Entamoeba invadens: Identification of ADF/cofilin and their expression analysis in relation to encystation and excystation
Entamoeba invadens: Identification of ADF/cofilin and their expression analysis in relation to encystation and excystation
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DOI：
10.1016/j.exppara.2010.07.018
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发表时间：
2011-01-01
影响因子：
2.1
通讯作者：
Takeuchi, Tsutomu
中科院分区：
文献类型：
--
作者：
Makioka, Asao;Kumagai, Masahiro;Takeuchi, Tsutomu
内阿米巴原虫的胞外和胞内分化过程是感染和完成其生命周期所必需的，这一过程需要细胞运动及其通过肌动蛋白细胞骨架重组来控制。本研究研究了内阿米巴侵染虫中肌动蛋白细胞骨架重组的重要分子——肌动蛋白解聚因子(ADF)/cofilin （Cfl）家族蛋白与胞内和胞外的关系。用无性系培养系统诱导胞内和胞外。同源性检索和分子克隆鉴定出3个ADF/Cfl家族蛋白（简称EiCfl-1、EiCfl-2和EiCfl-3）。这与其他内阿米巴原虫不同，即溶组织内阿米巴原虫和异速内阿米巴原虫，它们各自只有一个ADF/Cfl家族蛋白。这些ADF/Cfl不含Ser3（丝氨酸位于第一个蛋氨酸的第三位），类似于E. histolytica， E. dispar， Saccharomyces cerevisiae和Schizosaccharomyces pombe，尽管ADF/Cfl的活性在后生动物中受到Ser3磷酸化的负调控。系统发育分析显示，内阿米巴形成了一个独特的分支，与其他生物分开，树的分支分为两部分，与Ser3的存在和不存在一致。兔抗EiCfl-2血清与所有重组EiCfl及囊、滋养体和后囊变形虫裂解物中的EiCfl均有反应。该抗血清免疫荧光染色显示EiCfl与肌动蛋白在细胞膜下的共定位贯穿整个生命阶段。这两种蛋白均富含滋养体伪足和后囊变形虫。Real-time RT-PCR显示EiCfl-2和actis mRNA高表达，EiCfl-1和EiCfl-3 mRNA表达较少。在囊化过程中，EiCfl-2和actis的mRNA水平显著降低。所有三个EiCfls和actis在诱导增殖后都开始转录。在细胞松弛素d的作用下，只有EiCfl-1和EiCfl-3的mrna显著升高。这些结果表明，EiCfl-2和肌动素在滋养体和包囊以及假足丰富的后囊变形虫的细胞膜下共定位，EiCfl-1和EiCfl-3仅在诱导脱落后才表达，细胞松弛素的增强与EiCfl-1和EiCfl-3的高表达分离。(C) 2010爱思唯尔公司版权所有。
The differentiation processes of excystation and encystation of Entamoeba are essential for infection and completion of their life-cycle, and the processes need cell motility and its control by actin cytoskeletal reorganization. This study investigated actin depolymerizing factor (ADF)/cofilin (Cfl) family proteins, which are important molecules in actin cytoskeletal reorganization, in Entamoeba invadens in relation to the encystation and excystation. Axenic culture systems were used to induce encystation and excystation. A homology search of the E. invadens genome database and molecular cloning identified three ADF/Cfl family proteins of the parasite (named for short as EiCfl-1, EiCfl-2, and EiCfl-3). This is different from other Entamoeba species, i.e. Entamoeba histolytica and Entamoeba dispar, each of which has only one ADF/Cfl family protein. These ADF/Cfl of E. invadens do not have Ser3 (serine locates third from first methionine), similar to E. histolytica, E. dispar, Saccharomyces cerevisiae and Schizosaccharomyces pombe, although the activity of ADF/Cfl is negatively regulated by phosphorylation of the Ser3 in metazoans. Phylogenetic analysis revealed that Entamoeba Cfl formed a distinctive clade that is separate from other organisms, and the branches of the tree were separated in two consistent with the presence and absence of Ser3. Rabbit anti-EiCfl-2 serum reacted with all recombinant EiCfls and EiCfl in lysates of cysts, trophozoites and metacystic amoebae. Immunofluorescence staining with this antiserum showed co-localization of EiCfl with actin beneath the cell membrane through the life stages. Both proteins proved to be rich in pseudopodia of trophozoites and metacystic amoebae. Real-time RT-PCR showed that mRNAs of EiCfl-2 and actins were highly expressed, but there were few mRNA of EiCfl-1 and EiCfl-3. Remarkably decreased mRNA levels were observed in EiCfl-2 and actins during encystation. All three EiCfls and actins became transcribed after the induction of excystation. The mRNAs of only EiCfl-1 and EiCfl-3 increased remarkably when the excystation was induced in the presence of cytochalasin D. These findings demonstrate that EiCfl-2 and actins co-localize beneath the cell membrane in trophozoites and cysts as well as metacystic amoebae being rich in pseudopodia, that EiCfl-1 and EiCfl-3 are expressed only after the induction of excystation, and that enhanced excystation by cytochalasin Disassociated with high expression of EiCfl-1 and EiCfl-3. (C) 2010 Elsevier Inc. All rights reserved.