Identification of host-induced pathogen genes by differential fluorescence induction reporter systems.
The ability to monitor a pathogen's gene expression program in response to the host environment is central to understanding host-microbe interactions. This protocol describes the application of a fluorescence-based promoter trap strategy, termed differential fluorescence induction (DFI), to identify and characterize bacterial genes that are preferentially expressed in infected tissues. In this approach, animals are infected with a library of bacteria expressing random GFP transcriptional gene fusions, and fluorescent bacteria are recovered directly from host tissues using fluorescence-activated cell sorting (FACS). This methodology allows for the identification of bacterial promoters induced in distinct anatomical sites and at different stages of infection. Furthermore, unlike other methodologies, the use of the GFP reporter allows for single cell, temporal and spatial monitoring of pathogen gene expression in infected animals. Library construction, promoter identification and analysis can be done in 4-8 weeks.
Bumann
Dirk
D
Junior Research Group Mucosal Infections OE 9421, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
Valdivia
Raphael H
RH
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins