be susceptible to the toxin.

Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit

“…Shiga toxin overlay showed binding of the toxin to the Gb3 extracted from the HeLa-derived EVs. Gb3 on blood cell-derived EVs has been previously shown [ 26 ].…”

“…Certain HeLa cells were treated with D-threo-1-Phenyl-2-palmitoylamino -3-morpholino-1-propanol (PPMP, Abcam, Cambridge, UK) as previously described [ 26 ], in order to reduce Gb3 expression.…”

Shiga Toxin (Stx)-Binding Glycosphingolipids of Primary Human Renal Cortical Epithelial Cells (pHRCEpiCs) and Stx-Mediated Cytotoxicity

“…Stx-containing microvesicles are taken up by glomerular endothelial cells in vitro and undergo endocytosis, leading to inhibition of protein synthesis and cell death [ 33 , 34 ]. Importantly, Stx-bearing microvesicles exert a cytotoxic effect on recipient cells only when the cells do express the toxin’s receptor GSL Gb3Cer [ 35 ]. Moreover, an association of vesicular Stx2 in blood to the development of HUS, i.e., in the transition period from hemorrhagic colitis to HUS, was found in STEC-infected children [ 36 ].…”

Shiga Toxins: An Update on Host Factors and Biomedical Applications

“…A variety of human cell lines and primary cells have been utilized in studying Stx, such as colorectal adenocarcinoma cell lines HT-29 [ 54 ], Caco-2 and HCT-8 [ 55 ], glomerular endothelial cells [ 56 ], renal tubular epithelial cells [ 57 ], vascular endothelial cells [ 58 ], cervical cancer HeLa cells [ 59 ], Burkitt lymphoma cells [ 60 ], and macrophage-like THP-1 Cells [ 61 ]. Some cell lines can be sensitized to Stx by ectopic expression of exogenous Gb3 synthase [ 62 , 63 ].…”

“…The sensitivity of different cell lines to Stx depends not only on Gb3 expression levels but also on membrane microdomains, as well as other host factors involved in toxin trafficking [ 14 , 62 , 65 , 66 , 67 , 68 ]. For example, ACHN cells and Caki-2 cells, which are two human renal tubular adenocarcinoma cell lines, showed drastically different levels of sensitivity to Stx2: ACHN cells are highly sensitive while Caki-2 cells are not.…”