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。致病的主要原因是 严重疟疾是原生动物寄生虫恶性疟原虫。其毒力的关键是它有效地利用了 在颠覆宿主免疫系统中的抗原变异。恶性疟原虫表达一种跨膜蛋白， 被带出寄生虫，进入寄生虫所在的红细胞膜。这种蛋白质，PfEMP1， 对寄生虫来说是必不可少的，因为它介导了受感染的红细胞与宿主血管系统的结合，从而 防止被脾清除。同时，PfEMP1具有免疫原性，宿主将装载一个 针对这种蛋白的适应性免疫反应。寄生虫通过 免疫上不同的PfEMP1亚型的表达，每个亚型由var的不同成员编码 多拷贝基因家族。在任何一种寄生虫的基因组中，大约有40-90个var基因编码。 孤立，它们以一种相互排斥的方式表达。虽然表观遗传机制控制着 Var基因的激活和沉默已经被确定，关于var基因的调控知之甚少。 基因之间的转录转换。由于寄生虫内可用的var基因数量有限 基因组，基因之间的纯粹随机切换可能会导致抗原的过早消耗 曲目。数学算法已经提供了提出优化交换网络的模型，该模型 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 模型建议网络中的“汇聚节点”扮演一个协调转录切换的角色。 越来越多的证据表明，一种高度保守的独特的var基因var2csa是一种假设 “汇聚节点”。Var2csa编码与妊娠相关疟疾有关的PfEMP1。令人惊讶的是，很高 在未怀孕的个体中发现了var2csa转录本的水平，这表明该基因还有额外的作用。 当培养的恶性疟原虫中的var2csa基因座缺失时，所产生的寄生虫不能进行变种。 基因转录开关，表明var2csa有可能作为“汇聚节点”。这份提案看起来 以揭示var2csa在协调切换中履行这第二个角色的机制。Var2csa是唯一的 因为它是唯一一个具有高度保守的上游开放阅读框架(UORF)的var基因。出现了一个 已知uORF可影响转录稳定性，减少下游翻译，并可诱导基因沉默。 具体目的1旨在研究uORF在调节var2csa mRNA稳定性和 通过无意义介导的衰变(NMD)途径进行转录激活。《特定目标2》将考察 起始因子构象在调控var2csa转录本翻译中的作用。磷酸化 EIF2α的状态可以对改变的营养水平做出反应，导致翻译启动的变化。总而言之，这 提案将决定uORF在调节var2csa拟议的双重功能方面的作用。

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.