Shiga toxin (Stx)-producing Escherichia coli (STEC) express Stx1 and/or Stx2 (or a variant of Stx2) and harbor a 90kb plasmid. STEC 0157:H7, which also express the adhesin intimin from the eae locus, are food- and water-borne pathogens that are the most common infectious cause of bloody diarrhea in the U. S. Moreover, the hemolytic uremic syndrome (HUS), as a sequela of 0157:H7 infection, is the most frequent basis for acute kidney failure in U.S. children. The long term goals of this project are to define the pathogenic mechanisms by which STEC cause disease and to develop strategies for the prevention and treatment of STEC-mediated HUS.
The specific aims are to: 1) examine the reason for the striking virulence of eae- STEC 091:H21 strain B2F1 for streptomycin (str)-treated, orally-infected CD-1 mice by assessing whether virulence correlates with intestinal mucus activation of Stx2d2 produced by B2F1 and related STEC, by identifying the Stx2d2-activating substance in mucus, and by characterizing poorly adherent or mouse-virulence attenuated mini-TnphoA mutants of B2F1; 2) investigate the basis for differences in toxicity among Stx family members by creating various Stx1/Stx2 hybrid molecules and by defining both the structural changes that occur in Stx2d2 after activation and the amino acids in the Stx2d2 A2 domain required for activation; 3) analyze stx2d2 regulation by characterizing a gene encoding a repressor of Stx2d2 expression, inactivating the repressor gene in B2F1 and B2Flstx2d2::cat, and measuring the effect of that mutation on toxin production or chloramphenicol acetyl transferase (CAT) expression in vitro and on CAT levels expressed from the mutants in str-treated mice; 4) characterize the interaction between 86-24 intimin and the host cell by identifying a fragment of 86-24 intimin that can elicit anti-EHEC and anti-Enteropathogenic E. coli (EPEC) adherence blocking antibodies, defining environmental signals that regulate 86-24 intimin expression, and attempting to isolate the mammalian cell receptor for 86-24 intimin-mediated adherence; and, 5) develop potential vaccine candidates that will elicit antibodies that inhibit colonization of the host by EHEC 0157:H7 and EPEC and neutralize Stx, Stx1, and Stx2, e.g., intimin or intimin fragments combined with toxoids.
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