Enterohemorrhagic Escherichia coli (EHEC) O157:H7 belongs to a subset of Shiga toxin-producing E coli (STEC) that makes Shiga toxin (Stx) type 1 or type 2 (or a variant thereof), or both toxins, harbors pO157 (or a related plasmid) and expresses the adhesin intimin. Intimin is the product of the eae gene that is contained within a -43 kb pathogenicity island called the locus of enterocyte effacement or LEE. EHEC O157:H7 is the most common cause of bloody diarrhea (also called hemorrhagic colitis or HC) in the U.S. with an estimated incidence of 73,480 cases per annum. Moreover, the hemolytic uremic syndrome (HUS), a sequela of O 157:H7 infection, is the most frequent basis for acute kidney failure in U.S. children. The incidence of non-O157:H7 disease in this country is about half that of O157:H7, or 36,740 cases per year. Worldwide, the single most common serotype of STEC associated with human illness is O157:H7 but other serotypes have also been linked to development of HC and the HUS. The majority of such non-O157 isolates from humans are intimin positive, but intimin-negative strains of STEC have also been incriminated as causes of serious human disease. Because of the potential severity of STEC infection and because O157:H7 has a very low infectious dose 50% and can be spread from person to person, the bacterium is considered a category B biological threat by CDC. The long-term goals of this project are to define at the molecular, cellular, and whole animal levels the pathogenic mechanisms by which STEC cause disease and to develop strategies for prevention and treatment of STEC-mediated HC and HUS.
The specific aims are to: 1. assess the cell biology and distribution of the elastase-activatable, highly potent, phage-encoded Stx2d toxin; 2. define the structure function relationship between intimin-gamma of E. coli O157:H7 and the eukaryotic receptor nucleolin and determine whether nucleolin also engages other types of intimin; 3. test the theory that the interaction between intimin-y and nucleolin on the host cell surface activates an intracellular response that is essential for EHEC O157:I-'17 adherence; 4. explore the hypotheses that O157:H7 strain 86-24 can induce intestinal A/E lesions in orally-infected mice if the strain is altered to express Citrobacter rodentium intimin alone or in concert with other C. rodentium LEE-encoded proteins and that such a strain more efficiently delivers Stx2 systemically than does wild-type and is, therefore, more virulent, and; 5. continue to evaluate as vaccine candidates the C-terminal fragments of intimin-gamma from EHEC O 157:H7 and intimin-alpha from enteropathogenic E. coli (EPEC) as well as Stxl and Stx2 toxoids for capacity to elicit antibodies that inhibit adherence of EHEC O157:H7 and EPEC to tissue culture cells and to neutralize Stxl and Stx2, respectively, and to assess the protective efficacy of these immunogens alone or in combination when given to animals either parenterally or orally in transgenic plant cells.
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