Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis:: Modulation of gp63 level in promastigotes and their infection of macrophages in vitro
Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis:: Modulation of gp63 level in promastigotes and their infection of macrophages in vitro
复制标题
DOI：
10.1128/iai.68.1.80-86.2000
复制
发表时间：
2000-01-01
影响因子：
3.1
通讯作者：
Chang, KP
中科院分区：
文献类型：
--
作者：
Chen, DQ;Kolli, BK;Chang, KP
亚马逊利什曼原虫主要表面糖蛋白(Gp63)是一种与哺乳动物巨噬细胞感染有关的金属蛋白酶。通过调节gp63基因在野生型克隆中的表达水平，进一步研究了gp63的表达及其在此次感染中的作用。将gp63基因以两种不同的方向分别导入利什曼原虫特异表达载体，获得了gp63正、反义RNA的表达。随着选择压力的增加，有正义转录子的转染者细胞表面gp63表达增强，而反义转录子转录者细胞表面gp63表达被抑制到很低的水平。因此，来自染色体重复基因的gp63的表达在蛋白质水平上没有受到严格的调控，并且可以通过单拷贝的异构体反义转录来显著减少。两种转染体只有在gp63的水平上存在显著差异，从而可以在体外对该分子在巨噬细胞利什曼病毒感染中的特异性进行评估。体外感染的动力学研究表明，gp63不仅在寄生虫与巨噬细胞的结合中发挥作用，而且在其在巨噬细胞内的存活和复制中也发挥作用。
The major surface glycoprotein (gp63) of Leishmania amazonensis is a metalloprotease implicated in the infection of mammalian macrophages. The expression of gp63 and its participation in this infection were further examined by modulating the level of this molecule in a virulent gp63-abundant wild-type clone. Promastigotes were transfected with gp63 genes cloned into a Leishmania-specific vector in two different orientations, leading to the expression of gp63 sense and antisense RNAs. With increasing selective pressure, cell surface gp63 was increasingly augmented in the transfectants with sense transcripts and suppressed to a very low level in those with antisense transcripts. Thus, the expression of gp63 from chromosomal, repetitive genes is not stringently regulated at the protein level and can be substantially reduced by episomal antisense transcription of a single copy. The transfectants differed significantly only in the level of gp63, thereby allowing specific evaluation of this molecule in leishmanial infection of macrophages in vitro. Kinetic studies of infection in vitro indicate that gp63 plays a role not only in the binding of this parasite to these macrophages but also in its intramacrophage survival and replication.