Malaria Parasite Development, Drug Resistance, Pathogenesis, and Genomics

2013-2014年，我们继续关注三个主要研究领域：1)联合治疗和传播阻断药物的筛选和表征；2)利用约氏疟原虫/小鼠模型研究疟疾发病的分子机制；3)卵囊发育缺陷的分子基础。 与NCATS的科学家合作，我们已经完成了对药物组合的大规模筛选。有希望的药物组合已经确定，数据已经提交发表。我们还在评估钙离子和钠离子通道阻滞剂对寄生虫对青蒿素反应的影响。 利用啮齿动物疟疾寄生虫P.yoelii，我们在研究寄生虫与宿主的相互作用方面取得了良好的进展，方向如下： 1)我们继续定位和评估与宿主细胞因子/趋化因子反应相关的基因座上的候选基因，从而发表在《基因与免疫》(Pattaradilokrat Gene Immun)上。2014；15：145-152)。我们目前正在测试候选基因中氨基酸替换的寄生虫及其与细胞因子水平和疾病表型的关系。 2)我们发现I型干扰素在控制鼠类疟疾寄生虫约氏疟原虫N67感染的原虫血症中起着重要作用；这一结果最近发表(Wu等人，PNAS)。2014年；111：E511-20)。现在我们关注的是另一种疾病表型-感染另一种寄生虫后的组织坏死。 3)我们开发并测试了一个用于约氏疟原虫基因组基因分型的微阵列平台，导致最近发表了一篇论文(Nair等人，Mol Biochem Parasitol 2014；194：9-15)。这个基因分型平台正被用于对遗传杂交后代的DNA样本进行分型。 4)在上一篇报道中，我们将卵囊发育缺陷与约氏疟原虫6号染色体上的一个基因座相关联。我们已经敲除了候选基因，并证明了该基因确实在卵囊发育中发挥了关键作用。 5)去年，我们对寄主对一个遗传杂交后代的感染反应进行了全基因组连锁分析，发现了数百个与多个寄主基因的反应连锁的寄生虫遗传位点。我们已经开始使用基因敲除和其他方法测试一些选定的基因。 6)对黑杨N67C和约氏黄杨进行了多次杂交，获得了52个后代。在鉴定了后代的基因类型和表型后，我们确定了一些候选基因。我们正在使用寄生虫的基因转化来测试一个主要候选者。 7)我们还与伯氏疟原虫Anna和P.berghei NK165杂交，获得了几个后代，用于鉴定与脑型疟疾相关的基因。我们正在对这些后代的基因组进行测序，以确定其他遗传标记的多态，其中一些可能是致病突变。 8)最后，我们还研究了约氏疟原虫感染后细胞凋亡和坏死性下垂的分子机制。一些候选宿主基因正在进行评估。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。

In the year 2013-2014, we continued to focus on three major research area: 1) screening and characterizing drugs for combination therapy and transmission blocking; 2) studying molecular mechanisms of malaria pathogenesis using Plasmodium yoelii/mouse model; 3) molecular basis of an oocyst development defect. In collaboration with scientists in NCATS, we have completed a large-scale screening of drug combinations. Promising drug combinations were identified, and the data have been submitted for publication. We are also evaluating the effect of Ca++ and Na+ channel blockers on parasite response to artemisinin. Using rodent malaria parasite P. yoelii, we have made good progresses in studying parasite-host interaction in several directions: 1) We continued to map and evaluate candidate genes in the loci linked to host cytokine/chemokine response, leading to a publication in Gene and Immunity (Pattaradilokrat Genes Immun. 2014; 15: 145-152). We are currently testing parasites with amino acid substitutions in candidate genes and their association to cytokine levels and disease phenotypes. 2) We showed that type I interferon (IFN-I) played an important role in controlling parasitemia of rodent malaria parasite Plasmodium yoelii nigeriensis N67 infection; the results were published recently (Wu et al., PNAS. 2014; 111:E511-20). Now we are focusing on another disease phenotype---tissue necrosis after infection with another parasite. 3) We developed and tested a microarray platform for genotyping P. yoelii genome, leading to a publication recently (Nair et al, Mol Biochem Parasitol 2014; 194: 9-15). This genotyping platform is being used to type DNA samples from progeny of genetic crosses. 4) We linked an oocyst development defect to a locus on chromosome 6 of P. yoelii in our last report. We have knocked out the candidate gene and demonstrated the gene indeed plays a critical role in oocyst development. 5) Last year, we performed a genome-wide linkage analysis on host response to infection of progeny from a genetic cross and identified hundreds of parasite genetic loci linked to responses of many host genes. We have started testing some selected genes using genetic knockout and other methods. 6) We performed several additional crosses of P. y. negeriensis N67C and P. y. yoelii YM and have obtained 52 progeny. After characterizing the genotypes and phenotypes of the progeny, we identified some candidate genes. We are testing a primary candidate using genetic transformation of the parasites. 7) We have also crossed P. berghei ANNA and P. berghei NK165 and obtained several progeny to identify genes linked to cerebral malaria. We are sequencing the genomes of these progeny to identify polymorphisms for additional genetic markers, some of which could be the causative mutations. 8) Finally, we are also studying the molecular mechanism of apoptosis and necroptosis after P. yoelii infection. Some candidate host genes are being evaluated. Understanding the molecular mechanism of host-parasite interaction will allow development of effective measures for controlling parasite development and the disease it causes.