Genomic, proteomic, and transcriptomic analysis of virulent and avirulentRickettsia prowazekiireveals 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.

Two Protein Lysine Methyltransferases Methylate Outer Membrane Protein B from Rickettsia

“…Proteins were obtained by slow dialysis at stepwise, decreasing concentrations of urea. SET7 methyltransferase and the histone peptide substrate H3 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) were from New England Biolabs. N,N=-diallyltartardiamide (DATD) was from Bio-Rad.…”

“…Genomic sequence and transcription analyses showed that the mutation of RP027/RP028 in the Madrid E strain reverts back to the wild type (39). The gene encoding one of the putative methyltransferases RP027/RP028 was found to have a frameshift mutation in the avirulent strain, resulting in two split genes, the RP027 and RP028 genes, which are detectable only in avirulent strains (4). The putative methyltransferases and their native substrates in Rickettsia have not been identified or characterized, and the mechanism of action of protein lysine methyltransferases in the virulence and the pathogenesis of Rickettsia is not well understood.…”

Vasculitis: Endothelial Dysfunction During Rickettsial Infection

“…NF-κB is a transcription factor that triggers an inflammatory response during rickettsial infection of the endothelial cells. Interestingly, in an in vitro system where endothelial cells were infected with a virulent R. prowazekii strain, the expression of proapoptotic genes, such as Bcl 2, caspase 8 and Naip was decreased; moreover, expression of the interferon type I (IFN-I)-inducible genes was inhibited [Bechah et al, 2010]. This response suggests that the survival of rickettsiae within their host cells depends on a combination of several mechanisms.…”

Differential Proteomic Analysis of Rickettsia prowazekii Propagated in Diverse Host Backgrounds

“…This reductive evolution has resulted in a pseudogene-riddled genome with a high proportion of noncoding DNA (4). Characterizing rickettsial protein expression under various conditions and environments is critical to our understanding of how R. prowazekii, with its relatively small protein repertoire, exploits the intracellular niche in host cells as diverse as those of the louse vector and the human host.Several groups have evaluated rickettsial protein expression using both gel-based and mass spectrometry (MS)-based methods (9,10,19,24,26,27). Although gel-based methods offer certain advantages, the use of mass spectrometry offers the opportunity to evaluate comprehensive differential protein expression in complex samples.…”

“…Several groups have evaluated rickettsial protein expression using both gel-based and mass spectrometry (MS)-based methods (9,10,19,24,26,27). Although gel-based methods offer certain advantages, the use of mass spectrometry offers the opportunity to evaluate comprehensive differential protein expression in complex samples.…”