Plasmodium Recombination of Machinery

全世界每年有3 - 5亿人感染疟疾，1-2百万人死亡。然而，有效的疫苗仍然难以捉摸，部分原因是抗原多样性和寄生虫的免疫逃避策略。免疫机制与抗原变异密切相关，抗原变异是一种对于针对疟原虫等原生动物寄生虫的疫苗开发具有极其重要意义的现象。拟议研究的长期目标是研究与抗原变异等现象相关的遗传重排机制。提出的工作的一个基本原则是，了解疟原虫中的重组机制将为开发治疗方法提供更好的机会。在其他真核生物中，同源重组（HR）在染色体重排中起着重要作用，而细菌RecA重组酶的真核对应物Rad 51和Dmc 1蛋白是有丝分裂和减数分裂期间参与HR的中心分子。在真核生物中，RPA和Rad 54等其他蛋白质在功能上与Rad 51合作，介导HR和受损染色体的修复。提出的研究假设是Rad 51和其他相互作用的蛋白质作为HR的介质，在寄生虫的生长和发育过程中发挥关键作用，并促进恶性疟原虫中的基因重排。Rad 51和Dmc 1同源物在恶性疟原虫的分子鉴定和重组PfRad 51的酶性质，如DNA链交换和ATP酶活性的最新表征，强烈表明PfRad 51在这些生物体中的保守功能作用。我们将使用生物化学和遗传学方法来验证我们的假设。在修订后的具体目标1和2的研究将导致在体外表征的蛋白质参与HR，从而探讨在寄生虫重组和基因重排的生化基础。PfRad 51基因敲除研究将直接评估PfRad 51在寄生虫红细胞生长中的重要性，并分析var基因转录物的库。此外，关于Dmc 1破坏的研究（修订的具体目标3）将评估减数分裂特异性重组酶在疟疾传播过程中的作用。本研究结果对阐明恶性疟原虫重组机制及重组和基因重排的分子遗传基础具有重要意义。

Malaria parasites, worldwide are responsible for 300-500 million new infections and 1-2 million deaths each year. An effective vaccine, however, remains elusive, partly due to antigenic diversity and the immune evasion strategies of the parasite. Recombination mechanisms are intimately linked with antigenic variation, a phenomenon of utmost significance for vaccine development against protozoan parasites like the malaria parasite. The long term objective of the proposed studies is to investigate mechanism(s) of genetic rearrangements associated with phenomenon like antigenic variation. An underlying tenet of the work proposed is that understanding the recombination mechanisms in Plasmodium will provide improved opportunities for the development of therapies. In other eukaryotes, homologous recombination (HR) plays a major role in chromosomal rearrangements, and Rad51 and Dmc1 proteins, the eukaryotic counterparts of bacterial RecA recombinase, are central molecules involved in HR during mitosis and meiosis. In eukaryotes, additional proteins like RPA and Rad54 functionally cooperate with Rad51 and mediate HR and the repair of damaged chromosomes. The hypothesis underlying proposed studies is that the Rad51 and other interacting proteins, as mediators of HR, play critical role(s) during growth and development of the parasite and facilitate gene rearrangements in P. falciparum. Molecular identification of Rad51 and Dmc1 homologues in P. falciparum and recent characterization of enzymatic properties of recombinant PfRad51, such as DNA strand exchange and ATPase activities, have strongly indicated a conserved functional role for PfRad51 in these organisms. We will test our hypothesis using biochemical as well as genetic approaches. Studies in the revised specific aims 1 and 2 will lead to in vitro characterization of the proteins involved in HR and thus probe into the biochemical basis of recombination and gene rearrangements in the parasites. PfRad51 gene knockout studies in the revised specific aim 3 will directly evaluate importance of PfRad51 in the erythrocytic growth of the parasite and analysis of repertoire of var gene transcripts. Moreover, studies on Dmc1 disruption (revised specific aim 3) will evaluate the role of meiosis specific recombinase during malaria transmission. The results of this study should be important in unraveling the recombination machinery and molecular and genetic basis for recombination and genetic rearrangements in P. falciparum.