Abstract
AB5 protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB5 toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.
Highlights
Particular Shiga toxin (Stx)-producing Escherichia coli (STEC) strains produce, in addition to Stx, another AB5 toxin, the subtilase cytotoxin (SubAB) [1,2]
For StxA2a-His, the expression in E. coli C43 and auto-induction medium at 37 ◦ C initial growth and 20 ◦ C incubation overnight resulted in the highest protein levels in the soluble fraction of the bacterial cell
StxB2a-His revealed the highest amount of protein in the culture supernatant when expressed with E. coli C41 cultivated in LB broth at initially 37 ◦ C, and 20 ◦ C
Summary
Particular Shiga toxin (Stx)-producing Escherichia coli (STEC) strains produce, in addition to Stx, another AB5 toxin, the subtilase cytotoxin (SubAB) [1,2]. The production of one or more Stx variants is a major pathogenicity factor of enterohemorrhagic E. coli (EHEC). EHEC comprise a STEC subgroup being able to cause gastrointestinal diseases and extraintestinal sequelae such as the life-threatening hemolytic-uremic syndrome (HUS) [3]. The A-subunit StxA causes an irreversible removal of an adenine residue from the 28S rRNA in eukaryotic cells [5]. Due to this rRNA N-glycosidase activity, protein synthesis is inhibited and apoptosis of the affected cell is initiated [6]. The 32 kD A-subunit is a single polypeptide chain that consists of the two domains A1 and A2, which are inter-connected by a disulfide bond. The 27.5 kD A1-domain exhibits the catalytic activity, whereas the
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