Genomic, proteomic, and transcriptomic analysis of virulent and avirulent Rickettsia prowazekii reveals its adaptive mutation capabilities

Abstract

Rickettsia prowazekii, the agent of epidemic typhus, is an obligate intracellular bacterium that is transmitted to human beings by the body louse. Several strains that differ considerably in virulence are recognized, but the genetic basis for these variations has remained unknown since the initial description of the avirulent vaccine strain nearly 70 yr ago. We use a recently developed murine model of epidemic typhus and transcriptomic, proteomic, and genetic techniques to identify the factors associated with virulence. We identified four phenotypes of R. prowazekii that differed in virulence, associated with the up-regulation of antiapoptotic genes or the interferon I pathway in the host cells. Transcriptional and proteomic analyses of R. prowazekii surface protein expression and protein methylation varied 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) in eight genes in an avirulent strain that split and inactivated these genes. These included recO, putative methyltransferase, and exported protein. Passage of the avirulent Madrid E strain in cells or in experimental animals was associated with a cascade of gene reactivations, beginning with recO, that restored the virulent phenotype. An area of genomic plasticity appears to determine virulence in R. prowazekii and represents an example of adaptive mutation for this pathogen.

Figure 1.

Scheme of R. prowazekii strains origin and evolution. The Breinl strain and the most recent isolate, Rp22, are considered highly virulent. The Madrid I strain was isolated in 1941 during the Madrid outbreak of epidemic typhus. After passages in embryonated eggs, Madrid I has lost its virulence and has been used under the name of Madrid E as a vaccine in humans since 1944. When it was inoculated to small rodents, Madrid E recovered some virulence (Evir). From Madrid E (300–600 passages in eggs), we have recently generated a new isolate by cultivating them in L 929 cells (Erus). (Breinl and Rp22) Virulent for humans and animals and replicates efficiently in L929 cells; (Evir) virulent for animals and replicates efficiently in L929 cells; (Erus) avirulent for animals, but replicates with L929; (Madrid E) avirulent for humans and animals and grows slowly in L929 cells.

Figure 2.

Transcriptional response of human endothelial cells infected by different strains of R. prowazekii. (A) Venn diagram illustrating the number of genes of endothelial cells differentially expressed in response to different R. prowazekii strains as compared with uninfected cells. (B) Transcriptional response analyzed by RNA microarrays. Modulated genes (fold change ≥ 2) were compared by unsupervised hierarchic clustering analysis.

Figure 3.

Circular representation of R. prowazekii Rp22 genome. The circles show the G-C content and skews, CDSs, rRNA, tRNA, and miscellaneous RNA (other).

Figure 4.

Sequence alignments of three R. prowazekii genes compared between Rp22 (rpr22) and Madrid E (rpr) strains. Split genes in Madrid E were due to stop codon (A), poly(A) (B), or poly(T) (C).

Figure 5.

Transcriptional profile of Rp22 vs. Erus cultured on L929 cells and analyzed by RNA microarray. Regulated genes (fold change ≥ 1.5) in Rp22 as compared with Erus were represented using cluster analysis software.

Figure 6.

Lysine methylation in Rp22 (A,C,E) and Erus (B,D,F) strains. Two-dimensional Western blots were performed, and lysine methylation was visualized in pH range 4.0–7.0 (A,B) and pH range 6.0–11.0 (C,D). The zone that differed among both bacterial strains is boxed in C and D. Arrows in E and F indicate methylated spots of Rp22 and Erus strains that were identified as putative methyltransferase (RP789).