Biochemical and cell biological effects of the single A-subunit of the bacterial subtilase cytotoxin

Abstract

The subtilase cytotoxin is secreted by shiga toxin-producing E. coli strains and is mainly transmitted to humans through contaminated food and water. The toxin belongs to the family of AB5 toxins with a typical structure of a SubB pemtamer, which is responsible for toxin binding and uptake, and an A-subunit, which represents the enymatically active toxin moiety. Research of our group together with that of Prof. Schmidt (Hohenheim) could show for the first time that SubA induces cytotoxic effects in the absence of the B-domain. This discovery was completely unexpected, as it was previously assumed in expert circles that an intact holotoxin was necessary for this. In my PhD. thesis I investigated the cellular and molecular mechanisms underlying the single SubA effect in more detail. Since the previous work was mainly done with human cervical cancer ephithelial cells (HeLa), I first investigated whether human colon cancer-derived epithelial cells (HCT116), are target cells for the cytotoxic effects caused by SubA alone. These cells represent a more physiological cell line for an intestinal pathogen to study the autonomous effects of this toxin component. Having confirmed the cytotoxic effect of SubA, without SubB, on HCT116 cells, the question arose whether these effects were mediated intracellularly or extracellularly. Autonomous SubA uptake could be visualized by fuorescence microscopy and showed that SubA accumulates in the endoplasmic reticulum even in the absence of SubB. This observation was confirmed by Western blot results. These showed that SubA alone is capable of cleaving and thus inactivating the substrate GRP78/BiP in a time- and concentration-dependent manner. Furthermore, a series of inhibitor experiments showed that SubA, like SubAB, enters the ER through a retrograde transport pathway. These results suggested that SubA must carry an independent ER target sequence and indeed a sequence at the C-terminus, the SEEL motif, was identified as a critical motif for ER transport. Taken together, SubA shows similar cellular uptake and cytotoxic mode of action on eukaryotic cells as SubAB holotoxin. However, it should be noted that uptake of SubA and/or its transport into the ER is less efficient in the absence of SubB. This suggests that SubB is not essential for causing cytotoxicity, but that the presence of SubB enormously enhances the cytotoxic effect, which may allow the toxin to act at lower concentrations. However, the SubA effect alone should not be underestimated, since SubA subunits secreted by bacteria that have not found a SubB pentamer to form a holotoxin could extend and enhance the pathogenic potential of subtilase-producing E. coli strains.