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

Faburay B, Liu H, Peddireddi L, Ganta RR - BMC Microbiol. (2011)

Bottom Line: In recent studies, we demonstrated significant host-specific differences in protein expression in E. chaffeensis originating from its tick and vertebrate host cells.Our 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 study marks the beginning to broadly characterize the mechanisms controlling the transcription by Anaplasmataceae pathogens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.

ABSTRACT

Background: Ehrlichia 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.

Results: In 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.

Conclusions: Our 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.

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EMSA using short segments of three biotin-labeled probes of p28-Omp14 (panel A) and one p28-Omp19 (panel B) promoter segments. Addition of E. chaffeensis protein extracts (MQ-Ehr P) and unlabeled promoter DNA as a cold competitor (Competitor) or bovine serum albumin (BSA) as a non-specific protein control are indicated in captions at the bottom of the Figure for both the panels.
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Figure 9: EMSA using short segments of three biotin-labeled probes of p28-Omp14 (panel A) and one p28-Omp19 (panel B) promoter segments. Addition of E. chaffeensis protein extracts (MQ-Ehr P) and unlabeled promoter DNA as a cold competitor (Competitor) or bovine serum albumin (BSA) as a non-specific protein control are indicated in captions at the bottom of the Figure for both the panels.

Mentions: Electrophoretic mobility shift assay (EMSA) experiments utilizing the complete promoter regions of the p28-Omp14 and p28-Omp19 of E. chaffeensis showed promoter-specific binding of tick cell- or macrophage-derived E. chaffeensis proteins (not shown). Addition of 50 ng of specific competitor DNAs consisting of unlabeled full length promoter DNA of p28-Omp14 or p28-Omp19 abolished the shift of DNA-protein complex migration for both promoter regions. To further assess the interactions of Ehrlichia proteins with putative upstream sequences, five biotin-labelled short upstream DNA segments of p28-Omp14 (probes P1 to P5) (Figure 8A) and two DNA segments of p28-Omp19 (P6 and P7) (Figure 8B) promoters were prepared and used in the EMSA experiments. The promoter sequences of genes 14 and 19 included direct repeats and palindromic sequences [25]. The probes included one or more of the sequences. Three of the five probes for the p28-Omp14 promoter region exhibited significant shift in mobility in the presence of protein lysate from macrophage derived E. chaffeensis compared to the controls which contained probe alone with no lysate added or when non-specific protein was added to the probe fragments (Figure 9A). A shift in mobility was also noted in the interaction with one probe segment of the p28-Omp19 promoter region when the protein lysate was added (Figure 9B). Addition of a 50-fold excess of unlabeled specific-competitors in the binding reactions significantly reduced the mobility shift of the probes. Densitometry analysis of the mobility shifted fragments differed for each probe compared to the non-shifted fragments. The P1 probe had 84% shift which reduced to 29% when competitor DNA was added; P2 and P3 probes had about 31%, and 27% shifts, respectively, and the shifts for these probes were completely abolished in the presence of specific competitors. The p28-Omp19 promoter region probe had about 23% shift which was reduced to 10% in the presence of specific competitor.


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

Faburay B, Liu H, Peddireddi L, Ganta RR - BMC Microbiol. (2011)

EMSA using short segments of three biotin-labeled probes of p28-Omp14 (panel A) and one p28-Omp19 (panel B) promoter segments. Addition of E. chaffeensis protein extracts (MQ-Ehr P) and unlabeled promoter DNA as a cold competitor (Competitor) or bovine serum albumin (BSA) as a non-specific protein control are indicated in captions at the bottom of the Figure for both the panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108270&req=5

Figure 9: EMSA using short segments of three biotin-labeled probes of p28-Omp14 (panel A) and one p28-Omp19 (panel B) promoter segments. Addition of E. chaffeensis protein extracts (MQ-Ehr P) and unlabeled promoter DNA as a cold competitor (Competitor) or bovine serum albumin (BSA) as a non-specific protein control are indicated in captions at the bottom of the Figure for both the panels.
Mentions: Electrophoretic mobility shift assay (EMSA) experiments utilizing the complete promoter regions of the p28-Omp14 and p28-Omp19 of E. chaffeensis showed promoter-specific binding of tick cell- or macrophage-derived E. chaffeensis proteins (not shown). Addition of 50 ng of specific competitor DNAs consisting of unlabeled full length promoter DNA of p28-Omp14 or p28-Omp19 abolished the shift of DNA-protein complex migration for both promoter regions. To further assess the interactions of Ehrlichia proteins with putative upstream sequences, five biotin-labelled short upstream DNA segments of p28-Omp14 (probes P1 to P5) (Figure 8A) and two DNA segments of p28-Omp19 (P6 and P7) (Figure 8B) promoters were prepared and used in the EMSA experiments. The promoter sequences of genes 14 and 19 included direct repeats and palindromic sequences [25]. The probes included one or more of the sequences. Three of the five probes for the p28-Omp14 promoter region exhibited significant shift in mobility in the presence of protein lysate from macrophage derived E. chaffeensis compared to the controls which contained probe alone with no lysate added or when non-specific protein was added to the probe fragments (Figure 9A). A shift in mobility was also noted in the interaction with one probe segment of the p28-Omp19 promoter region when the protein lysate was added (Figure 9B). Addition of a 50-fold excess of unlabeled specific-competitors in the binding reactions significantly reduced the mobility shift of the probes. Densitometry analysis of the mobility shifted fragments differed for each probe compared to the non-shifted fragments. The P1 probe had 84% shift which reduced to 29% when competitor DNA was added; P2 and P3 probes had about 31%, and 27% shifts, respectively, and the shifts for these probes were completely abolished in the presence of specific competitors. The p28-Omp19 promoter region probe had about 23% shift which was reduced to 10% in the presence of specific competitor.

Bottom Line: In recent studies, we demonstrated significant host-specific differences in protein expression in E. chaffeensis originating from its tick and vertebrate host cells.Our 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 study marks the beginning to broadly characterize the mechanisms controlling the transcription by Anaplasmataceae pathogens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.

ABSTRACT

Background: Ehrlichia 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.

Results: In 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.

Conclusions: Our 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.

Show MeSH
Related in: MedlinePlus