Protein translation as major target for gene expression control of O. tsutsugamushi. Protein translation as major target for gene expression control of O. tsutsugamushi

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. Overall design: 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.

恙虫病东方体（Orientia tsutsugamushi）是一种专性胞内细菌，亦是恙虫病（Scrub typhus）的致病原。该细菌可在其节肢动物宿主以及包括人类在内的哺乳动物体内增殖。目前，关于真核宿主细胞内该细菌的基因表达调控机制仍知之甚少。此前本团队已有研究报道，恙虫病东方体基因组是目前已测序细菌基因组中重复序列密度最高的，这源于移动遗传元件（mobile genetic elements, MGEs）的大量增殖，提示其在胞内生存环境中存在独特的基因组演化模式。
本研究采用基因芯片（microarray）与蛋白质组学方法，对真核细胞内恙虫病东方体的全基因组基因表达情况进行了检测。上述方法共鉴定出643个表达基因，约占基因组编码基因总数的30%。大部分表达基因隶属于多个功能类别，包括蛋白质翻译、蛋白质加工/分泌以及复制/修复相关基因。本研究同时对不同恙虫病东方体菌株的基因组进行了保守序列块（conserved sequence blocks, CSBs）检索，以筛选出不受移动遗传元件增殖影响的基因区域。尽管两株不同菌株的基因组序列发生了广泛的重排，研究仍鉴定出204个保守序列块，其长度范围为1 kbp至29 kbp，覆盖了基因组48%的区域。
将保守序列块与全基因组基因表达数据相结合后可见，恙虫病东方体基因组中的保守序列块与表达基因的位置高度吻合，尤其在蛋白质水平上更为显著，这提示基因表达与基因组位置之间存在功能保守性。最后，本研究通过基因芯片分析，对比了感染成纤维细胞与巨噬细胞的恙虫病东方体的全基因组基因表达情况。具有已知功能的基因表达主要变化为：与翻译、蛋白质加工及分泌相关的基因表达下调，这导致细菌的翻译速率显著降低，并使其在巨噬细胞内的增殖能力减弱。上述结果表明，恙虫病东方体的增殖主要受细菌翻译及后续蛋白质加工/分泌相关基因的表达调控。
实验整体设计：本研究采用CombiMatrix公司CustomArray™ 4X2基因芯片（CombiMatrix），按照该公司公开的标准实验流程（详见http://www.combimatrix.com/products_custom4x2.htm，文档编号PTL005）进行芯片实验。针对恙虫病东方体基因组的1472个编码序列（CDS），设计了长度为27至40个碱基的寡核苷酸探针（共1613条探针），对应GenBank（基因序列数据库）登录号AM494475。每个编码序列对应1至6条探针，部分探针在芯片上重复排布，单张芯片总斑点数为2187个。设置阴性对照：分别从植物与噬菌体中衍生出5条和15条寡核苷酸探针，每张芯片上各在53个位点进行点样。本研究采用局部背景扣除法处理数据，将扣除背景后的信号值导入Avadis Prophetic软件（版本3.3，Strand Genomics公司）或Expander软件（版本4.1，http://acgt.cs.tau.ac.il/expander/）进行数据分析。采用Expander软件内置的分位数归一化法（quantile normalization）完成基因表达数据的全局归一化。利用Avadis软件的默认参数计算对数化数据的标准差与p值。转录组分析设置3个生物学重复用于未感染样本（未感染的L929细胞），以及5个生物学重复用于感染样本（感染恙虫病东方体48小时的L929细胞）。为对比成纤维细胞（L929细胞）与巨噬细胞（J774A.1细胞）来源的恙虫病东方体转录组，每个细胞系均采用2个生物学重复样本进行分析。