Escherichia coli can cause diarrhea by several distinct mechanisms. A newly recognized group of E. coli cause hemorrhagic colitis and the hemolytic uremic syndrome. These enterohemorrhagic E. coli (EHEC) produce elevated levels of Shiga-like toxin I (SLT-I) or Shiga-like toxin II (SLT-II) or both cytotoxins. SLT-I and SLT-II are antigenically distinct members of a family of cytotoxins that are related to the Shiga toxin of Shigella dysenteriae 1. These toxins inhibit protein synthesis in eucaryotic cells by a mechanism identical to that of the plant toxin ricin; the toxins act as 28 S rRNA glycosidases. Moreover, Shiga toxin, SLT-I, and SLT-II appear to have the same eucaryotic cell receptor, a glycolipid called GB3. The genes for Shiga toxin, SLT-I, and SLT-II have been cloned in this laboratory and their nucleotide and deduced amino acid sequences determined. The results of these studies indicate that SLT-I and Shiga toxin are >99% homologous, whereas SLT-II shares 55-60% amino acid homology with Shiga toxin/SLT-I. The long-term goals of this project are to analyze the synthesis and biologic activity of the Shiga/SLT toxin family at the molecular levels and to determine if these have essential or accessory roles in the pathogenesis of bacillary dysentery, hemorrhagic colitis, and the hemolytic uremic syndrome.
The specific aims designed to achieve these objectives are to: 1) investigate the relationships between the structure and biological activities of Shiga toxin/SLT- I and SLT-II by using site-directed mutagenesis, monoclonal antibodies, and synthetic peptides; 2) compare the regulation of the Shiga toxin/SLT-I and SLT-II operons by characterizing SLT-II transcripts, mapping and defining the relative strengths of the promoters for the Shiga toxin/SLT-I and SLT-II operons, identifying and mapping other Shiga toxin/SLT-I and SLT-II regulatory genes, and locating the transcription termination sequences for the Shiga toxin/SLT-I and SLT-II operons; 3) assess the role of Shiga toxin in the virulence of S. dysenteriae 1 and of SLT-I and SLT-II in the pathogenicity of EHEC by constructing non-toxinogenic mutants and comparing them with isogenic toxinogenic strains in appropriate animals models; 4) characterize the SLT operon in strains of E. coli that produce low levels of SLT by cloning and sequencing the low-level SLT genes, analyzing the regulation of the low-level SLT operon, and investigating the function of low-level SLT in E. coli.
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