Malaria Parasite Development, Drug Resistance, and Genomics

疟疾寄生虫的发育、耐药性和基因组学

• 批准号: 8555855
• 负责人: Xinzhuan Su
• 金额: $ 90.01万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555855
• 关键词: ATP-Binding Cassette Transporters; Animals; Antimalarials; Antisense RNA; Area; Artemisinins; Candidate Disease Gene; Culicidae; Development; Disease; Drug resistance; Exons; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Crosses; Genomics; Goals; Growth; Human; Immune response; Infection; Introns; Legal patent; Libraries; Macaca mulatta; Malaria; Malaria Vaccines; Manuscripts; Measures; Metabolism; Methods; Molecular; Mus; Oocysts; Parasite Control; Parasitemia; Parasites; Pathogenesis; Pharmaceutical Preparations; Phenotype; Plasmodium cynomolgi; Plasmodium falciparum; Plasmodium yoelii; Play; Process; Proteins; RNA Splicing; Relapse; Research; Resistance; Resources; Rodent; Role; Screening procedure; Sequence Analysis; Severity of illness; Staging; Study models; Testing; Time; Transcript; Virulence; Work; artemisinine; chemokine; cytokine; disease phenotype; functional genomics; gene function; genome database; genome wide association study; knockout gene; mortality; mouse model; novel; parasite genome; response; small molecule libraries; transmission process; vaccine candidate

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.

2011-2012年，我们重点开展了三个主要研究领域：1）筛选和鉴定阻断疟疾传播的药物; 2）研究寄生虫分子和寄生虫对抗疟药物的反应机制，特别是青蒿素及其衍生物; 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，如表征一种新的疟疾候选疫苗和分析恶性疟原虫不同发育阶段的反义转录物。 为了鉴定可以阻断蚊子体内寄生虫发育的药物，我们破坏了一个编码推定的ABC转运蛋白（PfABCG 2）的基因，该基因在性阶段高度表达。然后，我们对含有2，816种批准用于人类或动物的药物的小分子文库筛选了基因敲除的寄生虫和野生型3D 7寄生虫，并鉴定了一种化合物，该化合物在阻断恶性疟原虫和啮齿类寄生虫约氏疟原虫的卵囊发育方面非常有效。结构相关化合物的测试还鉴定了在阻断寄生虫传播方面具有类似活性的其他化合物。此外，该化合物似乎对恒河猴中食蟹猴疟原虫的复发具有一定活性。这项工作已导致专利申请，并已提交手稿。 在我们之前的全基因组关联研究中，确定了与青蒿素和其他抗疟药物反应相关的几个候选基因，我们现在已经在遗传上破坏了一个候选基因，并表明没有该基因的寄生虫对青蒿素更具抗药性。进一步的功能鉴定和蛋白定位正在进行，以阐明该基因的功能及其在青蒿素代谢或抗性中的作用。这项工作可能提供有关青蒿素在寄生虫体内转运或代谢机制的重要信息。 我们在利用啮齿类疟原虫约氏疟原虫研究寄生虫-宿主相互作用方面取得了良好进展。我们已经筛选和比较了宿主对不同寄生虫株感染的反应，并确定了宿主先天免疫反应、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，我们进行了多个遗传杂交，以确定寄生虫分子，导致宿主反应和疾病表型的差异。我们已经确定了几个遗传基因座和候选基因，并正在验证候选基因的功能或对表型的贡献。我们也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制定有效的措施来控制寄生虫的发展及其引起的疾病。 为了更好地理解基因表达和寄生虫发育的关系，我们对来自3D 7恶性疟原虫的环状、早期和晚期滋养体、鞭毛体、配子体II、配子体V和动合子的7个双向文库以及来自晚期滋养体、鞭毛体、配子体II和配子体V的4个链特异性文库进行了测序。经过序列分析，我们确定了大量的阶段特异性反义转录和新的内含子-外显子剪接连接。我们的研究结果表明，更多的基因在一个方向上表达的配子体比在寄生虫和反义RNA可能发挥重要作用的基因表达调控和寄生虫的发展。这些观察结果将有助于我们更好地了解疟原虫基因表达和调控的机制。