Malaria Parasite Development, Drug Resistance, Pathogenesis, and Genomics

在2014-2015年，我们继续专注于三个主要研究领域：1）筛选和表征联合治疗和传播阻断药物; 2）使用约氏疟原虫/小鼠模型研究疟疾发病机制的分子机制; 3）确定卵囊发育缺陷的分子基础。 1)我们与NCATS的科学家合作，合成并测试了一种有前途的化合物的一些衍生物，这种化合物与许多现有的抗疟药物相互作用。其中两种衍生物在杀死有性和无性寄生虫方面表现出更高的效力（20倍）。我们正在制造和测试其他衍生物，并致力于将这些化合物用于动物和临床试验。我们还计划在实地测试另一种化合物（酮替芬）的传播阻断作用。我们的抗疟疗法联合筛选的手稿已提交给《科学报告》。 2)利用啮齿类疟疾寄生虫约氏疟原虫，我们在研究寄生虫-宿主相互作用方面取得了以下几个方面的进展： I.去年，我们对宿主对约氏疟原虫遗传杂交后代感染的反应进行了全基因组连锁分析，并确定了数百个与许多宿主基因反应相关的寄生虫遗传位点。我们还聚类宿主基因的基础上，其全基因组模式的LOD分数（GPLS）在寄生虫感染，这使得基因功能的准确预测。然后，我们测试了15个基因（并验证了14个）预测发挥作用的I型干扰素反应，使用各种功能测定。这项工作开发了一种新的方法来预测响应寄生虫感染的宿主基因功能，以及预测在相关途径中发挥作用的基因簇的数据库（Cell Rep 2015; 12：661-672）。 二.我们进行了几次额外的P.y.杂交。negeriensis N67和P.y. yoelii YM获得了43个独立的重组子。在表征后代的基因型和表型后，我们确定了与各种表型相关的一些候选基因，包括编码推定的E3泛素连接酶的基因。虽然我们还不能破坏寄生虫中的基因，但我们能够在5个非翻译区（5 UTR）插入质粒，并表明插入改变了基因表达水平和寄生虫生长模式。我们正在进行额外的实验来修改基因，以更好地表征基因功能。 三.去年，我们还将伯氏疟原虫ANKA和伯氏疟原虫NK 165杂交，获得了7个后代，以鉴定与脑型疟疾相关的基因。我们对后代的基因组进行了测序，并确定了20个多态性。已经获得了更多的后代，正在进行分析。 四.我们正在研究约氏疟原虫感染后细胞凋亡和坏死性凋亡的分子机制。 我们现在专注于IFN-γ介导的促炎反应，并正在解剖寄生虫感染后导致炎症的分子相互作用和细胞类型。 3)我们完成了一个关于与卵囊发育缺陷相关的基因的定位和功能表征的项目。通过连锁作图、基因敲除（KO）和基因互补等方法，我们发现寄生虫性特异性小亚基核糖体RNA（D-ssu）在卵囊发育中起着重要作用。此外，我们证明约氏疟原虫D-ssu可以以菌株和阶段特异性的方式调节其卵囊发育，这表明核糖体RNA除了蛋白质翻译之外还可以调节基因表达（mBio 2015，6：e00117-15）。 此外，我们与中国厦门大学的科学家和学生合作，解决了一个关于鸟类血液寄生虫白细胞虫的百年难题。由于禽类红细胞具有细胞核，并且寄生虫感染会显著改变宿主细胞的形态，因此很难识别受感染的细胞。长期以来，被白细胞体配子体侵入的宿主细胞被认为是红细胞和/或白色血细胞。我们最近使用细胞类型特异性抗体的研究表明，Leucocytozoon配子体感染血小板（相当于人类血小板的细胞），而不是其他细胞类型（PLoS ONE 2015; 10：e0133478）。

In the year 2014-2015, we continued to focus on three major research areas: 1) screening and characterizing drugs for combination therapy and transmission blocking; 2) studying the molecular mechanisms of malaria pathogenesis using Plasmodium yoelii/mouse model; 3) determining the molecular basis of an oocyst development defect. 1) In collaboration with scientists in NCATS, we synthesized and tested some derivatives of a promising compound that interacts with many current antimalarial drugs. Two of the derivatives showed higher potency (20 fold) in killing both sexual and asexual parasites. We are making and testing additional derivatives and are working towards bringing the compounds for animal and clinical trials. We have also been planning to test another compound (ketotifen) for transmission blocking in the field. Our manuscript from our combinational screening for antimalarial therapies has been submitted to Scientific Reports. 2) Using the rodent malaria parasite Plasmodium yoelii, we have made progresses in studying parasite-host interaction in several directions: I. Last year, we performed a genome-wide linkage analysis on host responses to infection of progeny from a P. yoelii genetic cross and identified hundreds of parasite genetic loci linked to responses of many host genes. We also clustered host genes based on their genome-wide patterns of LOD scores (GPLS) in response to parasite infections, which allowed accurate prediction of gene function. We then tested 15 genes (and verified 14) predicted to play a role in type I interferon response using various functional assays. This work developed a novel approach to predict host gene functions in response to parasite infection and a database with clusters of genes predicted to function in related pathways (Cell Rep 2015; 12: 661-672). II. We performed several additional crosses of P. y. negeriensis N67 and P. y. yoelii YM and have obtained 43 independent recombinant progeny. After characterizing the genotypes and phenotypes of the progeny, we identified some candidate genes linked to various phenotypes, including a gene encoding a putative E3 ubiquitin ligase. Although we have not been able to disrupt the gene in the parasite, we were able to insert a plasmid at 5 untranslated region (5UTR) and showed that the insertion changed the gene expression level and parasite growth pattern. We are performing additional experiments to modify the gene to better characterize the gene function. III. We also crossed P. berghei ANKA and P. berghei NK165 and obtained seven progeny to identify genes linked to cerebral malaria last year. We sequenced the genomes of the progeny and identified 20 polymorphisms. Additional progeny have been obtained and are being analyzed. IV. We are studying the molecular mechanism of apoptosis and necroptosis after P. yoelii infection. We are now focusing on pro-inflammatory responses mediated by IFN-gamma and are dissecting the molecular interactions and cell types contributing to inflammation after parasite infection. 3) We finished a project on mapping and functional characterization of a gene linked to an oocyst development defect. Through linkage mapping, gene knockout (KO) and gene complementation, we showed that the parasites sexual specific small subunit ribosomal RNA (D-ssu) plays an essential role in oocyst development. Additionally, we demonstrated that the P. yoelii D-ssu could regulate its oocyst development in a strain- and stage-specific manner, showing that ribosomal RNAs can regulate gene expression in addition to protein translation (mBio 2015, 6: e00117-15). Additionally, in collaboration with scientists and students at Xiamen University, China, we solved a century-old puzzle on a bird blood parasite Leucocytozoon sabrazesi. Because avian red blood cells have nuclei, and the parasite infection dramatically change the morphology of the host cells, it has been difficult to identify the infected cells. For a long time, the host cells invaded by Leucocytozoon gametocytes were believed to be red and/or white blood cells. Our recent study using cell type specific antibodies demonstrated that the Leucocytozoon gametocytes infect thrombocytes, a cell equivalent to human platelets, not other cell types (PLoS ONE 2015; 10: e0133478).