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

ABSTRACT: Ehrlichia 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.

SUBMITTER: Cheng C

PROVIDER: S-EPMC4505860 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Cheng Chuanmin C Nair Arathy D S AD Jaworski Deborah C DC Ganta Roman R RR

PloS one 20150717 7

Ehrlichia 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), b ...[more]

PMID: 26186429

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Project description:The tick-borne rickettsial pathogen, Ehrlichia chaffeensis, causes monocytic ehrlichiosis in people and other vertebrate hosts. Mutational analysis in E. chaffeensis genome aids in better understanding of its infection and persistence in host cells and in the development of attenuated vaccines. Our recent RNA deep sequencing study revealed that three genomic mutations caused global changes in the gene expression patterns, which in turn affect the ability of pathogen's survival in a host and the host's ability to induce protection against the pathogen. In this follow-up study, we document the impact of mutations on the pathogen's global protein expression and the influence of protein abundance on a mutant's attenuation and protection of vertebrate host against infection. iTRAQ labeling and mass spectrometry analysis of E. chaffeensis wildtype and mutants identified 564 proteins covering about 63% of the genome. Mutation in ECH_0379 gene encoding for an antiporter protein, causing attenuated growth in vertebrate hosts, led to overexpression of p28 outer membrane proteins, molecular chaperons, and metabolic enzymes, while a mutation downstream to the ECH_0490 gene that caused minimal impact on the pathogen's in vivo growth resulted in major changes in the expression of outer membrane proteins, transcriptional regulators and T4SS proteins. ECH_0660 gene mutation, causing the pathogen's rapid clearance and offering protection against wild type infection challenge in a vertebrate host, had a minimal impact on proteome similar to our prior observations from transcriptome analysis. While the global proteome data revealed fewer translated proteins compared to the transcripts identified from RNA deep sequencing analysis, there is a great deal of correlation noted between the global proteome and transcriptome analysis. Further, global proteome analysis, including the assessment of 2D resolved total and immunoproteomes revealed greater variations in the highly immunogenic p28-Omp proteins.

Project description:Human monocytotropic ehrlichiosis is an emerging life-threatening zoonosis caused by obligately intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is transmitted by the lone star tick, Amblyomma americanum, and replicates in mononuclear phagocytes in mammalian hosts. Differences in the E. chaffeensis transcriptome in mammalian and arthropod hosts are unknown. Thus, we determined host-specific E. chaffeensis gene expression in human monocyte (THP-1) and in Amblyomma and Ixodes tick cell lines (AAE2 and ISE6) using a whole genome microarray.The majority (?80%) of E. chaffeensis genes were expressed during infection in human and tick cells. There were few differences observed in E. chaffeensis gene expression between the vector Amblyomma and non-vector Ixodes tick cells, but extensive host-specific and differential gene expression profiles were detected between human and tick cells, including higher transcriptional activity in tick cells and identification of gene subsets that were differentially expressed in the two hosts. Differentially and host-specifically expressed ehrlichial genes encoded major immunoreactive tandem repeat proteins (TRP), the outer membrane protein (OMP-1) family, and hypothetical proteins that were 30-80 amino acids in length. Consistent with previous observations, high expression of p28 and OMP-1B genes was detected in human and tick cells, respectively. Notably, E. chaffeensis genes encoding TRP32 and TRP47 were highly upregulated in the human monocytes and expressed as proteins; however, although TRP transcripts were expressed in tick cells, the proteins were not detected in whole cell lysates demonstrating that TRP expression was post transcriptionally regulated.Ehrlichia gene expression is highly active in tick cells, and differential gene expression among a wide variety of host-pathogen associated genes occurs. Furthermore, we demonstrate that genes associated with host-pathogen interactions are differentially expressed and regulated by post transcriptional mechanisms.

Project description:Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

Dataset Information

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

Publications

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

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