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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Unknown (R22)
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Bacteriology and Mycology Subcommittee 1 (BM)
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U.S. Uniformed Services University of Health Science
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Bunger, Joshua C; Melton-Celsa, Angela R; Maynard, Ernest L et al. (2015) Reduced Toxicity of Shiga Toxin (Stx) Type 2c in Mice Compared to Stx2d Is Associated with Instability of Stx2c Holotoxin. Toxins (Basel) 7:2306-20
Boisen, Nadia; Hansen, Anne-Marie; Melton-Celsa, Angela R et al. (2014) The presence of the pAA plasmid in the German O104:H4 Shiga toxin type 2a (Stx2a)-producing enteroaggregative Escherichia coli strain promotes the translocation of Stx2a across an epithelial cell monolayer. J Infect Dis 210:1909-19
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Melton-Celsa, Angela; Mohawk, Krystle; Teel, Louise et al. (2012) Pathogenesis of Shiga-toxin producing escherichia coli. Curr Top Microbiol Immunol 357:67-103
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Zumbrun, Steven D; Hanson, Leanne; Sinclair, James F et al. (2010) Human intestinal tissue and cultured colonic cells contain globotriaosylceramide synthase mRNA and the alternate Shiga toxin receptor globotetraosylceramide. Infect Immun 78:4488-99
Panda, Aruna; Tatarov, Ivan; Melton-Celsa, Angela R et al. (2010) Escherichia coli O157:H7 infection in Dutch belted and New Zealand white rabbits. Comp Med 60:31-7
Smith, Michael J; Melton-Celsa, Angela R; Sinclair, James F et al. (2009) Monoclonal antibody 11E10, which neutralizes shiga toxin type 2 (Stx2), recognizes three regions on the Stx2 A subunit, blocks the enzymatic action of the toxin in vitro, and alters the overall cellular distribution of the toxin. Infect Immun 77:2730-40
Smith, Michael J; Carvalho, Humberto M; Melton-Celsa, Angela R et al. (2006) The 13C4 monoclonal antibody that neutralizes Shiga toxin Type 1 (Stx1) recognizes three regions on the Stx1 B subunit and prevents Stx1 from binding to its eukaryotic receptor globotriaosylceramide. Infect Immun 74:6992-8

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