| The Gram-negative bacterial pathogen Escherichia coli O157:H7 accounts for an estimated 110,000 cases of disease each year in the United States. The pathogenicity of E. coli O157:H7 is due in large part to the production of one or more members of the Shiga toxin (Stx) family. Stx is an AB5 toxin. The A-subunit possesses RNA N-glycohydrolase activity and mediates host cell damage by cleaving a nucleotide from the 28S ribosomal RNA, inhibiting protein synthesis. The A-subunit is noncovalently associated with a ring-like structure comprised of five identical B-subunits which surrounds the carboxyl-terminus of the A-subunit. The B-pentamer binds to the glycolipid globotriaosylceramide (Gb3) on the surface of host cells and delivers the A-subunit into the cell. There are two immunologically distinct forms of Stx (Stx1 and Stx2) which share 57% amino acid sequence identity and a highly conserved structure. While strains of E. coli O157:H7 can produce Stx1, Stx2, or both, recent data has indicated that Stx2 is far more potent than Stx1 in vivo, and epidemiologic studies suggest that the vast majority of fatal disease cases in humans result from Stx2-producing E. coli strains. However, the molecular basis for the difference in potency between Stx1 and Stx2 is unclear.;Neutrophils have been proposed to act as carriers for Stxs in the bloodstream, and controversial reports suggest that differences in the interactions of Stx1 and Stx2 with human neutrophils occur. To clarify the existing controversy in the literature, the effect of Stx1 and Stx2 on human neutrophils was examined, and it was demonstrated that neither Stx1 nor Stx2 interacts directly with neutrophils.;Differences in the glycan binding specificities of Stx1 and Stx2 have been demonstrated, and it is possible that the toxins may interact with different receptors or co-receptors in vivo, thus mediating the difference in potency. To yield insight into the factors governing Stx binding specificity, binding of Stx1, Stx2, and receptor binding site mutants of the toxins to receptor analogues was examined across different binding platforms, receptor densities, and in the presence of heterogeneous glycan populations. Glycan preference was mapped to binding site 2, since reciprocal mutation of a single amino acid (asparagine 32 of Stx1B/serine 31 of Stx2B) reversed binding preference.;Differences in the relative stabilities of Stx1 and Stx2 could also account for the difference in potency, as a less stable toxin might more readily disassemble and deliver toxic components to the cell, or expose a new binding interface. In the present study, a comparative analysis of the solution-state assembly and stability of Stx1B, Stx2B, and B-subunit mutants revealed that Stx2B is approximately 50-fold less stable than Stx1B, and that a single amino acid (leucine 41 of Stx1B/glutamine 40 of Stx2B) mediates the difference in stability.;Potency of the Stx1 N32S (receptor binding) and Stx1 L41Q (stability) mutants was assessed in mice. Neither single amino acid mutation in Stx1 affected potency in vivo; however, it is possible that potency is co-dependent upon both receptor binding and staibility, and future experiments will address this hypothesis. |
