The enzyme subunit SubA of Shiga toxin-producing E. coli strains demonstrates comparable intracellular transport and cytotoxic activity as the holotoxin SubAB in HeLa and HCT116 cells in vitro

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.

Novel Aspects of the SubA Subunit of the Subtilase Cytotoxin

“…However Western blot experiments by Funk et al showed a concentration-dependent SubA binding to HeLa cells in the absence of SubB [8]. Cell surface binding of SubA was confirmed by flow cytometry [9], but to the best of our knowledge, a specific SubA receptor has yet not been identified. Since BiP/GRP78, the molecular substrate of SubA protease in the ER, is also present at the cell surface [20], it can be speculated that cell surface bound GRP78 might be a potential SubA receptor.…”

“…In the ER, SubA exhibits its enzyme activity and cleaves BiP/GRP78. However, BiP/GRP78 cleavage was delayed when cells were treated with SubA alone compared to SubAB [8,9]. To reach the ER, SubA alone seems to take the same overall route as SubAB via the retrograde trafficking pathway [9] (Figure 2).…”

“…Alternatively, a genome-wide CRISPR/Cas9 knockout screen for SubA-induced cell death, similar to how it was performed for SubAB, where O-glycans were identified as a further SubAB receptor [21], could provide new information. After binding of SubA, it is autonomously taken up into the host cell and causes similar cell morphological changes in several eukaryotic cell lines similar to SubAB [9]. However, cell death induced with SubA alone required higher concentrations of SubA compared to the amount of SubA in the SubAB holotoxin [9].…”

“…After binding of SubA, it is autonomously taken up into the host cell and causes similar cell morphological changes in several eukaryotic cell lines similar to SubAB [9]. However, cell death induced with SubA alone required higher concentrations of SubA compared to the amount of SubA in the SubAB holotoxin [9]. In vitro and in vivo experiments by Paton et al identified the endoplasmic Hsp70 chaperone BiP/GRP78 as cellular target for SubAB.…”

“…Until now, it was a major scientific paradigm that only a complete AB 5 holotoxin complex can cause cytotoxicity. However, recent publications show that the A subunit of the subtilase cytotoxin (SubA) can cause cell death in the absence of its corresponding B subunit (SubB) [8,9].…”