Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
Malar J. 2021 Jun 5;20(1):247. doi: 10.1186/s12936-021-03773-4.
Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites.
In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5' UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey.
Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC) values of blood stages measured by NanoLuc activity proved comparable to IC values measured by the standard SYBR Green method.
All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.
目前在马来西亚，疟原虫 knowlesi 已成为人类疟疾的主要病原体，这使得疟疾防控工作变得更加复杂，因为必须同时消灭多种疟原虫。近期，疟原虫 knowlesi 红内期寄生虫的体外培养、外源 DNA 的转化以及来自培养物的配子体感染蚊子等方面取得的进展，为无需资源密集型和高成本的非人类灵长类动物（NHP）模型即可开展这种病原体的研究开辟了道路。为了进一步了解和开发寄生虫转化方法，用于疟疾研究，本研究检测了各种跨物种转录控制序列的活性，以及疟原虫 vivax 着丝粒（pvcen）重复序列对质粒转染疟原虫 knowlesi 寄生虫的影响。
用包含疟原虫 vivax 或疟原虫 falciparum 5'UTR 的质粒构建体转染体外培养的疟原虫 knowlesi 寄生虫，该 5'UTR 驱动生物发光标记物（萤火虫荧光素酶或 Nanoluc）的表达。通过生物发光评估启动子活性，并使用抗叶酸剂选择表达人抗性等位基因二氢叶酸还原酶的质粒转化的寄生虫。通过在恒河猴、蚊子和第二只恒河猴体内进行完整的寄生虫生命周期，通过生物发光评估携带 pvcen 稳定的内粒体的转化体的稳定性。
荧光素酶表达评估表明，某些在进化上与疟原虫 falciparum 关系较远的疟原虫 vivax 启动子区域可驱动疟原虫 knowlesi 中转基因的表达。此外，pvcen 重复序列可能提高疟原虫 knowlesi 中质粒的稳定性，并支持在恒河猴到致倦库蚊再到猴的整个寄生虫生命周期中检测到 Nanoluc 表达元件。在氯喹、G418 和 WR9910 的药物反应测定中，通过 NanoLuc 活性测量的血阶段抗疟药物半数抑制浓度（IC）值与通过标准 SYBR Green 方法测量的 IC 值相当。
在本研究中测试的所有 3 种疟原虫 vivax 启动子均可在疟原虫 knowlesi 中发挥作用，而其中 2 种在疟原虫 falciparum 中无活性。Nanoluc 表达、着丝粒稳定的质粒可能支持对疟原虫 knowlesi 的高通量筛选，以寻找新的抗疟药物，包括可阻断蚊子和/或肝期寄生虫发育的化合物。
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