Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1. Shiga toxin-producing Escherichia coli (STEC) is an emerging foodborne and waterborne pathogen. STEC infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), with potentially lethal consequences (1). Due to a very low infectious dose and ease of person-to-person spread, STEC infection is the leading cause of death from foodborne bacterial infection in children (2). Presently, there are no postexposure therapeutics or vaccines available for STEC infection. Due to recent outbreaks of E. coli O157:H7 in the United States and the emergence of highly virulent new strains, such as E. coli O104:H4, which caused the deadliest HUS outbreak in Germany in 2011, STEC remains a major challenge for food safety and public health (3-6).The primary virulence factors of STEC, Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), are AB 5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits and are known as type II ribosome-inactivating proteins (RIPs). The A subunits of Stx1 and Stx2 consist of the catalytically active A1 (residues 1 to 251 in Stx1 and 1 to 250 in Stx2) and A2 (residues 252 to 293 in Stx1 and 251 to 297 in Stx2) chains, which are cleaved by the protease furin and kept together by a disulfide bond (7). The B subunits bind ...

Fusarium head blight (FHB) reduces crop yield and results in contamination of grains with trichothecene mycotoxins. We previously showed that mitochondria play a critical role in the toxicity of a type B trichothecene. Here, we investigated the direct effects of type A and type B trichothecenes on mitochondrial translation and membrane integrity in Saccharomyces cerevisiae. Sensitivity to trichothecenes increased when functional mitochondria were required for growth, and trichothecenes inhibited mitochondrial translation at concentrations, which did not inhibit total translation. In organello translation in isolated mitochondria was inhibited by type A and B trichothecenes, demonstrating that these toxins have a direct effect on mitochondrial translation. In intact yeast cells trichothecenes showed dose-dependent inhibition of mitochondrial membrane potential and reactive oxygen species, but only at doses higher than those affecting mitochondrial translation. These results demonstrate that inhibition of mitochondrial translation is a primary target of trichothecenes and is not secondary to the disruption of mitochondrial membranes.

The A1 subunits of Shiga toxin 1 (Stx1A1) and Shiga toxin 2 (Stx2A1) interact with the conserved C termini of ribosomal-stalk P-proteins to remove a specific adenine from the sarcin/ricin loop. We previously showed that Stx2A1 has higher affinity for the ribosome and higher catalytic activity than Stx1A1. To determine if conserved arginines at the distal face of the active site contribute to the higher affinity of Stx2A1 for the ribosome, we mutated Arg172, Arg176, and Arg179 in both toxins. We show that Arg172 and Arg176 are more important than Arg179 for the depurination activity and toxicity of Stx1A1 and Stx2A1. Mutation of a single arginine reduced the depurination activity of Stx1A1 more than that of Stx2A1. In contrast, mutation of at least two arginines was necessary to reduce depurination by Stx2A1 to a level similar to that of Stx1A1. R176A and R172A/R176A mutations eliminated interaction of Stx1A1 and Stx2A1 with ribosomes and with the stalk, while mutation of Arg170 at the active site reduced the binding affinity of Stx1A1 and Stx2A1 for the ribosome, but not for the stalk. These results demonstrate that conserved arginines at the distal face of the active site are critical for interactions of Stx1A1 and Stx2A1 with the stalk, while a conserved arginine at the active site is critical for non-stalk-specific interactions with the ribosome. Arginine mutations at either site reduced ribosome interactions of Stx1A1 and Stx2A1 similarly, indicating that conserved arginines are critical for ribosome interactions but do not contribute to the higher affinity of Stx2A1 for the ribosome. Shiga toxin (Stx)-producing Escherichia coli (STEC) is an emerging foodborne and waterborne pathogen responsible for hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC), which are the leading causes of acute renal failure and mortality in children in the United States (1). STEC serotypes, such as E. coli O157:H7, are associated with severe disease (2). Antibiotics are known to exacerbate the disease symptoms, and at present, there are no FDA-approved vaccines or therapeutics against STEC infection (3-5). STEC produces a family of structurally and functionally related virulence factors called Shiga toxins, the most predominant ones being Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) (6). Stx2 and Stx1 have one prototype (Stx1a and Stx2a) and several subtypes. Stxs are type II ribosome-inactivating proteins (RIPs) with a catalytically active A subunit attached to a pentamer of B subunits. The B subunits facilitate the endocytosis of the toxins into the cell by binding to a common receptor globotriaosylceramide (GB3 or CD77). The toxin travels in a retrograde manner from the endosome to the endoplasmic reticulum (ER) via the Golgi network (7). In order to intoxicate the cell, the A subunit is cleaved into the A1 fragment and the A2 fragment, which remain together via a disulfide bond. After reduction of the disulfide bond, the A1 fragment is translocated into the cytosol from the ER, where it refolds into an active confor...

Shiga toxin producing Escherichia coli O157:H7 (STEC) is one of the leading causes of food-poisoning around the world. Some STEC strains produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2) or variants of either toxin, which are critical for the development of hemorrhagic colitis (HC) or hemolytic uremic syndrome (HUS). Currently, there are no therapeutic treatments for HC or HUS. E. coli O157:H7 strains carrying Stx2 are more virulent and are more frequently associated with HUS, which is the most common cause of renal failure in children in the US. The basis for the increased potency of Stx2 is not fully understood. Shiga toxins belong to the AB5 family of protein toxins with an A subunit, which depurinates a universally conserved adenine residue in the α-sarcin/ricin loop (SRL) of the 28S rRNA and five copies of the B subunit responsible for binding to cellular receptors. Recent studies showed differences in the structure, receptor binding, dependence on ribosomal proteins and pathogenicity of Stx1 and Stx2 and supported a role for the B subunit in differential toxicity. However, the current data do not rule out a potential role for the A1 subunits in the differential toxicity of Stx1 and Stx2. This review highlights the recent progress in understanding the differences in the A1 subunits of Stx1 and Stx2 and their role in defining toxicity.

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