Project description:Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of Scrub typhus. The bacterium can replicate both in its arthropod host and in mammals, including humans. The control mechanisms for bacterial gene expression within eukaryotic host cells are largely unknown. Previously, we reported that the O. tsutsugamushi genome has the highest repeat density of any sequenced bacterial genome due to the extraordinary proliferation of mobile genetic elements (MGEs), suggesting a unique genomic evolution in intracellular niches. In this study, the global gene expression of O. tsutsugamushi within eukaryotic cells was examined using a microarray and proteomic approaches. These approaches identified 643 genes, corresponding to approximately 30% of genes encoded in the genome. The majority of expressed genes belonged to several functional categories including protein translation, protein processing/secretion, and replication/repair. We also searched the conserved sequence blocks (CSBs) in the genomes of different O. tsutsugamushi strains to identify gene blocks impermeable to proliferating MGEs. Although extensive shuffling of genomic sequences was observed between two different strains, 204 CSBs with sizes ranging from 1 kbp to 29 kbp, covering 48% of the genome, were identified. When combining the data of the CSBs and global gene expression, CSBs in the O. tsutsugamushi genomes correlates well with the location of expressed genes, especially in protein level, suggesting the functional conservation between gene expression and genomic location. Finally, we compared global gene expression of the bacteria infecting fibroblasts and macrophages using microarray analysis. Major changes in the expression of genes with known functions were the downregulation of genes involved in translation, protein processing and secretion, which resulted in significant reduction in bacterial translation rates and growth within macrophages. These results suggest that the replication of O. tsutsgamushi is controlled primarily by the expression of genes involved in bacterial translation and subsequent protein processing/secretion.

Project description:Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of Scrub typhus. The bacterium can replicate both in its arthropod host and in mammals, including humans. The control mechanisms for bacterial gene expression within eukaryotic host cells are largely unknown. Previously, we reported that the O. tsutsugamushi genome has the highest repeat density of any sequenced bacterial genome due to the extraordinary proliferation of mobile genetic elements (MGEs), suggesting a unique genomic evolution in intracellular niches. In this study, the global gene expression of O. tsutsugamushi within eukaryotic cells was examined using a microarray and proteomic approaches. These approaches identified 643 genes, corresponding to approximately 30% of genes encoded in the genome. The majority of expressed genes belonged to several functional categories including protein translation, protein processing/secretion, and replication/repair. We also searched the conserved sequence blocks (CSBs) in the genomes of different O. tsutsugamushi strains to identify gene blocks impermeable to proliferating MGEs. Although extensive shuffling of genomic sequences was observed between two different strains, 204 CSBs with sizes ranging from 1 kbp to 29 kbp, covering 48% of the genome, were identified. When combining the data of the CSBs and global gene expression, CSBs in the O. tsutsugamushi genomes correlates well with the location of expressed genes, especially in protein level, suggesting the functional conservation between gene expression and genomic location. Finally, we compared global gene expression of the bacteria infecting fibroblasts and macrophages using microarray analysis. Major changes in the expression of genes with known functions were the downregulation of genes involved in translation, protein processing and secretion, which resulted in significant reduction in bacterial translation rates and growth within macrophages. These results suggest that the replication of O. tsutsgamushi is controlled primarily by the expression of genes involved in bacterial translation and subsequent protein processing/secretion. Microarray analysis using the Combimatrix CustomArrayTM 4X2 microarray (CombiMatrix) was performed according to the standard CombiMatrix protocol described in detail at http://www.combimatrix.com/ products_custom4x2.htm (PTL005). Oligonucleotide probes (27 to 40-mers, 1613 probes) were designed for 1472 CDSs of the O. tsutsugamushi genome (GeneBank accession no. AM494475). 1 to 6 probes per CDS were designed and some probes were duplicated on the microarray (total 2187 spots on an array). As negative controls, 5 and 15 oligonucleotide probes derived from plant and bacteriophage respectively were spotted on the array at 53 sites per array. A local background subtraction method was used and the background-subtracted signal values were imported into the Avadis Prophetic software (ver. 3.3, Strand Genomics) or Expander (ver. 4.1, http://acgt.cs.tau.ac.il/expander/) for data analysis. . Global normalization of gene expression data was performed using the quantile normalization method embedded in the Expander software. The standard deviations of log data and the p-values were calculated using default settings in the Avadis software. Transcriptome analysis was carried out using three biological replicates for uninfected samples (uninfected L929 cells) and five biological replicates for infected samples (L929 cells infected with O. tsutsugamushi for 48 h). For comparison of the O. tsutsugamushi transcriptome from fibroblasts (L929 cells) and macrophages (J774A.1 cells), two biological replicates from each cell line were used.

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Orientia tsutsugamushi, the agent of scrub typhus, induces an inflammatory program in human macrophages

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