title: Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions.
abstract: The green fluorescent protein (GFP) from Aequorea victoria is a novel fluorescent marker that has potential use in the study of bacterial pathogenicity. To explore some of the potential applications of GFP to the study of host-parasite interactions, we constructed two GFP expression vectors suitable for different facultative intracellular bacterial pathogens. The first expression vector was tested in the enteric pathogens, Salmonella typhimurium and Yersinia pseudotuberculosis, and the second vector tested in Mycobacterium marinum (Mm). Both expression vectors were found to be stable and to direct high levels of GFP synthesis. Standard epifluorescence microscopy was used to detect all three bacterial pathogenic species during the early and late stages of infection of live mammalian cells. Mm expressing gfp was also visualized in infected animal tissues. gfp expression did not adversely affect bacterial survival, nor did it compromise entry into mammalian cells or their survival within macrophages. In addition, all three gfp-expressing bacterial pathogens could be detected and sorted in a flow cytometer, either alone or in association with epithelial cells or macrophages. Therefore, GFP not only provides a convenient tool to image pathogenic bacteria, but allows the quantitative measurement of bacterial association with mammalian cells.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305-5402, USA. valdivia@cmgm.stanford.edu
authors.forename: R H
authors.initials: RH
authors.lastname: Valdivia
authors.forename: A E
authors.initials: AE
authors.lastname: Hromockyj
authors.forename: D
authors.initials: D
authors.lastname: Monack
authors.forename: L
authors.initials: L
authors.lastname: Ramakrishnan
authors.forename: S
authors.initials: S
authors.lastname: Falkow
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D008099:Liver / Q000382:microbiology / Q000473:pathology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000382:microbiology; D051379:Mice; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000302:isolation & purification / Q000472:pathogenicity / Q000502:physiology*; D011894:Rana pipiens; D012486:Salmonella typhimurium / Q000302:isolation & purification / Q000472:pathogenicity / Q000502:physiology*; D037401:Scyphozoa; D013154:Spleen / Q000382:microbiology / Q000473:pathology; D014407:Tumor Cells, Cultured; D015011:Yersinia pseudotuberculosis / Q000302:isolation & purification / Q000472:pathogenicity / Q000502:physiology*
chemical_list: D017931:DNA Primers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins