Transcriptional silencing of multiple genes in trophozoites of Entamoeba histolytica.
Transcriptional silencing of multiple genes in trophozoites of Entamoeba histolytica.
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DOI：
10.1371/journal.ppat.0020048
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发表时间：
2006-05
期刊：
影响因子：
6.7
通讯作者：
Mirelman D
中科院分区：
文献类型：
--
作者：
Bracha R;Nuchamowitz Y;Anbar M;Mirelman D
在以前的工作中，我们描述了阿米巴A（AP-A）基因（Ehap-a）的溶组织内阿米巴菌株HM-1：IMSS的转录沉默。用含有Ehap-a 5′上游区（473 bp）的质粒转染后发生沉默，所述Ehap-a 5′上游区（473 bp）包括短散布核元件（SINE 1）的截短片段（140 bp）。即使在去除质粒（克隆G3）后，沉默仍然有效。在基因沉默的滋养体中，没有检测到短干扰RNA和甲基化DNA，但Ehap-a的染色质结构域被修饰。另外两个类似的基因（Ehap-b和一个编码Saposin样蛋白SAPLIP 1）也被沉默。在目前的工作中，我们证明了沉默的第二个基因的选择，一个编码的轻亚基的半乳糖/GalNAc可凝集素（Ehlgl 1）和其他半胱氨酸蛋白酶5（EhCP-5）。这种沉默发生在用质粒转染的G3滋养体中，其中473 bp的5′上游Ehap-a片段直接连接到第二个基因。在转基因和染色体基因中都发生了转录沉默。SINE 1序列是必不可少的，因为Ehap-a上游区域和第二个基因的开放阅读框的开始之间有直接的连接。基因沉默没有发生在菌株HM-1：IMSS与任何这些质粒构建体。两个基因沉默的滋养体的毒力减弱，是克隆G3。此外，不表达Lgl 1和AP-A蛋白的滋养体在与凝集素重（170 kDa）亚基抗体孵育时，将Gal/GalNAc-凝集素加帽至尿状体区域的能力显著降低。缺乏半胱氨酸蛋白酶5和AP-A蛋白的滋养体溶解产物的半胱氨酸蛋白酶活性比HM-1：IMSS菌株或G3克隆的溶解产物低30%。G3阿米巴中其他基因的沉默可以为研究它们的各种功能提供一个模型。此外，双基因沉默，毒力减弱的滋养体可能是疫苗开发的重要工具。人类肠道寄生虫溶组织内阿米巴有许多编码毒力的基因。沉默单个基因的表达有助于确定它们的作用。在以前的工作中，作者证明了编码阿米巴的基因的沉默，阿米巴负责杀死人类细胞。他们用一种质粒转染阿米巴滋养体，该质粒含有与阿米巴基因启动子区同源的DNA序列，以及一部分重复DNA元件（称为短散布核元件）。该构建体诱导染色质的修饰并阻止基因的表达。质粒的去除导致稳定的，阿米巴孔缺陷型寄生虫具有低毒力。在目前的工作中，Bracha和他的同事发现，在转染大肠杆菌后，其他基因沉默。溶组织菌滋养体已经在阿米巴中被含有直接连接到阿米巴基因上游区域的第二基因的质粒沉默。鉴定了将沉默从质粒转移到染色体基因拷贝所必需的DNA序列。作者不可逆沉默的其他毒力基因是编码介导寄生虫粘附宿主细胞的表面凝集素亚基和在肠道炎症和入侵中起作用的半胱氨酸蛋白酶的基因。
In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5′ upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5′ upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development. The human intestinal parasite Entamoeba histolytica has numerous genes that code for virulence. Silencing the expression of individual genes is useful to determine their roles. In previous work the authors demonstrated the silencing of the gene coding for amoebapore, which is responsible for killing of human cells. They transfected amoebic trophozoites with a plasmid that contained DNA sequences homologous to the promoter region of the amoebapore gene, as well as a portion of a repetitive DNA element (called a short interspersed nuclear element). This construct induced a modification of the chromatin and prevented the expression of the gene. Removal of the plasmid resulted in stable, amoebapore-deficient parasites possessing low virulence. In the present work, Bracha and colleagues show silencing of additional genes following transfection of E. histolytica trophozoites already silenced in amoebapore with a plasmid containing the second gene directly ligated to the upstream region of the amoebapore gene. The DNA sequences that are essential for transferring the silencing from the plasmid to the chromosomal gene copy were identified. Additional virulence genes that the authors irreversibly silenced are those encoding a subunit of a surface lectin that mediates the adherence of the parasite to host cells, and a cysteine proteinase that plays a role in inflammation and invasion of the intestine.