Malaria Parasite Development, Drug Resistance, and Genomics

在2011-2012年间，我们集中在三个主要研究领域：1)筛选和表征可以阻断疟疾传播的药物；2)研究寄生虫分子和寄生虫对抗疟疾药物，特别是青蒿素及其衍生物的反应机制；3)利用约氏疟原虫/小鼠模型研究疟疾发病的分子机制。我们还完成了几个较小的项目，如鉴定一种新的疟疾候选疫苗，分析恶性疟原虫不同发育阶段的反义转录。 为了确定可以阻止蚊子寄生虫发展的药物，我们破坏了一个编码假定的ABC转运蛋白(PfABCG2)的基因，该转运蛋白在有性阶段高度表达。然后，我们对带有基因敲除的寄生虫和野生型3D7寄生虫进行了筛选，以对抗包含2816种经批准用于人或动物的药物的小分子库，并确定了一种高度有效的化合物，可以阻止蚊子中恶性疟原虫和啮齿动物寄生虫的卵囊发育。对结构相关化合物的测试还发现，还有一些化合物具有类似的阻断寄生虫传播的活性。此外，该化合物似乎有一定的活性，以防止食蟹猴疟原虫在恒河猴体内复发。这项工作已经申请了专利，并已提交了手稿。 在我们之前的全基因组关联研究确定了几个与青蒿素和其他抗疟疾药物相关的候选基因之后，我们现在已经从基因上扰乱了一个候选基因，并表明没有该基因的寄生虫对青蒿素的抗药性更强。目前正在进行进一步的功能鉴定和蛋白质定位，以阐明该基因的功能及其在青蒿素新陈代谢或抗药性中的作用。这项工作可能提供有关青蒿素在寄生虫内的运输或代谢机制的重要信息。 我们在利用啮齿动物疟疾寄生虫约氏疟原虫研究寄生虫与宿主的相互作用方面取得了良好的进展。我们筛选和比较了宿主对不同寄生虫株感染的反应，并确定了宿主天然免疫反应、寄生虫生长和疾病严重程度的差异。我们测量了随时间推移的寄生虫血症、小鼠死亡率以及未感染和感染小鼠的细胞因子/趋化因子水平。然后，我们进行了多个基因杂交，以确定导致宿主反应和疾病表型差异的寄生虫分子。我们已经确定了几个遗传位点和候选基因，并正在验证候选基因的功能或对表型的贡献。我们还在研究宿主对疟疾感染的先天免疫反应的机制。了解宿主-寄生虫相互作用的分子机制将有助于开发有效的措施来控制寄生虫的发展及其引起的疾病。 为了更好地了解基因表达与寄生虫发育的关系，我们从3D7恶性疟原虫的环状、早期和晚期滋养体、裂殖体、配子体II、配子体V和运动细胞中测序了7个双向文库，以及从晚期滋养体、裂殖体、配子体II和配子体V中获得的4个链特异性文库。经过序列分析，我们鉴定了大量的阶段特异性反义转录本和新的内含子-外显子剪接连接。我们的结果表明，在配子体中单向表达的基因比在裂殖体中表达的基因更多，反义RNA可能在基因表达调控和寄生虫发育中发挥重要作用。这些观察将有助于我们更好地了解疟疾寄生虫中基因表达和调控的机制。

In the 2011-2012, we focused on three major research areas: 1) screening and characterizing drugs that can block malaria transmission; 2) studying parasite molecules and mechanisms of parasite response to antimalarial drugs, particularly artemisinin and derivatives; and 3) studying the molecular mechanism of malaria pathogenesis using Plasmodium yoelii/mouse model. We also finished several smaller projects such as characterizing a new malaria vaccine candidate and analyzing antisense transcripts in different Plasmodium falciparum developmental stages. To identify drugs that can block parasite development in mosquitoes, we disrupted a gene encoding a putative ABC transporter (PfABCG2) that is highly expressed in sexual stages. We then screened parasites with the gene knockout and the wild-type 3D7 parasite against a library of small molecules containing 2,816 drugs approved for human or animal use and identified a compound that was highly potent in blocking oocyst development of P. falciparum and the rodent parasite P. yoelii in mosquitoes. Tests of structurally related compounds also identified additional compounds having similar activities in blocking parasite transmission. Additionally, the compound appeared to have some activity against relapse of Plasmodium cynomolgi in rhesus monkeys. This work has led to a patent application, and a manuscript has been submitted. Following our previous genome-wide association study that identified several candidate genes associated with response to artemisinin and other antimalarial drugs, we now have genetically disrupted a candidate gene and showed that parasites without the gene became more resistant to artemisinin. Further functional characterization and protein localization are being conducted to elucidate the function of the gene and its role in metabolism or resistance of artemisinin. This work can potentially provide important information about the mechanism of artemisinin transport or metabolism within the parasite. We have made good progress in studying parasite-host interactions using rodent malaria parasite P. yoelii. We have screened and compared host responses to infection of different parasite strains and identified differences in host innate immune response, parasite growth, and disease severity. We measured parasitemia over time, mouse mortality, and cytokine/chemokine levels of non-infected and infected mice. We then performed multiple genetic crosses to identify parasite molecules that cause the differences in host response and disease phenotypes. We have identified several genetic loci and candidate genes and are in the process of verifying the functions or the contribution of the candidate genes to the phenotypes. We are also studying the mechanism of host innate immune response to malaria infection. Understanding the molecular mechanism of host-parasite interaction will allow development of effective measures to control parasite development and the disease it causes. To better understand the relationship of gene expression and parasite development, we sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 P. falciparum parasite. After analysis of the sequences, we identified large numbers of stage-specific antisense transcripts and novel intron-exon splicing junctions. Our results suggested that more genes are expressed in one direction in gametocytes than in schizonts and that antisense RNA may play an important role in gene expression regulation and parasite development. These observations will help us better understand the mechanism of gene expression and regulation in malaria parasites.