Identification of amino acids critical for the cytotoxicity of Shiga toxin 1 and 2 in Saccharomyces Cerevisiae

Abstract

Shiga toxins (Stx1 and Stx2) are produced by E. coli O157:H7, which is a leading cause of foodborne illness. The A subunits of Stx1 (Stx1A) and Stx2 (Stx2A) are ribosome inactivating proteins (RIPs) that inhibit translation by removing an adenine from the highly conserved α-sarcin ricin loop (SRL) of the large rRNA. Here, we used mutagenesis in S. cerevisiae to identify residues critical for cytotoxicity of Stx1A and Stx2A. The A subunits depurinated the SRL, inhibited translation and caused apoptotic-like cell death in yeast. Single mutations in Asn75, Tyr77, Glu167 and Arg176 reduced the cytotoxicity of both toxins around 10-fold. However, Asn75 and Tyr77 were more critical for the depurination activity of Stx2A, while Arg176 was more critical for the depurination activity of Stx1A. The crystal structures of the two proteins lack electron density for some surface loops, including one which is adjacent to the active site in both molecules. Modeling these loops changed neither the secondary nor the tertiary structures of the rest of the protein. Analysis of solvent accessible surface areas indicated that Asn75 and Tyr77 are more exposed in Stx2A, while Arg176 is more exposed in Stx1A, indicating that residues with higher surface exposure were more critical for enzymatic activity. Double mutations at Glu167 and Arg176 eliminated the depurination activity and cytotoxicity of both toxins. C-terminal deletions of A chains eliminated cytotoxicity of both toxins, but showed functional differences. Unlike Stx1A, cytotoxicity of Stx2A was lost before its ability to depurinate ribosomes. These results identify residues that affect enzymatic activity and cytotoxicity of Stx1A and Stx2A differently and demonstrate that the function of these residues can be differentiated in yeast. The extent of ribosome depurination and translation inhibition did not correlate with the extent of cell death, indicating that depurination of the SRL and inhibition of translation are not entirely responsible for cell death.

The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

“…To compare the cytotoxicities of the A1 subunits of Stx1 and Stx2 in vivo, the mature A1 subunits were expressed in the yeast S. cerevisiae, with the V5 and 10ϫHis epitope tags under the tightly controlled GAL1 promoter. Previously reported results indicated that the V5 and 10ϫHis epitope tags do not affect the activity of the A subunits (39,40). Irreversible growth inhibition by Stx1A1 and Stx2A1 was determined by plating cells onto medium containing 2% dextrose after galactose induction for the indicated times in liquid medium.…”

“…Its interaction with the negatively charged RNA may reduce the availability of the active-site residues to perform catalysis. Previously reported results showed that mutations of residues at the active sites of Stx1A and Stx2A affect cytotoxicity and catalytic activity differently (39). The residues previously shown to be involved in the depurination reaction in Stx1 and Stx2 (E167 and R170) are more exposed in Stx2A1 than in Stx1A1 (39).…”

“…Previously reported results showed that mutations of residues at the active sites of Stx1A and Stx2A affect cytotoxicity and catalytic activity differently (39). The residues previously shown to be involved in the depurination reaction in Stx1 and Stx2 (E167 and R170) are more exposed in Stx2A1 than in Stx1A1 (39). Further studies are needed to understand if these differences are responsible for the affinity of the Shiga toxins for the RNA and their catalytic activity.…”

“…Moreover, the role of the A1 subunits in differential toxicity was not investigated further because Stx1a and Stx2a showed similar translation-inhibitory activities in a cell-free system (24). We have developed the yeast Saccharomyces cerevisiae as a powerful tool to examine the toxicity and the ribosome interactions of the A subunits of Stx1a and Stx2a independent of the B subunits in vivo and have identified amino acids critical for toxicity (39). The depurination activity of the A subunits of Stx1a and Stx2a was reduced in the yeast P-protein mutants, and Stx1a and Stx2a differed in their requirements for the stalk proteins in vivo (40).…”

Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome

“…Early studies using the holotoxins identified Glu167 and Arg170 of the A1 subunits as important for catalytic activity (33)(34)(35)(36). The corresponding Arg180 in RTA is proposed to promote cleavage of the adenine by protonating position N3 of the adenine (37)(38)(39), while the corresponding Glu177 polarizes a surrounding water molecule to produce a hydroxide ion that aids in the cleavage of adenine.…”