Project description:Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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:Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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. A microarray (4-plex) study using E. chaffeensis cultivated in each cell line (THP-1, AAE2 and ISE6), three biological replicates/cell line. For each cell line, RNA was also extracted from uninfected cells (negative controls) and was processed similar to the infected cells; these samples were used for background subtraction during data analysis.
Project description:This study is to determine and compare the transcriptomes in two eukaryotic hosts (mammalian host - DH82 canine cell line and tick vector - ISE6 Ixodes scapularis cell line) of eight Ehrlichia chaffeensis strains in three divergent genetic groups, including Arkansas, Heartland, Jax, Liberty, Osceola, St. Vincent, Wakulla, and West Paces, and one Ehrlichia sp. HF strain.
Project description:Ehrlichia chaffeensis is an obligatory intracellular organism, and causes human monocytic ehrlichiosis (HME), a tick-borne illness. Clinical signs of HME vary from asymptomatic infection to severe mobility that requires hospitalization or death. After whole genome sequencing of this bacterium, 425 open reading frames (38%) of 1115 in the genome were found as hypothetical gene with unknown function. A little is known about pathogenicity gene(s) and strain variation of this bacterium to date. Previously several E. chaffeensis strains were shown to be different based on comparison of limited gene sequences (p120, OMP-1[p28]s, and VLPT). To elucidate strain genetic variations and associated virulency, we carried out comparative genome hybridization (CGH) of E. chaffeensis Arkansas, Wakulla and Liberty strains, since these strains are most distinct in the previous studies. The tiling array containing approximately 300,000 probes of 29 nt spaced as dense as 8 bp on both strands of the genome of E. chaffeensis Arkansas was used. Keywords: Comparative genome hybridization
Project description:This study is to determine and compare the transcriptomes in two eukaryotic hosts (mammalian host - DH82 canine cell line and tick vector - ISE6 Ixodes scapularis cell line) of eight Ehrlichia chaffeensis strains in three divergent genetic groups, including Arkansas, Heartland, Jax, Liberty, Osceola, St. Vincent, Wakulla, and West Paces, and one Ehrlichia sp. HF strain. RNA samples for the Arkansas (reference), Heartland, Jax, Liberty, Osceola, St. Vincent, Wakulla, and West Paces strains in both tick and canine cell lines, including two uninfected controls, will be isolated in triplicate. As such 108 libraries will be constructed and sequenced corresponding to (8 strains + 1 control) x 2 conditions (tick cells + dog cells) x 3 biological triplicates x 2 molecules sequenced (eukaryotic RNA + prokaryotic RNA). The transcriptomes are obtained with Illumina HiSeq reads obtained from paired end libraries. The bacterial reads will be mapped against the reference genome and compared through a differential expression analysis. The eukaryotic reads from the same cell types (i.e. tick or canine) will be assembled with Trinity. The reads will then be mapped back to this assembly for the differential expression analysis.
Project description:Ehrlichia chaffeensis is an obligately intracellular bacterium that establishes infection in mononuclear phagocytes through largely undefined reprogramming strategies. Recently, E. chaffeensis effectors Ank200, TRP120, and TRP32 have been shown to function as nucleomodulins that enter the host nucleus and directly modulate transcription of genes implicated in cellular processes such as transcription regulation, apoptosis, phosphorylation, and immune cell activation. In this study, we found that E. chaffeensis TRP47 enters the host cell nucleus and binds regulatory regions of multiple host genes relevant to infection.
Project description:Host genes interacting with ankyrin repeat-containing protein, p200, in Ehrlichia chaffeensis-infected monocytes. Chromatin immunoprecipitation (ChIP) together with microarray (chip) analysis demonstrated enrichment of relatively small subset (n=456) of host cell genes associated with molecular and biological processes, many of which are known to be altered during ehrlichial infection. Keywords: ChIP-chip
Project description:Host genes interacting with ankyrin repeat-containing protein, p200, in Ehrlichia chaffeensis-infected monocytes. Chromatin immunoprecipitation (ChIP) together with microarray (chip) analysis demonstrated enrichment of relatively small subset (n=456) of host cell genes associated with molecular and biological processes, many of which are known to be altered during ehrlichial infection. Keywords: ChIP-chip Detection of genes with high signal enrichment by comparing the ratios of p200/input