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    A large genetic locus of enteropathogenic Escherichia coli sufficient to confer the attaching and effacing virulence phenotype in vitro

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    Author
    McDaniel, Timothy Keith
    Advisor
    Kaper, James B.
    Date
    1996
    Type
    dissertation
    
    Metadata
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    Abstract
    Enteropathogenic Escherichia coli (EPEC), an agent of severe infant diarrhea in the less developed world, causes attaching and effacing lesions on epithelial cells of the human intestine and on cultured human cells. These lesions are marked by localized rearrangements of the actin cytoskeleton, resulting in microvillus destruction and the formation of shallow protrusions from the apical surface that intimately bind to the bacteria. The genetics of the attaching and effacing phenotype were investigated by mapping previously-generated transposon insertions that abolish the phenotype. This mapping resulted in the discovery of the locus of enterocyte effacement (LEE), a {dollar}\sim{dollar}35.5-kb cluster of DNA, containing two genes previously shown necessary for AE lesion formation and many hitherto undescribed sequences. DNA probes derived from sequences throughout the LEE hybridized under high stringency to a panel of EPEC and other enteric pathogens that cause AE lesions, but not to non-AE strains of the same species. The nucleotide sequence of 23 kb of the LEE was determined and found to contain 32 novel open reading frames, including nine similar to genes encoding components of specialized secretory systems that secrete virulence factors in a variety of other bacterial pathogens. The {dollar}\rm G+C{dollar} nucleotide composition of the LEE is significantly lower than the E. coli genome as a whole, suggesting that it was acquired by horizontal gene transfer. To see if the LEE contains all genes necessary for the AE phenotype, its 35-kb sequence and approximately 750 bp of flanking DNA were isolated on a cosmid cloning vector. Addition of the LEE-containing cosmid, but not the vector alone, to a variety of avirulent laboratory E. coli strains conferred the ability to secrete at least one EPEC protein from the bacterium, trigger host signal transduction pathways, and induce AE lesions on cultured epithelial cells. These results show that the LEE is a functional genetic unit containing all factors necessary to confer AE lesion formation in vitro.
    Description
    University of Maryland, Baltimore. Molecular Medicine. Ph.D. 1996
    Keyword
    Biology, Molecular
    Biology, Cell
    Biology, Microbiology
    Health Sciences, Pathology
    attaching and effacing lesions
    Enteropathogenic Escherichia coli--genetics
    Genetic Loci
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1448
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    Theses and Dissertations School of Medicine
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