Vanchinathan Padmini, Brewer Jeremy L, Harb Omar S, Boothroyd John C, Singh Upinder
Department of Internal Medicine, Division of Infectious Diseases, Stanford University School of Medicine, California 94305-5124, USA.
Infect Immun. 2005 Oct;73(10):6680-8. doi: 10.1128/IAI.73.10.6680-6688.2005.
During its life cycle in intermediate hosts, Toxoplasma gondii exists in two interconverting developmental stages: tachyzoites and bradyzoites. This interconversion is essential for the survival and pathogenicity of the parasite, but little is known about the genetic mechanisms that control this process. We have previously generated tachyzoite-to-bradyzoite differentiation (Tbd(-)) mutants using chemical mutagenesis and a green fluorescent protein-based selection strategy. The genetic loci responsible for the Tbd(-) phenotype, however, could not be identified. We have now used an insertional mutagenesis strategy to generate two differentiation mutants: TBD-5 and TBD-6 that switch to bradyzoites at 10 and 50% of wild-type levels, respectively. In TBD-6 there is a single insertion of the mutagenesis vector 164 bp upstream of the transcription start site of a gene encoding a zinc finger protein (ZFP1). Disruption of this locus in wild-type parasites reproduces the decreased stage conversion phenotype. ZFP1 is targeted to the parasite nucleolus by CCHC motifs and significantly altered expression levels are toxic to the parasites. This represents the first identification of a gene necessary for efficient conversion of tachyzoites to bradyzoites.
在中间宿主的生命周期中，刚地弓形虫存在于两种相互转化的发育阶段：速殖子和缓殖子。这种相互转化对于寄生虫的生存和致病性至关重要，但对于控制这一过程的遗传机制知之甚少。我们之前使用化学诱变和基于绿色荧光蛋白的筛选策略产生了速殖子向缓殖子分化（Tbd(-)）突变体。然而，负责Tbd(-)表型的基因位点无法确定。我们现在使用插入诱变策略产生了两个分化突变体：TBD-5和TBD-6，它们分别以野生型水平的10%和50%转变为缓殖子。在TBD-6中，诱变载体在编码锌指蛋白（ZFP1）的基因转录起始位点上游164 bp处有一个单一插入。在野生型寄生虫中破坏这个位点会重现阶段转化减少的表型。ZFP1通过CCHC基序定位于寄生虫核仁，显著改变的表达水平对寄生虫有毒性。这代表了首次鉴定出一个对于速殖子有效转化为缓殖子所必需的基因。
