Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes

<p>Abstract</p> <p>Background</p> <p><it>Ehrlichia chaffeensis </it>is a rickettsial agent responsible for an emerging tick-borne illness, human monocytic ehrlichiosis. Recently, we reported that <it>E. chaffeensis </it>protein expression is influenced by macrophage and tick cell environments. We also demonstrated that host response differs considerably for macrophage and tick cell-derived bacteria with delayed clearance of the pathogen originating from tick cells.</p> <p>Results</p> <p>In this study, we mapped differences in the promoter regions of two genes of p28-Omp locus, genes 14 and 19, whose expression is influenced by macrophage and tick cell environments. Primer extension and quantitative RT-PCR analysis were performed to map transcription start sites and to demonstrate that <it>E. chaffeensis </it>regulates transcription in a host cell-specific manner. Promoter regions of genes 14 and 19 were evaluated to map differences in gene expression and to locate RNA polymerase binding sites.</p> <p>Conclusion</p> <p>RNA analysis and promoter deletion analysis aided in identifying differences in transcription, DNA sequences that influenced promoter activity and RNA polymerase binding regions. This is the first description of a transcriptional machinery of <it>E. chaffeensis</it>. In the absence of available genetic manipulation systems, the promoter analysis described in this study can serve as a novel molecular tool for mapping the molecular basis for gene expression differences in <it>E. chaffeensis </it>and other related pathogens belonging to the <it>Anaplasmataceae </it>family.</p

Transcriptional analysis and promoter characterization of two differentially expressed outer membrane protein genes of Ehrlichia chaffeensis

Doctor of PhilosophyDepartment of Diagnostic Medicine/PathobiologyRoman Reddy R. GantaEhrlichia chaffeensis is a Gram negative, rickettsial organism responsible for human monocytic ehrlichiosis, an emerging disease in people. E. chaffeensis infection to a vertebrate host occurs when the pathogen is inoculated by an infected tick, Amblyomma americanum. White-tailed deer is a reservoir host for this pathogen. The strategies employed by E. chaffeensis in support of its dual host adaptation and persistence are not clear. One of the possible mechanisms by which the pathogen adapts and persists, is by altering its gene expression in response to its host cell environments. Recently, we reported that E. chaffeensis protein expression including that from a 28 kDa outer membrane protein multigene locus (p28-Omp), is influenced by macrophage and tick cell environments. E. chaffeensis expresses p28-Omp gene 14 product predominantly when it is grown in tick cells and p28-Omp gene 19 protein in macrophages. We hypothesize that E. chaffeensis achieves its host-specific gene expression by employing transcriptional regulation by sensing the host cell signals. In support of this hypothesis, transcriptional analysis of 14 and 19 genes was performed utilizing several RNA analysis methods. The results supported our hypothesis that the gene regulation occurs at mRNA level in a host cell-specific manner. This analysis also identified transcription start sites and located putative promoters for the p28-Omp genes 14 and 19. Promoter regions of genes 14 and 19 were mapped to identify gene-specific differences, RNA polymerase binding sequences and the putative regulatory elements that may influence the promoter activities. Electrophoretic mobility shift assays revealed interaction of E. chaffeensis proteins with gene 14 and 19 promoters. Several E. chaffeensis putative regulatory proteins were expressed as recombinants and their effects on a p28-Omp gene promoter activity were evaluated. In summary, we demonstrated that the differences in the E. chaffeensis p28-Omp genes 14 and 19 are the result of their regulation at transcriptional level in response to the host cell environment. We also identified RNA polymerase binding regions and several DNA sequences that influenced promoter activity. This is the first description of a transcriptional machinery of E. chaffeensis. The data from these studies provide important insights about molecular mechanisms of gene regulation in E. chaffeensis

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

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.</p

Influenza C Virus in Cattle with Respiratory Disease, United States, 2016–2018

We identified influenza C virus (ICV) in samples from US cattle with bovine respiratory disease through real-time PCR testing and sequencing. Bovine ICV isolates had high nucleotide identities (≈98%) with each other and were closely related to human ICV strains (≈95%). Further research is needed to determine bovine ICV’s zoonotic potential

Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: &sigma;32 and &sigma;70. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing &sigma;70 (HU, P28-Omp14 and P28-Omp19) and &sigma;32 (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions

Differential Clearance and Immune Responses to Tick Cell-Derived versus Macrophage Culture-Derived Ehrlichia chaffeensis in Mice

Human monocytic ehrlichiosis is caused by a tick-transmitted rickettsia, Ehrlichia chaffeensis. We recently reported that E. chaffeensis grown in tick cells expresses different proteins than bacteria grown in macrophages. Therefore, we tested the hypothesis that immune responses against E. chaffeensis would be different if the mice are challenged with bacteria grown in macrophages or tick cells. We assessed the E. chaffeensis clearance from the peritoneum, spleen, and liver by C57BL/6J mice using a TaqMan-based real-time reverse transcription-PCR assay. Macrophage-grown E. chaffeensis was cleared in 2 weeks from the peritoneum, whereas the pathogen from tick cells persisted for nine additional days and included three relapses of increasing bacterial load separated by three-day intervals. Tick cell-grown bacteria also persisted in the livers and spleens with higher bacterial loads compared to macrophage-grown bacteria and fluctuated over a period of 35 days. Three-day periodic cycles were detected in T-cell CD62L/CD44 ratios in the spleen and bone marrow in response to infections with both tick cell- and macrophage-grown bacteria and were accompanied by similar periodic cycles of spleen cell cytokine secretions and nitric oxide and interleukin-6 by peritoneal macrophages. The E. chaffeensis-specific immunoglobulin G response was considerably higher and steadily increased in mice infected with the tick cell-derived E. chaffeensis compared to DH82-grown bacteria. In addition, antigens detected by the immunoglobulins were significantly different between mice infected with the E. chaffeensis originating from tick cells or macrophages. The differences in the immune response to tick cell-grown bacteria compared to macrophage-grown bacteria reflected a delay in the shift of gene expression from the tick cell-specific Omp 14 gene to the macrophage-specific Omp 19 gene. These data suggest that the host response to E. chaffeensis depends on the source of the bacteria and that this experimental model requires the most natural inoculum possible to allow for a realistic understanding of host resistance

Onset and duration of transient infections among antibody-diverse beef calves exposed to a bovine viral diarrhea virus persistently infected calf

Persistently infected (PI) cattle are the reservoir of bovine viral diarrhea virus (BVDV), yet data describing BVDV transient infections (TI) among non-PI populations are minimal. Study objectives consisted of: 1. Estimating the onset and duration of TI based on serum VI and rRT-PCR, and 2. Determination of the potential of TI cattle to shed BVDV. Two 21-day studies were performed where one PI calf was commingled with a confirmed non-PI cattle population with heterogeneous BVDV antibody status (n=12 and n=15, respectively). After PI exposure, virus isolation on serum and nasal swabs failed to detect BVDV among non-PI cattle. Despite minimal disease (n=1), BVDV transmission occurred as 78% (n=21) of non-PI calves displayed a four-fold rise in BVDV antibody titers, 81.5% (n=22) displayed a transient positive serum BVDV rRT-PCR outcome, and 74.1% (n=20) displayed a transient positive rRT-PCR result on nasal swabs. Median days of positive serum rRT-PCR onset and duration were 10.0 (range: 6-21) and 3.0 (range: 1-9) days, respectively. These data suggest that non-PI cattle can become TI with minimal clinical disease while possessing the potential to transmit BVDV. The speed with which exposed cattle become transiently infected and their potential ability to shed the virus may impact design and implementation of BVDV control programs