Shiga toxin attacks bacterial ribosomes as effectively as eucaryotic ribosomes.
Several pathogenic bacteria, including Shigelladysenteriae and certain strains of Escherichia coli, produce potent class 2 ribosome inhibiting proteins (RIPs) termed Shiga toxins (Stx). The toxins are bipartite molecules composed of a single A chain (StxA) noncovalently associated with a pentamer of receptor-binding B subunits (StxB). StxA and Stx1A from E. coli are protoxins. Proteolysis generates an A1 enzyme (28 kDa) and an A2 fragment (3 kDa), which remain bound, inactivating the enzyme, until a disulfide bond linking them is reduced. Efforts to express active recombinant Stx1A1 in the cytoplasm of E. coli were very difficult and led to the hypothesis that Stx1A1 is toxic to E. coli. We created the gene for a His-tagged Stx1A1 (cStx1A1) and expressed it in E. coli from a tightly controlled expression vector. About 1-2 mg of protein can be purified in a one-step isolation from 1 L of culture. cStx1A1, RTA, and PAP exhibited similar high toxicity against the Artemia ribosomes with IC50 values near 1 nM. Surprisingly, Stx1A1 had an IC50 of 0.8 nM against E. coli ribosomes, about the same as it had for Artemia ribosomes. This is about 250 times more active than PAP against bacterial targets, making Stx1A1 the most powerful RIP toxin presently known against E. coli ribosomes.
Suh
J K
JK
Institute of Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas, Austin 78712, USA.
Hovde
C J
CJ
Robertus
J D
JD
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001156:Artemia / Q000187:drug effects* / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000187:drug effects* / Q000378:metabolism; D012276:Ricin / Q000494:pharmacology; D022603:Shiga Toxins; D012762:Shigella dysenteriae / Q000502:physiology*; D012995:Solubility
D000998:Antiviral Agents; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D022603:Shiga Toxins; D012276:Ricin; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein