Regulation of H2O2 Stress-responsive Genes through a Novel Transcription Factor in the Protozoan Pathogen Entamoeba histolytica
Regulation of H2O2 Stress-responsive Genes through a Novel Transcription Factor in the Protozoan Pathogen Entamoeba histolytica
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DOI：
10.1074/jbc.m112.423467
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发表时间：
2013-02-08
影响因子：
4.8
通讯作者：
Singh, Upinder
中科院分区：
文献类型：
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作者：
Pearson, Richard J.;Morf, Laura;Singh, Upinder
感染的结果取决于入侵的病原体和宿主之间复杂的相互作用。作为宿主先天免疫反应的一部分，吞噬细胞释放活性氧和氮物质是建立感染的主要障碍。人体寄生虫溶组织内阿米巴存活活性氧和氮的能力是其致病潜力的核心，并有助于疾病的结果。为了确定与氧化应激相关的转录网络，我们利用MEME和MAST程序分析了57个阿米巴基因的启动子区域，这些基因在H2 O2暴露后特异性表达增加。我们在功能上表征了H2 O2-调节基序（HRM）（（1）AAACCTCAATGAAGA（15）），其在这些启动子中富集并特异性结合阿米巴核蛋白。启动子-荧光素酶融合的测定确立了关键残基的重要性，并且HRM基序直接影响H2 O2响应启动子驱动基因表达的能力。DNA亲和层析和质谱鉴定EHI_108720为HRM DNA结合蛋白。EHI_108720的过表达和下调证明了EHI_108720蛋白与HRM结合的特异性，并且过表达增加了H2 O2响应性野生型启动子的基础表达，但不增加其突变体的表达。因此，EHI_108720或HRM结合蛋白代表了E.溶组织因子，其控制与氧化应激相关的转录调节网络。EHI_108720的过表达增加了寄生虫的毒力。了解E. histolytica对氧化应激的反应增加了我们对这种重要的人类病原体如何建立侵袭性疾病的理解。
Outcome of infection depends upon complex interactions between the invading pathogen and the host. As part of the host's innate immune response, the release of reactive oxygen and nitrogen species by phagocytes represents a major obstacle to the establishment of infection. The ability of the human parasite Entamoeba histolytica to survive reactive oxygen and nitrogen species is central to its pathogenic potential and contributes to disease outcome. In order to define the transcriptional network associated with oxidative stress, we utilized the MEME and MAST programs to analyze the promoter regions of 57 amoebic genes that had increased expression specifically in response to H2O2 exposure. We functionally characterized an H2O2-regulatory motif (HRM) ((1)AAACCTCAATGAAGA(15)), which was enriched in these promoters and specifically bound amoebic nuclear protein(s). Assays with promoter-luciferase fusions established the importance of key residues and that the HRM motif directly impacted the ability of H2O2-responsive promoters to drive gene expression. DNA affinity chromatography and mass spectrometry identified EHI_108720 as an HRM DNA-binding protein. Overexpression and down-regulation of EHI_108720 demonstrated the specificity of EHI_108720 protein binding to the HRM, and overexpression increased basal expression from an H2O2-responsive wild-type promoter but not from its mutant counterpart. Thus, EHI_108720, or HRM binding protein, represents a new stress-responsive transcription factor in E. histolytica that controls a transcriptional regulatory network associated with oxidative stress. Overexpression of EHI_108720 increased parasite virulence. Insight into how E. histolytica responds to oxidative stress increases our understanding of how this important human pathogen establishes invasive disease.