GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8 T Cell Immunology in R. typhi Infection.

is 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 of with the pRAM18dRGA plasmid that originally derives from and encodes the expression of GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light). Transformed () bacteria are viable, replicate with kinetics similar to those of wild-type in cell culture, and stably maintain the plasmid and GFPuv expression under antibiotic treatment and during infection of mice. CB17 SCID mice infected with succumb to the infection with kinetics similar to those for animals infected with wild-type and 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 from -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, bacteria are a novel, potent tool to study infection with the pathogen and and the immune response to these bacteria.