Malaria Parasite Sexual Development, Drug Resistance, and Evolution

在过去的一年里，我们已经完成了两个芯片平台的开发和评估，用于大规模的恶性疟原虫基因分型。利用我们在前一年收集的单核苷酸多态性（SNP），我们开发了一种使用分子倒置探针（MIP）技术的阵列，并使用该阵列对100多种寄生虫进行分型。我们还评估了用于SNP检测和基因拷贝数变异的平铺阵列。我们证明了平铺阵列也是一种很好的基因分型工具。这两种阵列都已被用于对寄生虫基因组进行分型。我们正在测试寄生虫对不同抗疟药物的反应，以确定可能导致抗药性的基因。 这项研究预计将确定一些候选基因，可以进一步测试使用其他方法，如基因的遗传修饰。例如，我们在以前的一项研究中发现，几种转运蛋白与对CQ和QN的较高水平的抗性相关。现在，我们已经破坏了其中一个候选基因，并表明它确实可以影响寄生虫对CQ和QN的反应。我们已经完成了基因敲除寄生虫对药物和其他化学物质的反应测试。从这项研究中开发的资源将有助于研究其他寄生虫表型。 耐药基因的突变往往是有害的，寄生虫为了生存，可以对突变作出反应，在其基因组中发生补偿性变化。我们使用微阵列来寻找关键CQ抗性基因（pfcrt）突变后表达发生变化的基因。在pfcrt突变的寄生虫中，发现了一系列表达水平和拷贝数发生变化的基因。 与关联作图相关的另一个重要问题是准确的表型。我们调查了耐药和敏感寄生虫的混合感染（常见于田间样本）如何影响药物检测结果。我们发现，在敏感人群中10%（或更多）的耐药寄生虫的混合物可以极大地影响药物测试结果，为该领域的药物测试提供重要信息。 去年，我们发现墨西哥南部的间日疟原虫寄生虫由三个亚群组成，它们具有强烈的蚊子媒介偏好。鉴定介导蚊子特异性的分子可能为传播控制提供关键信息。我们已经对数百个寄生虫样本进行了基因分型，并正在研究基因型与复发/再感染的关系，以更好地了解寄生虫传播动态。 我们也对基因调控机制感兴趣，如染色质修饰或microRNA介导的机制。

In the past year, we have finished developing and evaluating two microarray platforms for large-scale genotyping of P. falciparum parasite. Using single nucleotide polymorphism (SNP) we collected in previous year, we have developed an array using Molecular Inversion Probe (MIP) technology and used the array to type more than 100 parasites. We also evaluated a tiling array for SNP detection and gene copy number variation. We showed that the tiling array was an excellent tool for genotyping too. Both arrays have been used to type parasite genomes. We are testing parasite responses to different antimalrial drugs to identify genes that may contribute to drug resistance. This study is expected to identify some candidate genes that can be further tested using other methods such as genetic modification of the genes. For example, we showed that several transporters were associated with higher levels of resistance to CQ and QN in one of our previous studies. Now we have disrupted one of the candidate genes and showed that it indeed could affect parasite response to both CQ and QN. We have finished testing the knockout parasites for their responses to drugs and other chemicals. The resources developed from this study will be useful for studying other parasite phenotypes. Mutations in drug resistance gene are often deleterious; and parasite can respond to the mutations with compensatory changes in their genome in order to survive. We used mcicroarray to search for genes that changed in expression after mutations in the key CQ resistant gene (pfcrt). A list of genes that changed in expression levels and in copy number were identified in parasite that had mutations in pfcrt. Another important issue associated with association mapping is accurate phenotyping. We investigated how mixed infections (often seen in field samples) of drug resistant and sensitive parasites affect drug test results. We found that a mixture of 10% (or more) resistant parasite in a sensitive population could greatly affect drug test results, providing important information for drug tests in the field. Last year, we found that P. vivax parasites in Southern Mexico consist of three subpopulations that had strong mosquito vector preferences. Identification of molecules that mediate mosquito specificity may provide critical information for transmission control. We have genotyped hundreds of more parasite samples and are investigating the relationship of genotypes and relapse/re-infections to better understand parasite transmission dynamics. We are also interested in gene regulation mechanisms such as chromatin modification or microRNA mediated mechanisms.