Molecular, Biochemical and Functional Analysis of Shiga Toxin and Subtilase Cytotoxin Subunits of Enterohemorrhagic Escherichia coli

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The observation, that the A-subunits of E. coli subtilase and Shiga toxin can cause cytotoxicity without their respective B-subunits, represents a change in a basic toxicological paradigm. Prompted by these recent findings, the main objective of the proposed project is to characterize this unexpected behavior of both AB5-toxins in detail. Therefore, we will further characterize the cell binding and intracellular transport of the SubA-subunit and analyze the molecular mechanisms underlying the cytotoxic effects of the Stx2 A-subunit (StxA2) in the absence of a binding/transport subunit in human cells. In detail, we will investigate binding, uptake and intracellular transport of StxA2 and directly compare these processes with the canonical holotoxins Stx2, and Stx1, consisting of 1 A- and 5 B-subunits, respectively. Furthermore, pharmacological inhibitors including human peptides that neutralize the cytotoxic effects of Stx2 and Stx1 will be discovered and characterized, as done before for SubAB. Moreover, the mode of expression, assembly and secretion of the mature toxin from bacterial cells leaves unresolved questions. Therefore, control of stx subunit gene transcription and subunit assembly will be characterized to understand whether free A-subunits are present in the supernatant of bacterial cultures. This work will contribute to a deeper understanding of the molecular and cellular mechanisms underlying the pathogenesis of EHEC-mediated diseases and should provide a starting point for novel therapeutic options.