Wang Zenglei, Liu Min, Liang Xiaoying, Siriwat Salil, Li Xiaolian, Chen Xiaoguang, Parker Daniel M, Miao Jun, Cui Liwang
Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America.
Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, United States of America; Department of Parasitology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong, P.R. China.
PLoS One. 2014 Apr 15;9(4):e93825. doi: 10.1371/journal.pone.0093825. eCollection 2014.
Malaria elimination/eradication campaigns emphasize interruption of parasite transmission as a priority strategy. Screening for new drugs and vaccines against gametocytes is therefore urgently needed. However, current methods for sexual stage drug assays, usually performed by counting or via fluorescent markers are either laborious or restricted to a certain stage. Here we describe the use of a transgenic parasite line for assaying drug sensitivity in all gametocyte stages.
A transgenic parasite line expressing green fluorescence protein (GFP) under the control of the gametocyte-specific gene α-tubulin II promoter was generated. This parasite line expresses GFP in all gametocyte stages. Using this transgenic line, we developed a flow cytometry-based assay to determine drug sensitivity of all gametocyte stages, and tested the gametocytocidal activities of four antimalarial drugs.
This assay proved to be suitable for determining drug sensitivity of all sexual stages and can be automated. A Z' factor of 0.79 ± 0.02 indicated that this assay could be further optimized for high-throughput screening. The daily sensitivity of gametocytes to three antimalarial drugs (chloroquine, dihydroartemisinin and pyronaridine) showed a drastic decrease from stage III on, whereas it remained relatively steady for primaquine.
A drug assay was developed to use a single transgenic parasite line for determining drug susceptibility of all gametocyte stages. This assay may be further automated into a high-throughput platform for screening compound libraries against P. falciparum gametocytes.
疟疾消除/根除运动强调将阻断寄生虫传播作为优先战略。因此，迫切需要筛选针对配子体的新药和疫苗。然而，目前用于性阶段药物检测的方法，通常通过计数或荧光标记进行，要么费力，要么局限于特定阶段。在此，我们描述了一种利用转基因寄生虫系来检测所有配子体阶段药物敏感性的方法。
构建了一种在配子体特异性基因α-微管蛋白II启动子控制下表达绿色荧光蛋白（GFP）的转基因寄生虫系。该寄生虫系在所有配子体阶段均表达GFP。利用此转基因系，我们开发了一种基于流式细胞术的检测方法来测定所有配子体阶段的药物敏感性，并测试了四种抗疟药物的杀配子体活性。
该检测方法被证明适用于测定所有性阶段的药物敏感性，并且可以实现自动化。Z'因子为0.79±0.02，表明该检测方法可进一步优化用于高通量筛选。配子体对三种抗疟药物（氯喹、双氢青蒿素和咯萘啶）的每日敏感性从III期开始急剧下降，而对伯氨喹则保持相对稳定。
开发了一种药物检测方法，利用单一转基因寄生虫系来测定所有配子体阶段的药物敏感性。该检测方法可进一步自动化成为一个高通量平台，用于筛选针对恶性疟原虫配子体的化合物文库。
