Malaria Parasite Sexual Development, Drug Resistance, and Evolution

在过去的一年里，我们已经完成了两个用于恶性疟原虫大规模基因分型的微阵列平台的开发和评估。利用去年收集的单核苷酸多态性（SNP），我们利用分子倒置探针（MIP）技术开发了一个阵列，并使用该阵列对100多种寄生虫进行了分型。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个很好的基因分型工具。这两种阵列都被用于寄生虫基因组的分型。我们正在测试寄生虫对不同抗疟药物的反应，以确定可能导致耐药性的基因。

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.