A Pseudouridine Synthase Homologue Is Critical to Cellular Differentiation in Toxoplasma gondii
A Pseudouridine Synthase Homologue Is Critical to Cellular Differentiation in Toxoplasma gondii
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DOI：
10.1128/ec.00329-08
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发表时间：
2009-03-01
期刊：
影响因子：
--
通讯作者：
Boothroyd, John C.
中科院分区：
文献类型：
--
作者：
Anderson, Matthew Z.;Brewer, Jeremy;Boothroyd, John C.
弓形虫是一种单倍体原生动物寄生虫，在美国大约每七人中就有一人感染。弓形虫在世界范围内流行的关键是它能够通过逃避免疫系统来建立终身的慢性感染，而这一点的核心是两种无性形式--速殖子和缓殖子--之间的发育转换。通过插入突变，建立了速殖子-缓殖子分化缺陷突变体库(TbD(-))。该文库包含的突变体与野生型相比，在阶段转化时的效率在20%到74%之间。鉴定出两个突变株，TBD5和TBD8，其编码假性尿苷合成酶PUS1的基因发生了突变。TbD8基因的突变位于PUS1基因的5‘端，似乎产生了一个零等位基因，在分化中有50%的缺陷。这与利用工程pus1缺失突变体(Delta Pus1)获得的开关效率大致相同。在TbD5中的插入在PUS1编码区内，这似乎导致了更极端的表型，仅类似于10%的开关效率。TbD8与基因组PUS1等位基因互补，恢复了野生型分化效率。小鼠感染pus1突变株后，急性期死亡率增加，慢性感染时包囊负担增加，体内由于pus1基因突变而表现出异常的分化表型。我们的结果表明，RNA修饰在这一生物过程中扮演着令人惊讶和重要的角色。
Toxoplasma gondii is a haploid protozoan parasite infecting about one in seven people in the United States. Key to the worldwide prevalence of T. gondii is its ability to establish a lifelong, chronic infection by evading the immune system, and central to this is the developmental switch between the two asexual forms, tachyzoites and bradyzoites. A library of mutants defective in tachyzoite-to-bradyzoite differentiation (Tbd(-)) was created through insertional mutagenesis. This library contains mutants that, compared to the wild type, are between 20% and 74% as efficient at stage conversion. Two mutants, TBD5 and TBD8, with disruptions in a gene encoding a putative pseudouridine synthase, PUS1, were identified. The disruption in TBD8 is in the 5' end of the PUS1 gene and appears to produce a null allele with a 50% defect in differentiation. This is about the same switch efficiency as obtained with an engineered pus1 deletion mutant (Delta pus1). The insertion in TBD5 is within the PUS1 coding region, and this appears to result in a more extreme phenotype of only similar to 10% switch efficiency. Complementation of TBD8 with the genomic PUS1 allele restored wild-type differentiation efficiency. Infection of mice with pus1 mutant strains results in increased mortality during the acute phase and higher cyst burdens during the chronic infection, demonstrating an aberrant differentiation phenotype in vivo due to PUS1 disruption. Our results suggest a surprising and important role for RNA modification in this biological process.