Abstract
The subtilase cytotoxin (SubAB) is secreted by certain Shiga toxin-producing Escherichia coli (STEC) strains and is composed of the enzymatically active subunit SubA and the pentameric binding/transport subunit SubB. We previously demonstrated that SubA (10 µg/ml), in the absence of SubB, binds and intoxicates the human cervix cancer-derived epithelial cell line HeLa. However, the cellular and molecular mechanisms underlying the cytotoxic activity of SubA in the absence of SubB remained unclear. In the present study, the cytotoxic effects mediated by SubA alone were investigated in more detail in HeLa cells and the human colon cancer cell line HCT116. We found that in the absence of SubB, SubA (10 µg/ml) is internalized into the endoplasmic reticulum (ER), where it cleaves the chaperone GRP78, an already known substrate for SubA after its canonical uptake into cells via SubB. The autonomous cellular uptake of SubA and subsequent cleavage of GRP78 in cells is prevented by treatment of cells with 10 µM brefeldin A, which inhibits the transport of protein toxins into the ER. In addition, by analyzing the SubA mutant SubAΔC344, we identified the C-terminal SEEL motif as an ER-targeting signal. Conclusively, our results strongly suggest that SubA alone shares the same intracellular transport route and cytotoxic activity as the SubAB holotoxin.
Highlights
Shiga toxin-producing Escherichia coli (STEC) strains are enteric bacterial pathogens and when transmitted to humans, they can cause severe enteric diseases ranging from serious
We have examined whether human colon cancer-derived epithelial cells are target cells for the cytotoxic effects caused by SubA alone, since these cells represent, for an intestinal pathogen, a more physiological cell line to investigate the autonomous effects of this toxin component
An overall lower concentration was used and it should be noted that fluorescently labeled SubA and non-labeled SubB were mixed in a molar ratio of 1:5
Summary
The cytotoxic effects of SubA in the absence of SubB have already been described for HeLa and Vero cells (Funk et al 2015). Treatment of cells with SubA alone for 48 h resulted in an obvious change in the morphology of HCT116 cells as well as HeLa cells, which were included in this experiment as a positive control for a SubA susceptible cell line This change in cell morphology represents an established specific and selective endpoint to monitor the cytotoxic effects of SubAB or SubA in cell-based experiments (Funk et al 2015). HeLa cells were incubated with ATTO647N-labeled SubA (30 μg/ml) and/or the AlexaFluor594-conjugated B-subunit of the cholera toxin (CTB), which was used as an established and specific marker for the ER. In contrast to the wildtype (shown in Fig. 1), the mutant S ubAΔC344 alone did neither cause morphological changes nor a decrease of the amount of HCT116 or HeLa cells (Fig. 4a). When cells were incubated with the combination of SubB and S ubAΔC344, GRP78 was cleaved in the
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