Department of Tropical Medicine, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo, 105-8461, Japan.
Parasitol Res. 2012 Jun;110(6):2095-104. doi: 10.1007/s00436-011-2735-3. Epub 2011 Dec 17.
Cell motility by actin cytoskeleton is essential for differentiation processes of excystation and encystation of Entamoeba. We recently studied an actin depolymerizing factor (ADF)/cofilin (Cfl) of Entamoeba invadens (Ei), and demonstrated its contribution to the encystation and excystation of E. invadens through actin cytoskeletal reorganization. Profilin is also an actin-binding protein but its function is different from that of Cfl in actin assembly. This study investigated E. invadens profilins in relation to encystation and excystation which were induced in axenic culture systems. A homology search of the E. invadens genome database and molecular cloning identified four profilins of the parasite named EiPFN1, EiPFN2, EiPFN3, and EiPFN4. There were also multiple genes of profilin in Entamoeba histolytica (Eh) and Entamoeba dispar (Ed), each of which had three profilins. A search for conserved domains revealed that these profilins of Entamoeba had actin, phosphoinositide, and poly-proline binding sites. Phylogenetic analysis revealed that EiPFN3 and EiPFN4 formed the same clades including EhPFN3 and EdPFN3, and EhPFN2 and EdPFN2, respectively, while EiPFN1 and EiPFN2 were separated from EhPFN1 and EdPFN1. Rabbit anti-EiPFN1 serum reacted with recombinant EiPFN3 and EiPFN4 but not EiPFN2, and also reacted with EiPFN in lysates of cysts and trophozoites. Immunofluorescence staining with this antiserum showed co-localization of EiPFN with actin beneath the cell membrane through the life stages and also showed cytoplasmic localization. Both proteins proved to be rich in pseudopodia of trophozoites. Real-time RT-PCR showed that the mRNA level of EiPFN1 and EiPFN4 in trophozoites was comparable but that of EiPFN2 and EiPFN3 was very low. During encystation, the mRNA expression of EiPFN1 and EiPFN4 increased remarkably in the early phase much higher than that of EiPFN2 and EiPFN3. Then, the expression of all four PFNs sharply decreased in the later phase. This was in contrast to the sharp decrease in the mRNA level of EiCfl-2 during encystation in our previous study. In cysts, EiPFN1 was most abundantly expressed and EiPFN4 was at a lower level, while the expressions of EiPFN2 and EiPFN3 were virtually absent. Following the induction of excystation, mRNA levels of EiPFN1, EiPFN2, and EiPFN4 in cysts 5 h after induction were significantly higher than those in cysts before induction, while that of EiPFN3 was slightly higher than before induction. The mRNAs of EiPFN1 increased most extensively when the excystation was induced in the presence of cytochalasin D. Small interfering RNA (siRNA) to EiPFN1 inhibited both encystation and excystation but not growth. These findings demonstrate different expression of EiPFNs and the contribution of EiPFN to the encystation and excystation.
细胞运动肌动蛋白细胞骨架是必不可少的分化过程中的逸出和包囊的 Entamoeba。我们最近研究了一种肌动蛋白解聚因子（ADF）/丝切蛋白（Cfl）的 Entamoeba invadens（Ei），并证明它通过肌动蛋白细胞骨架重组对 Entamoeba invadens 的逸出和逸出有贡献。前蛋白也是一种肌动蛋白结合蛋白，但它在肌动蛋白组装中的作用不同于 Cfl。本研究调查了 Entamoeba invadens 前蛋白与在无细胞培养系统中诱导的逸出和逸出的关系。对 Entamoeba invadens 基因组数据库的同源搜索和分子克隆鉴定了寄生虫的四种前蛋白，命名为 EiPFN1、EiPFN2、EiPFN3 和 EiPFN4。在 Entamoeba histolytica（Eh）和 Entamoeba dispar（Ed）中也有多个前蛋白基因，每个基因都有三种前蛋白。保守结构域搜索表明，这些 Entamoeba 的前蛋白具有肌动蛋白、磷酸肌醇和多脯氨酸结合位点。系统发育分析表明，EiPFN3 和 EiPFN4 形成了相同的分支，包括 EhPFN3 和 EdPFN3，以及 EhPFN2 和 EdPFN2，而 EiPFN1 和 EiPFN2 则与 EhPFN1 和 EdPFN1 分开。兔抗 EiPFN1 血清与重组 EiPFN3 和 EiPFN4 反应，但与 EiPFN2 不反应，也与胞囊和滋养体裂解物中的 EiPFN 反应。用该抗血清进行免疫荧光染色显示 EiPFN 与细胞膜下的肌动蛋白在生命阶段的共定位，也显示细胞质定位。两种蛋白都被证明富含滋养体的伪足。实时 RT-PCR 显示，在滋养体中 EiPFN1 和 EiPFN4 的 mRNA 水平相当，但 EiPFN2 和 EiPFN3 的水平非常低。在包囊形成过程中，EiPFN1 和 EiPFN4 的 mRNA 表达在早期阶段显著增加，远高于 EiPFN2 和 EiPFN3。然后，所有四种 PFNs 的表达在后期急剧下降。这与我们之前的研究中 EiCfl-2 在包囊形成过程中 mRNA 水平的急剧下降形成对比。在胞囊内，EiPFN1 表达最丰富，EiPFN4 表达水平较低，而 EiPFN2 和 EiPFN3 的表达几乎不存在。在诱导逸出后，诱导后 5 小时胞囊内 EiPFN1、EiPFN2 和 EiPFN4 的 mRNA 水平明显高于诱导前的水平，而 EiPFN3 的水平略高于诱导前的水平。当在细胞松弛素 D 的存在下诱导逸出时，EiPFN1 的 mRNA 增加最多。小干扰 RNA（siRNA）对 EiPFN1 的抑制作用既抑制包囊形成又抑制逸出，但不抑制生长。这些发现表明 EiPFNs 的不同表达和 EiPFN 对包囊形成和逸出的贡献。
