Analysis of Salmonella typhi genes expressed within macrophages using the green fluorescent protein.
Abstract Text
Salmonella enterica serovar Typhi (S. typhi) is the etiological agent of typhoid fever which is characterized by sustained fever, headache, and systemic infection. S. typhi is a human-restricted pathogen. Little is known about the virulence genes involved in the invasion and infection process, because of the lack of a suitable animal model. Thus, conclusions about S. typhi pathogenicity that have been drawn from the mouse model of typhoid fever using S. typhimurium, a broad host serovar, should be interpreted with reserve. S. typhimurium has a survival advantage in murine macrophage cells; however, in human macrophages, survival of S. typhi is favored. Our hypothesis is that S. typhi possesses genes that are involved in host specificity. The purpose of this project was to identify S. typhi genes expressed only in human macrophages. Screens for in vivo-induced genes were achieved by differential fluorescence induction (DFI). DFI is a promoter-trap strategy that uses the green fluorescent protein (GFP) as a reporter gene of in vivo activated promoters. First, a library was made by inserting a GFP promoterless transposon into the S. typhi genome, strain ISP 1820. Mutants were used to infect human macrophages for two hours. Macrophages that showed levels of fluorescence above those infected with wild-type (non-GFP) were collected by FACS and lysed. Fluorescent bacteria were collected and used to infect murine macrophages. The least fluorescent cells were selected and lysed. These bacteria were used for a final enrichment selection in human macrophages. We isolated 8 mutants in which GFP gene expression is under the control of promoters that were active only in human macrophages. A Southern blot allowed the discrimination between siblings. Finally, regions adjacent to the gfp containing transposons were sequenced and we have identified three ORFs, including a gene previously involved in pathogenesis. To further elucidate molecular mechanisms that control host-specificity, we are currently investigating the role of these genes in S. typhi survival within macrophages.
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