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Shiga-Like Toxin 2 of Enterohemorrhagic Escherichia Coli (EHEC): Genetic Organization and Effects of Toxin in a Murine Model of EHEC Infection

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Technical Report

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Uniformed Services University Of The Health Sciences Bethesda United States

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Enterohemorrhagic Escherichia coli EHEC strains associated with diarrhea, hemorrhagic colitis, and the hemolytic uremic syndrome in humans and E. coli strains responsible for edema disease of swine produce cytotoxins related to Shiga toxin of Shigella dysenteriae type 1. These Shiga-like toxins SLTs include Shiga-like toxin type I SLT-I, Shiga-like toxin type II SLT-II, and Shiga-like toxin type II variant SLT-llv and constitute a family whose members are related in structure and biological activities. The major objective of this study was to analyze the molecular genetics of Shiga-like toxin type II so as to better understand the relationship of SLT-II to other members of the Shiga toxin family. One specific aim of this study was to characterize the SLT-II operon. The promoter was mapped by primer extension and S1 nuclease protection analyses, and the transcription terminator was identified by nucleotide sequence homology computer search. In contrast to the Shiga toxin and SLT-I operon promoters, the SLT-II operon promoter was not regulated by the fur gene product and its iron co-repressor. The transcriptional efficiency of the SLT-II operon promoter was equivalent to the Shiga toxin operon promoter under low iron growth conditions. Northern blot analysis revealed that the SLT-II operon was transcribed as a single polycistronic mRNA. These data indicated that the difference in the level of production between Shiga toxinSLT-l and SLT-II as well as the single A subunit to multiple B subunit stoichiometry of the SLT-II holotoxin are not consequences of regulation at the transcriptional level.Another specific aim of this research was to evaluate the virulence of E. coli strains containing recombinant Shiga-like toxin plasmids in an animal model. In contrast to Shiga toxin-producing bacteria, SLT-ll-producing bacteria killed orally infected mice.

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