Cytotoxicity of enterohemorrhagic Escherichia coli correlates with Shiga toxin rRNA-N-glycosidase activity but not with toxin subunit ratios.

The expression of Shiga toxins (Stx) is the major pathogenicity factor of enterohemorrhagic Escherichia coli (EHEC). Stx structurally consist of one enzymatically active A-subunit (StxA) and a pentamer of non-covalently linked B-subunits (StxB). While the B-subunit mediates binding to the cell surface receptors globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4), as well as the cellular uptake of the toxin, the A-subunit exhibits a rRNA-N-glycosidase activity leading to inhibition of protein synthesis and consequently apoptosis of the eukaryotic target cells. Recent work has demonstrated that free A-subunits of Stx are able to intoxicate eukaryotic target cells even in absence of corresponding B-subunits and that such free A-subunits indeed occur in the natural context of EHEC wildtype strains. In a previous study, we demonstrated that Stx2a subunits are not produced in the expected 1:5 (StxA2a:StxB2a) stoichiometry, but in ratios of at least 1:1, indicating that substantial levels of free StxA2a are present in the bacterial environment. Therefore, in the present study, we investigated whether these differences in the Stx2a subunit ratios and the resulting amounts of free StxA2a determine the cytotoxicity of culture supernatants from EHEC wildtype strains towards Vero B4 cells. Analysis of the cytotoxic effects of EHEC culture supernatants by neutral red uptake assay and morphological analysis revealed that all investigated Stx2a-producing E. coli strains significantly reduced the viability of Vero B4 cells. However, neither the Stx2a subunit ratio nor the absolute StxA2a concentration correlated with the extent of cytotoxicity. Protein synthesis was reduced in Vero B4 cells after 18 h of exposure to the culture supernatants of the EHEC wildtype strains, with E. coli O26:H11 strain HUSEC018 and E. coli O113:H21 strain TS18/08 causing inhibition to a similar extent as diphtheria toxin. These differences did not correlate with the Stx2a subunit ratio or the overall StxA2a levels. Instead, the results of our study indicated that the catalytic activity of StxA2a determines the cytotoxicity of the investigated EHEC wildtype strains. In conclusion, neither the Stx2a subunit ratios nor the levels of free StxA2a directly determine the cytotoxicity in Stx2a-producing EHEC wildtype strains towards Vero B4 cells. Instead, the rRNA-N-glycosidase activity of StxA2a is closely associated with inhibition of protein synthesis and cytotoxicity and thus likely represents a major determinant of cytotoxicity, although additional strain-specific bacterial factors and target cell responses in this multistep intoxication process remain to be elucidated.