Key Laboratory of Zoonosis, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, 120 Dongling Road, Shenyang, 110866, People's Republic of China.
Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, 130062, People's Republic of China.
Malar J. 2018 Jun 18;17(1):232. doi: 10.1186/s12936-018-2388-0.
Plasmodium falciparum is the most virulent parasite of the five Plasmodium species that cause human malaria, and biological analysis of the parasite is critical for the development of novel strategies for disease control. DNA endonucleases are important for maintaining the biological activity, gene stability of the parasite and interaction with host immune systems. In this study, ten sequences of DNA endonucleases were found in the genome of P. falciparum 3D7 clone, seven of them were predicted to contain an endonuclease/exonuclease/phosphatase (IPR005135) domain which plays an important role in DNA catalytic activity. The seven DNA endonucleases of P. falciparum were systematically investigated.
Plasmodium falciparum 3D7 clone was cultured in human O RBCs, RNA was extracted at 8, 16, 24, 32, 40, and 48 h post invasion and real-time quantitative PCR was carried out to analyse the transcription of the seven DNA endonuclease genes in asexual stages. Immunofluorescence assay was carried out to confirm the location of the encoded proteins expressed in the erythrocytic stages. Finally, the catalytic activity of the DNA nucleases were tested.
Of the seven proteins analysed, two proteins were not soluble. Fragments derived from the rest five endonuclease sequences were successfully expressed as soluble proteins, and which were used to generate antisera for protein localization. The proteins were all located in the nucleus at ring and trophozoite stages. While at schizont stage, proteins encoded by PF3D7_1238600, PF3D7_0107200 and PF3D7_0319200 were in the punctuated forms in the parasite most likely around nuclei of the merozoites. But the proteins encoded by PF3D7_0305600 and PF3D7_1363500 were distributed around the infected erythrocyte membrane. The enzymatic activity of the recombinant GST-PF3D7_1238600 was very efficient without divalent iron, while the activity of the rest four enzymes was iron dependent. Further, divalent irons did not show any specific enhancement on the activity of GST-PF3D7_1238600, but the activity of GST-PF3D7_0107200, GST-PF3D7_1363500 and GST-PF3D7_0319200 were Cu dependent. The activity of GST-PF3D7_0305600 was dependent on Mg and Mn. Except GST-PF3D7_1363500, four of the GST tagged recombinant proteins hydrolysed the supercoiled circular plasmid DNA with or without divalent metal ions. The GST-PF3D7_1363500 protein only changed the supercoiled circular plasmid DNA into nicked plasmids, even with Cu.
Fragments derived from five of the endonuclease sequences of P. falciparum 3D7 clone were successfully expressed. The proteins displayed diverse cell distribution, biochemical and enzymatic activities, which indicated that they carried different biological function in the development of the parasite in the erythrocytes. The DNA repair and DNA degradation capacity of the DNA endonucleases in the biology of the parasite remained further studied.
恶性疟原虫是导致人类疟疾的五种疟原虫中最具毒性的寄生虫，对寄生虫的生物学分析对于开发新的疾病控制策略至关重要。DNA 内切酶对于维持寄生虫的生物活性、基因稳定性以及与宿主免疫系统的相互作用非常重要。在本研究中，在恶性疟原虫 3D7 克隆的基因组中发现了 10 个 DNA 内切酶序列，其中 7 个被预测含有内切酶/核酸外切酶/磷酸酶（IPR005135）结构域，该结构域在 DNA 催化活性中起着重要作用。本研究对 7 个恶性疟原虫 DNA 内切酶进行了系统研究。
在人类 O 型红细胞中培养恶性疟原虫 3D7 克隆，在入侵后 8、16、24、32、40 和 48 小时提取 RNA，并进行实时定量 PCR 分析无性阶段 7 个 DNA 内切酶基因的转录情况。通过免疫荧光试验确认在红细胞阶段表达的编码蛋白的位置。最后，测试了 DNA 核酶的催化活性。
在所分析的 7 种蛋白中，有 2 种蛋白不可溶。从其余 5 个内切酶序列衍生的片段成功表达为可溶性蛋白，并用于产生蛋白定位的抗血清。这些蛋白均位于环状和滋养体阶段的核内。而在裂殖体阶段，PF3D7_1238600、PF3D7_0107200 和 PF3D7_0319200 编码的蛋白可能在裂殖子的核周围呈点状形式。但 PF3D7_0305600 和 PF3D7_1363500 编码的蛋白则分布在感染的红细胞膜周围。重组 GST-PF3D7_1238600 的酶活性非常高效，无需二价铁，而其余 4 种酶的活性依赖于二价铁。此外，二价铁对 GST-PF3D7_1238600 的活性没有任何特定的增强作用，但 GST-PF3D7_0107200、GST-PF3D7_1363500 和 GST-PF3D7_0319200 的活性依赖于 Cu。GST-PF3D7_0305600 的活性依赖于 Mg 和 Mn。除了 GST-PF3D7_1363500 外，GST 标记的重组蛋白中的 4 种能够水解超螺旋环状质粒 DNA，无论是否存在二价金属离子。GST-PF3D7_1363500 蛋白仅将超螺旋环状质粒 DNA转化为缺口质粒，即使有 Cu 存在也是如此。
成功表达了恶性疟原虫 3D7 克隆中 5 个内切酶序列的片段。这些蛋白显示出不同的细胞分布、生化和酶活性，这表明它们在寄生虫在红细胞中的发育过程中具有不同的生物学功能。寄生虫生物学中的 DNA 内切酶的 DNA 修复和 DNA 降解能力有待进一步研究。
