Impact of Three Different Mutations in Ehrlichia chaffeensis in Altering the Global Gene Expression Patterns

Citation: Kondethimmanahalli, C., & Ganta, R. (2018). Impact of Three Different Mutations in Ehrlichia chaffeensis in Altering the Global Gene Expression Patterns. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-24471-3The rickettsial pathogen Ehrlichia chaffeensis causes a tick-borne disease, human monocytic ehrlichiosis. Mutations within certain genomic locations of the pathogen aid in understanding the pathogenesis and in developing attenuated vaccines. Our previous studies demonstrated that mutations in different genomic sites in E. chaffeensis caused variable impacts on their growth and attenuation in vertebrate and tick hosts. Here, we assessed the effect of three mutations on transcriptional changes using RNA deep-sequencing technology. RNA sequencing aided in detecting 66–80% of the transcripts of wildtype and mutant E. chaffeensis. Mutation in an antiporter gene (ECH_0379) causing attenuated growth in vertebrate hosts resulted in the down regulation of many transcribed genes. Similarly, a mutation downstream to the ECH_0490 coding sequence resulted in minimal impact on the pathogen’s in vivo growth, but caused major changes in its transcriptome. This mutation caused enhanced expression of several host stress response genes. Even though the ECH_0660 gene mutation caused the pathogen’s rapid clearance in vertebrate hosts and aids in generating a protective response, there was minimal impact on the transcriptome. The transcriptomic data offer novel insights about the impact of mutations on global gene expression and how they may contribute to the pathogen’s resistance and/or clearance from the host

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

Comparative Experimental Infection Study in Dogs with Ehrlichia canis, E. chaffeensis, Anaplasma platys and A. phagocytophilum

Citation: Nair, A. D. S., Cheng, C., Ganta, C. K., Sanderson, M. W., Alleman, A. R., Munderloh, U. G., & Ganta, R. R. (2016). Comparative Experimental Infection Study in Dogs with Ehrlichia canis, E. chaffeensis, Anaplasma platys and A. phagocytophilum. Plos One, 11(2), 21. doi:10.1371/journal.pone.0148239Dogs acquire infections with the Anaplasmataceae family pathogens, E. canis, E. chaffeensis, E. ewingii, A. platys and A. phagocytophilum mostly during summer months when ticks are actively feeding on animals. These pathogens are also identified as causing diseases in people. Despite the long history of tick-borne diseases in dogs, much remains to be defined pertaining to the clinical and pathological outcomes of infections with these pathogens. In the current study, we performed experimental infections in dogs with E. canis, E. chaffeensis, A. platys and A. phagocytophilum. Animals were monitored for 42 days to evaluate infection-specific clinical, hematological and pathological differences. All four pathogens caused systemic persistent infections detectible throughout the 6 weeks of infection assessment. Fever was frequently detected in animals infected with E. canis, E. chaffeensis, and A. platys, but not in dogs infected with A. phagocytophilum. Hematological differences were evident in all four infected groups, although significant overlap existed between the groups. A marked reduction in packed cell volume that correlated with reduced erythrocytes and hemoglobin was observed only in E. canis infected animals. A decline in platelet numbers was common with E. canis, A. platys and A. phagocytophilum infections. Histopathological lesions in lung, liver and spleen were observed in all four groups of infected dogs; infection with E. canis had the highest pathological scores, followed by E. chaffeensis, then A. platys and A. phagocytophilum. All four pathogens induced IgG responses starting on day 7 post infection, which was predominantly comprised of IgG2 subclass antibodies. This is the first detailed investigation comparing the infection progression and host responses in dogs after inoculation with four pathogens belonging to the Anaplasmataceae family. The study revealed a significant overlap in clinical, hematological and pathological changes resulting from the infections

A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis

Citation: Wang, Y., Wei, L., Liu, H., Cheng, C., & Ganta, R. R. (2017). A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis. Scientific Reports, 7(1). https://doi.org/10.1038/s41598-017-16023-yObligate intracellular bacteria (obligates) belonging to Rickettsiales and Chlamydiales cause diseases in hundreds of millions of people worldwide and in many animal species. Lack of an efficient system for targeted mutagenesis in obligates remains a major impediment in understanding microbial pathogenesis. Challenges in creating targeted mutations may be attributed to essential nature of majority of the genes and intracellular replication dependence. Despite success in generating random mutations, a method that works well in creating mutations in specific genes of interest followed by complementation remains problematic for obligates and is a highly sought-after goal. We describe protocols to generate stable targeted mutations by allelic exchange in Ehrlichia chaffeensis, an obligate intracellular tick-borne bacterium responsible for human monocytic ehrlichiosis. Targeted mutations in E. chaffeensis were created to disrupt two genes, and also to restore one gene by another allelic exchange mutation leading to the restoration of transcription and protein expression from the inactivated gene and the recovered organisms also express mCherry, which distinguishes from the wild type. We expect that the methods developed are broadly applicable to other obligates, particularly to rickettsial pathogens, to routinely perform targeted mutations to enable studies focused on protein structure-function analyses, host-pathogen interactions and in developing vaccines

Mutations in Ehrlichia chaffeensis Causing Polar Effects in Gene Expression and Differential Host Specificities

Citation: Cheng, C. M., Nair, A. D. S., Jaworski, D. C., & Ganta, R. R. (2015). Mutations in Ehrlichia chaffeensis Causing Polar Effects in Gene Expression and Differential Host Specificities. Plos One, 10(7), 13. doi:10.1371/journal.pone.0132657Ehrlichia chaffeensis, a tick-borne rickettsial, is responsible for human monocytic ehrlichiosis. In this study, we assessed E. chaffeensis insertion mutations impacting the transcription of genes near the insertion sites. We presented evidence that the mutations within the E. chaffeensis genome at four genomic locations cause polar effects in altering gene expressions. We also reported mutations causing attenuated growth in deer (the pathogen's reservoir host) and in dog (an incidental host), but not in its tick vector, Amblyomma americanum. This is the first study documenting insertion mutations in E. chaffeensis that cause polar effects in altering gene expression from the genes located upstream and downstream to insertion sites and the differential requirements of functionally active genes of the pathogen for its persistence in vertebrate and tick hosts. This study is important in furthering our knowledge on E. chaffeensis pathogenesis

Transcription of Ehrlichia chaffeensis genes is accomplished by RNA polymerase holoenzyme containing either sigma 32 or sigma 70

Citation: Liu, H., Ohlen, T. V., Cheng, C., Faburay, B., & Ganta, R. R. (2013). Transcription of Ehrlichia chaffeensis Genes Is Accomplished by RNA Polymerase Holoenzyme Containing either Sigma 32 or Sigma 70. PLOS ONE, 8(11), e81780. https://doi.org/10.1371/journal.pone.0081780Bacterial gene transcription is initiated by RNA polymerase containing a sigma factor. To understand gene regulation in Ehrlichia chaffeensis, an important tick-transmitted rickettsiae responsible for human monocytic ehrlichiosis, we initiated studies evaluating the transcriptional machinery of several genes of this organism. We mapped the transcription start sites of 10 genes and evaluated promoters of five genes (groE, dnaK, hup, p28-Omp14 and p28-Omp19 genes). We report here that the RNA polymerase binding elements of E. chaffeensis gene promoters are highly homologous for its only two transcription regulators, sigma 32 and sigma 70, and that gene expression is accomplished by either of the transcription regulators. RNA analysis revealed that although transcripts for both sigma 32 and sigma 70 are upregulated during the early replicative stage, their expression patterns remained similar for the entire replication cycle. We further present evidence demonstrating that the organism’s -35 motifs are essential to transcription initiations. The data suggest that E. chaffeensis gene regulation has evolved to support the organism’s growth, possibly to facilitate its intraphagosomal growth. Considering the limited availability of genetic tools, this study offers a novel alternative in defining gene regulation in E. chaffeensis and other related intracellular pathogens

Bayesian spatio-temporal analysis and geospatial risk factors of Human Monocytic Ehrlichiosis

Citation: Raghavan RK, Neises D, Goodin DG, Andresen DA, Ganta RR (2014) Bayesian Spatio-Temporal Analysis and Geospatial Risk Factors of Human Monocytic Ehrlichiosis. PLoS ONE 9(7): e100850. doi:10.1371/journal.pone.0100850Variations in spatio-temporal patterns of Human Monocytic Ehrlichiosis (HME) infection in the state of Kansas, USA were examined and the relationship between HME relative risk and various environmental, climatic and socio-economic variables were evaluated. HME data used in the study was reported to the Kansas Department of Health and Environment between years 2005–2012, and geospatial variables representing the physical environment [National Land cover/Land use, NASA Moderate Resolution Imaging Spectroradiometer (MODIS)], climate [NASA MODIS, Prediction of Worldwide Renewable Energy (POWER)], and socio-economic conditions (US Census Bureau) were derived from publicly available sources. Following univariate screening of candidate variables using logistic regressions, two Bayesian hierarchical models were fit; a partial spatio-temporal model with random effects and a spatio-temporal interaction term, and a second model that included additional covariate terms. The best fitting model revealed that spatio-temporal autocorrelation in Kansas increased steadily from 2005–2012, and identified poverty status, relative humidity, and an interactive factor, ‘diurnal temperature range x mixed forest area’ as significant county-level risk factors for HME. The identification of significant spatio-temporal pattern and new risk factors are important in the context of HME prevention, for future research in the areas of ecology and evolution of HME, and as well as climate change impacts on tick-borne diseases

Hierarchical Bayesian Spatio-Temporal Analysis of Climatic and Socio-Economic Determinants of Rocky Mountain Spotted Fever

Citation: Raghavan, R. K., Goodin, D. G., Neises, D., Anderson, G. A., & Ganta, R. R. (2016). Hierarchical Bayesian Spatio-Temporal Analysis of Climatic and Socio-Economic Determinants of Rocky Mountain Spotted Fever. Plos One, 11(3), 17. doi:10.1371/journal.pone.0150180This study aims to examine the spatio-temporal dynamics of Rocky Mountain spotted fever (RMSF) prevalence in four contiguous states of Midwestern United States, and to determine the impact of environmental and socio-economic factors associated with this disease. Bayesian hierarchical models were used to quantify space and time only trends and spatio-temporal interaction effect in the case reports submitted to the state health departments in the region. Various socio-economic, environmental and climatic covariates screened a priori in a bivariate procedure were added to a main-effects Bayesian model in progressive steps to evaluate important drivers of RMSF space-time patterns in the region. Our results show a steady increase in RMSF incidence over the study period to newer geographic areas, and the posterior probabilities of county-specific trends indicate clustering of high risk counties in the central and southern parts of the study region. At the spatial scale of a county, the prevalence levels of RMSF is influenced by poverty status, average relative humidity, and average land surface temperature (> 35 degrees C) in the region, and the relevance of these factors in the context of climate-change impacts on tick-borne diseases are discussed

Vaccination with an Attenuated Mutant of Ehrlichia chaffeensis Induces Pathogen-Specific CD4(+) T Cell Immunity and Protection from Tick-Transmitted Wild-Type Challenge in the Canine Host

Citation: McGill, J. L., Nair, A. D. S., Cheng, C. M., Rusk, R. A., Jaworski, D. C., & Ganta, R. R. (2016). Vaccination with an Attenuated Mutant of Ehrlichia chaffeensis Induces Pathogen-Specific CD4(+) T Cell Immunity and Protection from Tick-Transmitted Wild-Type Challenge in the Canine Host. Plos One, 11(2), 15. doi:10.1371/journal.pone.0148229Ehrlichia chaffeensis is a tick-borne rickettsial pathogen and the causative agent of human monocytic ehrlichiosis. Transmitted by the Amblyomma americanum tick, E. chaffeensis also causes disease in several other vertebrate species including white-tailed deer and dogs. We have recently described the generation of an attenuated mutant strain of E. chaffeensis, with a mutation in the Ech_0660 gene, which is able to confer protection from secondary, intravenous-administered, wild-type E. chaffeensis infection in dogs. Here, we extend our previous results, demonstrating that vaccination with the Ech_0660 mutant protects dogs from physiologic, tick-transmitted, secondary challenge with wild-type E. chaffeensis; and describing, for the first time, the cellular and humoral immune responses induced by Ech_0660 mutant vaccination and wild-type E. chaffeensis infection in the canine host. Both vaccination and infection induced a rise in E. chaffeensis-specific antibody titers and a significant Th1 response in peripheral blood as measured by E. chaffeensis antigen-dependent CD4(+) T cell proliferation and IFN. production. Further, we describe for the first time significant IL-17 production by peripheral blood leukocytes from both Ech_0660 mutant vaccinated animals and control animals infected with wild-type E. chaffeensis, suggesting a previously unrecognized role for IL-17 and Th17 cells in the immune response to rickettsial pathogens. Our results are a critical first step towards defining the role of the immune system in vaccine-induced protection from E. chaffeensis infection in an incidental host; and confirm the potential of the attenuated mutant clone, Ech_0660, to be used as a vaccine candidate for protection against tick-transmitted E. chaffeensis infection