title: GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8+T Cell Immunology in R. typhi Infection.
abstract: Rickettsia typhiis the causative agent of endemic typhus, a disease with increasing incidence worldwide that can be fatal. Because of its obligate intracellular life style, genetic manipulation of the pathogen is difficult. Nonetheless, in recent years, genetic manipulation tools have been successfully applied to rickettsiae. We describe here for the first time the transformation ofR. typhiwith the pRAM18dRGA plasmid that originally derives fromRickettsia amblyommatisand encodes the expression of GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light). TransformedR. typhi(R. typhiGFPuv) bacteria are viable, replicate with kinetics similar to those of wild-typeR. typhiin cell culture, and stably maintain the plasmid and GFPuv expression under antibiotic treatmentin vitroandin vivoduring infection of mice. CB17 SCID mice infected withR. typhiGFPuvsuccumb to the infection with kinetics similar to those for animals infected with wild-typeR. typhiand develop comparable pathology and bacterial loads in the organs, demonstrating that the plasmid does not influence pathogenicity. In the spleen and liver of infected CB17 SCID mice, the bacteria are detectable by immunofluorescence microscopy in neutrophils and macrophages by histological staining. Finally, we show for the first time that transformed rickettsiae can be used for the detection of CD8+T cell responses. GFP-specific restimulation of spleen cells fromR. typhiGFPuv-infected BALB/c mice elicits gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 2 (IL-2) secretion by CD8+T cells. Thus,R. typhiGFPuvbacteria are a novel, potent tool to study infection with the pathogenin vitroandin vivoand the immune response to these bacteria.
authors.affiliation: Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Hauptmann
authors.affiliation: Department of Entomology, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: Nicole
authors.initials: N
authors.lastname: Burkhardt
authors.affiliation: Department of Entomology, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Munderloh
authors.affiliation: Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
authors.forename: Svenja
authors.initials: S
authors.lastname: Kuehl
authors.affiliation: Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
authors.forename: Ulricke
authors.initials: U
authors.lastname: Richardt
authors.affiliation: Institute for Neuropathology, University Medical Center Eppendorf, Hamburg, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Krasemann
authors.affiliation: Institute for Neuropathology, University Medical Center Eppendorf, Hamburg, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Hartmann
authors.affiliation: Institute for Pathology, University Medical Center Eppendorf, Hamburg, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Krech
authors.affiliation: Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Fleischer
authors.affiliation: Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.|Institute of Virology, University Hospital Giessen and Marburg, Marburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Keller
authors.affiliation: Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany osterloh@bni-hamburg.de.
authors.forename: Anke
authors.initials: A
authors.lastname: Osterloh
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D016207:Cytokines / Q000276:immunology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008099:Liver / Q000382:microbiology; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D009504:Neutrophils / Q000382:microbiology; D010957:Plasmids; D012286:Rickettsia typhi / Q000235:genetics / Q000472:pathogenicity*; D013154:Spleen / Q000382:microbiology; D014169:Transformation, Bacterial; D014437:Typhus, Endemic Flea-Borne / Q000276:immunology* / Q000382:microbiology
chemical_list: D016207:Cytokines; D049452:Green Fluorescent Proteins
keywords: CD8+ T cell response; GFPuv; Rickettsia typhi; transformation