Dataset Information

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

ABSTRACT: Rickettsia typhi 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 R. typhi with the pRAM18dRGA plasmid that originally derives from Rickettsia amblyommatis and encodes the expression of GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light). Transformed R. typhi (R. typhiGFPuv) bacteria are viable, replicate with kinetics similar to those of wild-type R. typhi in cell culture, and stably maintain the plasmid and GFPuv expression under antibiotic treatment in vitro and in vivo during infection of mice. CB17 SCID mice infected with R. typhiGFPuv succumb to the infection with kinetics similar to those for animals infected with wild-type R. typhi 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 R. typhiGFPuv-infected BALB/c mice elicits gamma interferon (IFN-?), tumor necrosis factor alpha (TNF-?), and interleukin 2 (IL-2) secretion by CD8+ T cells. Thus, R. typhiGFPuv bacteria are a novel, potent tool to study infection with the pathogen in vitro and in vivo and the immune response to these bacteria.

SUBMITTER: Hauptmann M

PROVIDER: S-EPMC5442613 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Publications

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

Hauptmann Matthias M Burkhardt Nicole N Munderloh Ulrike U Kuehl Svenja S Richardt Ulricke U Krasemann Susanne S Hartmann Kristin K Krech Till T Fleischer Bernhard B Keller Christian C Osterloh Anke A

Infection and immunity 20170523 6

Rickettsia typhi 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 R. typhi with the pRAM18dRGA plasmid that originally derives from Rickettsia amblyommatis an ...[more]

PMID: 28289147

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Project description:The long-standing proposal that phospholipase A2 (PLA2) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA2 activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA2 activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA2 activity were reduced by PLA2 inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.

Dataset Information

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

Publications

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

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