Project description:Here we present the first microarray analysis of global gene expression changes in the obligate intracytoplasmic pathogen Rickettsia prowazekii using temperature upshift as a model stress condition. We describe a methodology for isolating highly-purified rickettsial RNA where a combination of differential centrifugation and use of Ambion’s MICROBEnrich reagent removed the majority (~90 %) of host cell RNA. A pool of rickettsiae-infected L-cells grown at 34 oC was split and half of the sample was challenged at 42 oC for 30 minutes, total RNA was isolated and converted to cDNA followed by hybridization to an oligonucleotide DNA array. We report data (duplicate hybridizations) from four, independent biological replicates. Twenty-three rickettsial transcripts were significantly increased by temperature upshift greater than two-fold and no transcripts demonstrated reproducible decreases. Notably, the GroESL, DnaK, and DnaJ members of the family of chaperones as well as the two R. prowazekii proteins annotated as putative heat shock proteins were induced. In addition, proteins annotated as proteases were also induced indicating that R. prowazekii mounts a response to heat-shock induced protein unfolding. These data are the first global analysis of the R. prowazekii transcriptome and provide insight into the mechanisms employed by rickettsiae to adapt to temperature upshifts.
Project description:We identified four virulence phenotypes of Rickettsia prowazekii (the deadly agent of epidemic typhus) that are associated with the upregulation of antiapoptotic genes (virulent strain) or the Interferon I pathway (avirulent). Transcriptional and proteomic analyses of R. prowazekii linked surface protein expression and methylation with virulence. By sequencing a virulent strain and using comparative genomics, we found hotspots of mutations in homopolymeric tracts of poly(A) and poly(T) that lead to gene split and inactivation and explain the loss of virulence in the vaccine strain. These areas of instability explains adaptive mutations leading to virulence recovery in the vaccine strain. Transcriptional analysis of two different strains growing in L929 cells. A virulent strain (Rp22) was compared to a avirulent strain (Erus). The experiment was performed with 3 independant biological replicates.
Project description:We identified four virulence phenotypes of Rickettsia prowazekii (the deadly agent of epidemic typhus) that are associated with the upregulation of antiapoptotic genes (virulent strain) or the Interferon I pathway (avirulent). Transcriptional and proteomic analyses of R. prowazekii linked surface protein expression and methylation with virulence. By sequencing a virulent strain and using comparative genomics, we found hotspots of mutations in homopolymeric tracts of poly(A) and poly(T) that lead to gene split and inactivation and explain the loss of virulence in the vaccine strain. These areas of instability explains adaptive mutations leading to virulence recovery in the vaccine strain.
Project description:We identified four virulence phenotypes of Rickettsia prowazekii (the deadly agent of epidemic typhus) that are associated with the upregulation of antiapoptotic genes (virulent strain) or the Interferon I pathway (avirulent). Transcriptional and proteomic analyses of R. prowazekii linked surface protein expression and methylation with virulence. By sequencing a virulent strain and using comparative genomics, we found hotspots of mutations in homopolymeric tracts of poly(A) and poly(T) that lead to gene split and inactivation and explain the loss of virulence in the vaccine strain. These areas of instability explains adaptive mutations leading to virulence recovery in the vaccine strain.
