SHIGA TOXIN MODE OF ACTION IN E. COLI O157:H7 DISEASE

Tom G. Obrig

Department of Microbiology and Immunology, University of Rochester, Box 672, 601 Elmwood Avenue, Rochester, NY 14642

Received 12/2/97 Accepted 12/8/97

3. BIOCHEMICAL FEATURES OF THE SHIGA TOXINS

3.1 Subunit protein structure

Structurally, toxins in the Stx family each consist of one A-subunit responsible for the inhibitory activity and five identical B-subunits that determine binding specificity to eukaryotic cells (2,8). These toxins have been the subject of comprehensive review articles (2,8,9). Stx has been purified from Shigella dysenteriae 1 and shown to be a 68,000 kDa multi-subunit complex Shiga toxin comprised of a single 32kDa "A-subunit" and 5 x 7.7kDa "B-subunits" (10,11). Shiga-like toxins ( Stx1, Stx2, and Stx2c) have also been purified from E. coli and appear similar to Shiga toxin (2). Despite minor differences between the A-subunit amino acid sequences of the Stx subspecies (Stx1, Stx2, Stx2c, etc.), all appear to exhibit identical enzymatic activity. However, differences in the B-subunit amino acid sequences are important in determining the receptor to which the Stx will bind, and thus the target cell type. Stx, Stx1, Stx2 and Stx2c all bind to the same receptor, the glycosphingolipid, Gb3 (9). Antigenically, Stx1 from E. coli is considered to be almost identical to Shiga toxin (Stx) from S. dysenteriae 1, but is distinct from E. coli Stx2, and Stx2c (2). Another member of the Stx family , Stx2e, is produced by unique isolates of E. coli that infect pigs, and thus is also called pig edema factor. Stx2e is not only antigenically distant from the other toxins, but prefers to bind to a different glycolipid, i.e. Gb4 (12,13).

3.2 Stx removal of a single base from 28S rRNA

The A-subunit of the Stx inhibits protein synthesis by inactivating ribsomes. The A subunit enzymatically removes a purine base from the 28S rRNA within the 60S ribosomal subunit (14). This modification of the ribosome prevents interaction of peptide elongation factors (EF-1 and EF-2) with the altered ribosome and stops protein synthesis at the level of peptide elongation (15,16).

3.3. Stx is a member of the ribosome inactivating protein family

Stx A-subunits work biochemically in a manner identical to that of a series of other ribosome inactivating proteins (RIPs) derived from higher plants. It has been 25 years since the first reports appeared defining the RIPs as ribosome-inactivating proteins (17,18). Primary amino acid structure is highly conserved within the enzymatic site of the RIPs (19). In contrast to the Stxs, most of the plant-derived RIPs exist as a single peptide without associated B-subunit proteins. An exception to this is the toxin, ricin. It should be emphasized that Stx and ricin are much more potent than the single peptide RIPs because their B-subunits direct the toxins to specific high affinity receptors on eukaryotic cells, whereas the single peptide RIPs interact weakly and less discriminately to certain sugar residues on cells. The natural importance of RIPs to bacteria is questionable with the exception that they may help in spread of the pathogen by increasing bacterial shedding during the disease process. Person-to-person spread of EHEC is a well-documented problem within a household and in day care centers (5). Plant-derived RIPs exhibit natural antiviral activity against plant RNA viruses, and likely serve a natural role in protecting plants from their viral pathogens. To date, attempts to harness the power of of RIPs by biotechnology for use as immunotoxins for antitumor purposes has been a general disappointment. Although there have been some partial successes, the failures far outnumber the successes.