Abstract
The cardinal virulence factor of human-pathogenic enterohaemorrhagic Escherichia coli (EHEC) is Shiga toxin (Stx), which causes severe extraintestinal complications including kidney failure by damaging renal endothelial cells. In EHEC pathogenesis, the disturbance of the kidney epithelium by Stx becomes increasingly recognised, but how this exactly occurs is unknown. To explore this molecularly, we investigated the Stx receptor content and transcriptomic profile of two human renal epithelial cell lines: highly Stx-sensitive ACHN cells and largely Stx-insensitive Caki-2 cells. Though both lines exhibited the Stx receptor globotriaosylceramide, RNAseq revealed strikingly different transcriptomic responses to an Stx challenge. Using RNAi to silence factors involved in ACHN cells’ Stx response, the greatest protection occurred when silencing RAB5A and TRAPPC6B, two host factors that we newly link to Stx trafficking. Silencing these factors alongside YKT6 fully prevented the cytotoxic Stx effect. Overall, our approach reveals novel subcellular targets for potential therapies against Stx-mediated kidney failure.
Highlights
The cardinal virulence factor of human-pathogenic enterohaemorrhagic Escherichia coli (EHEC) is Shiga toxin (Stx), which causes severe extraintestinal complications including kidney failure by damaging renal endothelial cells
ACHN cells were highly sensitive to Stx2a, showing a remarkable decrease in viability at 0.5 ng/mL Stx2a, where only 13.4% of cells survived; this was a drastic reduction from the cell viability of 86.8% at the low Stx2a concentration of 0.5 pg/mL
As ACHN cells only showed a comparable level of survival when exposed to the lowest concentration of 0.5 pg/mL Stx2a (86.8%), these results show that Caki-2 cells were 106 times less susceptible to the toxin
Summary
The cardinal virulence factor of human-pathogenic enterohaemorrhagic Escherichia coli (EHEC) is Shiga toxin (Stx), which causes severe extraintestinal complications including kidney failure by damaging renal endothelial cells. In EHEC pathogenesis, the disturbance of the kidney epithelium by Stx becomes increasingly recognised, but how this exactly occurs is unknown To explore this molecularly, we investigated the Stx receptor content and transcriptomic profile of two human renal epithelial cell lines: highly Stx-sensitive ACHN cells and largely Stx-insensitive Caki-2 cells. The Stx B subunit binds to Gb3Cer, the dominant receptor on human endothelial cells[12], after which Stx is internalised by clathrin-dependent and -independent endocytic processes[22,24] and intracellularly trafficked in a variety of cells. One possibility is that these differences may be related to Stx receptor content and/or how Stx is taken up or trafficked in these cell lines
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