Isolation and characterization of Ehrlichia chaffeensis RNA polymerase and its use in evaluating p28 outer membrane protein gene promoters

Abstract

BackgroundEhrlichia chaffeensis is a tick-transmitted rickettsial pathogen responsible for an important emerging disease, human monocytic ehrlichiosis. To date how E. chaffeensis and many related tick-borne rickettsial pathogens adapt and persist in vertebrate and tick hosts remain largely unknown. In recent studies, we demonstrated significant host-specific differences in protein expression in E. chaffeensis originating from its tick and vertebrate host cells. The adaptive response of the pathogen to different host environments entails switch of gene expression regulated at the level of transcription, possibly by altering RNA polymerase activity.ResultsIn an effort to understand the molecular basis of pathogen gene expression differences, we isolated native E. chaffeensis RNA polymerase using a heparin-agarose purification method and developed an in vitro transcription system to map promoter regions of two differentially expressed genes of the p28 outer membrane protein locus, p28-Omp14 and p28-Omp19. We also prepared a recombinant protein of E. chaffeensis σ70 homologue and used it for in vitro promoter analysis studies. The possible role of one or more proteins presents in E. chaffeensis lysates in binding to the promoter segments and on the modulation of in vitro transcription was also assessed.ConclusionsOur experiments demonstrated that both the native and recombinant proteins are functional and have similar enzyme properties in driving the transcription from E. chaffeensis promoters. This is the first report of the functional characterization of E. chaffeensis RNA polymerase and in vitro mapping of the pathogen promoters using the enzyme. This study marks the beginning to broadly characterize the mechanisms controlling the transcription by Anaplasmataceae pathogens.

Ehrlichia chaffeensis Infection in the Reservoir Host (White-Tailed Deer) and in an Incidental Host (Dog) Is Impacted by Its Prior Growth in Macrophage and Tick Cell Environments

“…Host cell free E. chaffeensis lysates were prepared from the organisms recovered from infected DH82 and ISE6 cultures [38] and the lysates were used for the ELISA analysis [18] . Briefly, host cell free Ehrlichia antigens were prepared by following the protocol involving sonication of 80–90% infected culture, low speed centrifugation at 400×g for 5 min to remove nuclei and unbroken host cells, filtration of the supernatant through a 2.7 µm filter followed by a 1.6 µm filter to recover the host cell-free Ehrlichia organisms, then the organisms were pelleted by high speed centrifugation at 15,557×g, and finally washed three times with SPK buffer to remove any residual host cell antigens.…”

Sequence determinants spanning -35 motif and AT-rich spacer region impactingEhrlichia chaffeensisSigma 70-dependent promoter activity of two differentially expressed p28 outer membrane protein genes

“…These genes were previously identified as transcribed by the E. chaffeensis σ 70 . 32 These gene promoter segments, cloned in front of a reporter gene for β-galactosidase in a plasmid, were used to transform the modified CAG20177 strain of E. coli ( Supplementary Fig. S2 ).…”

“… 30 The E. coli rpoH from this plasmid was replaced with the E. chaffeensis rpoD (Ech_ rpoD ) gene by digesting the plasmid with Afl II and Sal I, blunt ending the digested fragments with Klenow DNA polymerase (BioLabs, Ipswich, MA), and then ligating with the Ech_ rpoD sequence. Ech_ rpoD segment was generated by PCR from plasmid pET32-Ech_ rpoD 32 using Pfu DNA polymerase (Promega, Madison, WI). The modified plasmid is referred to as the pSAKT32-Ech_ rpoD .…”

“…The expression plasmids of E. chaffeensis wild-type (WT) σ 70 or its variants were constructed for preparing purified recombinant proteins using the E. chaffeensis σ 70 plasmid reported earlier. 32 Ehrlichia chaffeensis σ 70 variants within the 4.2 region of E. chaffeensis σ 70 were constructed by modifying the plasmid pET32a-Ech_ rpoD by using a QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, La Jolla, CA) and the modified expression constructs were then used to prepare modified recombinant proteins. The names of the modified pET32-Ech_ rpoD are provided in Supplementary Table S1 .…”

“…In vitro transcription reactions were performed in 10 μl reaction mixture containing 0.13 pmol each of the supercoiled plasmid DNA as the template and using RNAP holoenzyme containing either recombinant E. chaffeensis σ 70 or its derivatives. 32 The holoenzyme was prepared by mixing 0.5 μl of 1:10 diluted stock of E. coli core enzyme (Epicentre, Madison, WI) mixed with 10-fold molar excess of purified recombinant E. chaffeensis σ 70 or its derivatives and kept in ice for 30 min prior to using for the reactions. The transcription reactions were performed at 37 °C for 20 min, and the reactions were terminated by adding 7 μl of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol).…”