The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1.
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.
Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey, USA.
Debaleena
D
Basu
Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey, USA.
Xiao-Ping
XP
Li
Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey, USA.
Jennifer N
JN
Kahn
Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey, USA.
Kerrie L
KL
May
Department of Biochemistry and Microbiology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey, USA.
Peter C
PC
Kahn
Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey, USA tumer@aesop.rutgers.edu.
Nilgun E
NE
Tumer
D000818:Animals; D002460:Cell Line; D004927:Escherichia coli Infections / Q000382:microbiology; D057809:HEK293 Cells; D006463:Hemolytic-Uremic Syndrome / Q000382:microbiology; D006801:Humans; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics; D051381:Rats; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D022621:Shiga Toxin / Q000378:metabolism*; D022622:Shiga Toxin 1 / Q000378:metabolism*; D022641:Shiga Toxin 2 / Q000378:metabolism*; D054323:Shiga-Toxigenic Escherichia coli / Q000378:metabolism*
D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; C493898:Shiga toxin subunit A; D022621:Shiga Toxin