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 Gb3-positive 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.
Highlights
Shiga toxin 2 (Stx2) is an AB5 toxin that consists of a pentameric B-subunit, which mediates binding, and an enzymatically active A-subunit (Endo et al, 1988; Ling et al, 1998)
Results showed that Chinese hamster ovary (CHO)-Gb3 cells incubated with Stx2-positive microvesicles for 4 h had a significant decrease in protein synthesis compared to those incubated with Stx2-negative microvesicles (Figure 2A), in contrast to CHO-control and CHO native cells that displayed no difference in protein synthesis (Figures 2B,C)
CHO-Gb3 and HeLa cells that had been treated with Stx2-positive microvesicles had a significantly lower protein synthesis compared to CHO-control cells and PPMP-treated HeLa cells (P < 0.0001 and P < 0.001, respectively)
Summary
Shiga toxin 2 (Stx2) is an AB5 toxin that consists of a pentameric B-subunit, which mediates binding, and an enzymatically active A-subunit (Endo et al, 1988; Ling et al, 1998). In the ER the disulphide bonds are reduced and the A1-moiety is released into the cytosol, where it depurinates a specific base in the 28S rRNA of the ribosome, inhibiting protein translation, leading to cell death (Sandvig and van Deurs, 1996; Spooner and Lord, 2012). EHEC is a non-invasive bacterium that secretes virulence factors, including Stx, that gain access to the circulation (McKee and O’Brien, 1995). We have previously shown that these Stx2-positive blood cell-derived microvesicles circulate in EHECinfected patients and in EHEC-infected mice (Ståhl et al, 2015). The toxin is thereby transported in the systemic circulation and the microvesicles, with their toxic cargo, are taken up by kidney cells (Karpman et al, 2017). Once intracellular the toxin is released from the microvesicles and leads to inhibited protein synthesis (Ståhl et al, 2015)
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