A Molecular Switch Controlling Antigenic Variation in Malaria Parasites

摘要 疟疾是整个发展中世界发病率和死亡率的主要原因。的主要致病因素 严重的疟疾是原生动物寄生虫恶性疟原虫。其毒性的关键在于它有效地利用了 抗原变异破坏宿主免疫系统。恶性疟原虫表达一种跨膜蛋白， 被运输出寄生虫并进入它所居住的红细胞膜。这种蛋白质PfEMP 1 是寄生虫所必需的，因为它介导受感染的红细胞与宿主脉管系统的结合，从而 阻止脾脏清除。与此同时，PfEMP 1具有免疫原性，宿主会在体内产生一种免疫原性。 特异性针对该蛋白质的适应性免疫反应。寄生虫通过免疫系统逃避这种反应。 PfEMP 1的免疫学上不同的同种型的表达，每种同种型由不同成员编码。 多拷贝基因家族在任何给定的寄生虫的基因组中编码有大约40-90个var基因 它们是孤立的，并且以相互排斥的方式表达。虽然控制这些基因的表观遗传机制 已经鉴定了var基因的激活和沉默，但是关于var基因的调控知之甚少。 基因间的转录转换由于寄生虫体内的var基因数量有限 基因组中，基因之间的纯粹随机转换可能会导致抗原的过早消耗， 保留曲目。数学算法已经提供了提出优化的交换网络的模型， 使寄生虫能够在感染过程中有效地利用其全部var基因。这些 模型已经提出了网络内协调转录转换的“汇节点”的作用。 有越来越多的证据表明，一个高度保守的，独特的var基因，var 2csa，是假设 “sink-node”。var 2csa编码负责妊娠相关疟疾的PfEMP 1。令人惊讶的是，高 在非怀孕个体中发现了var 2csa转录物的水平，这表明该基因具有额外的作用。 当在培养的恶性疟原虫中缺失var 2csa基因座时，所产生的寄生虫不能经历var 2csa基因的突变。 基因转录开关，表明var 2csa作为“汇节点”的潜力。该提案看起来 以揭示VAR 2CSA在协调交换中实现该第二角色的机制。var 2csa是唯一的 因为它是唯一具有高度保守上游开放阅读框（uORF）的变异基因。的存在 已知uORF影响转录物稳定性，减少下游翻译，并可诱导基因沉默。 具体目的1被设计为研究uORF在调节var 2csa mRNA稳定性中的作用， 通过无义介导的衰变（NMD）途径的转录激活。具体目标2将检查 起始因子构象在调节var 2csa转录物翻译中的作用。磷酸化 eIF 2 α的状态可以响应改变的营养水平，导致翻译起始的变化。在一起，这 该提案将确定uORF在调节var 2csa的拟议双重功能中的作用。

Abstract Malaria is a major cause of morbidity and mortality across the developing world. The major causative agent of severe malaria is the protozoan parasite Plasmodium falciparum. Key to its virulence is its effective use of antigenic variation in subverting the host immune system. P. falciparum expresses a transmembrane protein that is trafficked out of the parasite and into the membrane of the erythrocyte in which it resides. This protein, PfEMP1, is essential to the parasite as it mediates binding of the infected erythrocyte to the host vasculature, thereby preventing clearance by the spleen. At the same time, PfEMP1 is immunogenic and the host will mount an adaptive immune response specifically targeting this protein. The parasite evades this response through the expression of immunologically distinct isoforms of PfEMP1, each encoded by different members of the var multicopy gene family. There are approximately 40-90 var genes encoded in the genome of any given parasite isolate and they are expressed in a mutually exclusive fashion. While the epigenetic mechanisms controlling the activation and silencing of var genes have been identified, little is known regarding the regulation of transcriptional switching between genes. Due to the limited number of var genes available within the parasite genome, purely stochastic switching between genes would likely result in premature expenditure of the antigenic repertoire. Mathematical algorithms have provided models proposing an optimized switching network that enables the parasite to efficiently utilize its full complement of var genes over the course of an infection. These models have proposed a role for a “sink-node” within the network that coordinates transcriptional switching. There is an increasing body of evidence that a highly conserved, unique var gene, var2csa, is the hypothesized “sink-node”. var2csa encodes the PfEMP1 responsible for pregnancy associated malaria. Surprisingly, high levels of var2csa transcripts are found in non-pregnant individuals, suggesting an additional role for this gene. When the var2csa locus is deleted in cultured P. falciparum, the resulting parasites are unable to undergo a var gene transcriptional switch, indicating the potential for var2csa to serve as the “sink-node.” This proposal looks to uncover the mechanisms by which var2csa fulfills this second role in coordinating switching. var2csa is unique in that it is the only var gene with a highly conserved upstream open reading frame (uORF). The presence of a uORF is known to influence transcript stability, reduce downstream translation and can induce gene silencing. Specific Aim 1 is designed to investigate the role of the uORF in modulating var2csa mRNA stability and transcriptional activation through the nonsense mediated decay (NMD) pathway. Specific Aim 2 will examine the role of initiation factor conformation in regulating translation of the var2csa transcript. The phosphorylation state of eIF2α can respond to altered nutrient levels, resulting in changes in translational initiation. Together, this proposal will determine the role of the uORF in regulating var2csa’s proposed dual function.