Microbial genes expressed exclusively during infection confer unique attributes to the organism. They are usually induced in response to specific signals encountered within the host and may encode factors that help a pathogen adapt to the host environment, establish itself in its niche, and cause disease. Consequently, such virulence genes are suitable targets for the development of diagnostic assays, prophylactic and therapeutic options against disease caused by that pathogen. Here, we propose the identification of Escherichia coli O157:H7 (O157) genes expressed only during human infection, using a recently described novel technique termed In Vivo-lnduced Antigen Technology (IVIAT). This technique involves the sequential adsorption of convalescent sera from a patient who had hemolytic uremic syndrome (HUS) against whole cells, cell-lysates, and heat-denatured cell-lysates of the cognate pathogen grown under standard laboratory conditions. The adsorption process selectively removes antibodies against antigens expressed during in vitro growth, and enriches for those antibodies against in vivo-expressed antigens. Here, we will pool convalescent sera from three patients who recovered from HUS, adsorb them as described above against whole cells, cell lysates and heat-denatured cell lysates of three O157 strains isolated from the same patients who were the source of convalescent sera. The adsorbed convalescent serum pool will then be used to screen three individual O157 DNA expression libraries made from genomic DNA of the O157 strain isolated from the each of the above three patients. The genes contained in reactive clones will be identified by BLAST, against the genomes of O157 EDL 933 and Sakai strains, and other DNA sequences available in the National Center for Biotechnology Information (NCBI) non-redundant database and The Institute for Genomic Research (TIGR) database. Because of time and budgetary constraints, we will focus on only two or three of the IVIAT antigens. Previously unidentified antigens encoded by genes located on genomic sequences unique to O157 that are not transcribed during in vitro growth, will be purified via nickel affinity chromatography. Antibody responses against such antigens will be quantified in individual convalescent phase serum from the three patients described above. Ideal antigens for further study are those that are the target of robust antibody responses across all three patients. Such antigens (pending further studies) may help in the development of prophylactic options and could potentially serve as markers of ongoing or recent O157 disease in stool specimens. Broad, robust antibody responses against such antigens, could also serve as the basis for the development of serological diagnostic assays for recent O157 infection. ? ? ?
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