Tohidi Farideh, Babaei Zahra, Kazemi Bahram, Bandehpour Mojgan, Sharifi Iraj, Rabiei Mohammad Reza, Dezaki Ebrahim Saedi
Department of Medical Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman; Infectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, Iran.
Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran.
J Vector Borne Dis. 2019 Apr-Jun;56(2):98-104. doi: 10.4103/0972-9062.263718.
BACKGROUND & OBJECTIVES: Leishmania parasites cause various clinical symptoms in humans such as cutaneous ulcers and fatal visceral diseases. These parasites cannot synthesize purine rings de novo and must uptake purines from their hosts via salvage. Salvage is regulated by permeases in the cell membrane. There are hundreds of membrane transporter proteins to receive nutrients in Leishmania. Nucleoside transporter 4 (NT4) is one of the purine transporters that is involved in enhancing the uptake of adenine in Leishmania major. They are important new drug targets for the treatment of leishmaniasis because they can be used to transport toxic purine analogs to kill parasitic cells, thus preventing the progression of the infection. The present study was conducted to silence the NT4 nucleobase involved in the salvage pathway to interrupt purine nucleotide membrane transport in the cells of L. major.
In this study, a 502 bp segment of NT4 gene sequence was selected and designed as antisense transcripts after insertion in the parasite. The NT4 construct was transfected into L. major promastigotes for in vitro study of gene expression. Then, BALB/c mice infected with transgenic Leishmania and wild-type strain along with the number and size of lesions were studied in vivo.
The study showed that relative expression of NT4 gene in mutant Leishmania was lower than in the control on Day 3 to 20. The percentages and the number of amastigotes in infected macrophages with wild-type strain L. major were more than infected macrophages with mutant parasites. Infected BALB/c mice with transgenic Leish- mania showed a lower number and size of lesions than the BALB/c mice infected with wild-type strain.
INTERPRETATION & CONCLUSION: The results of the study indicated that the use of antisense RNA reduced NT4 gene expression in L. major. Further, studies are needed to ascertain that the use of antisense can be considered as a new treatment for leishmaniasis.
利什曼原虫可导致人类出现多种临床症状，如皮肤溃疡和致命的内脏疾病。这些寄生虫无法从头合成嘌呤环，必须通过补救途径从宿主摄取嘌呤。补救途径由细胞膜中的通透酶调节。利什曼原虫中有数百种膜转运蛋白来摄取营养物质。核苷转运蛋白4（NT4）是嘌呤转运蛋白之一，参与增强硕大利什曼原虫对腺嘌呤的摄取。它们是治疗利什曼病的重要新药物靶点，因为可用于转运有毒嘌呤类似物来杀死寄生细胞，从而阻止感染的进展。本研究旨在使参与补救途径的NT4核碱基沉默，以中断硕大利什曼原虫细胞中的嘌呤核苷酸膜转运。
本研究选择了NT4基因序列的502 bp片段，插入寄生虫后设计为反义转录本。将NT4构建体转染到硕大利什曼原虫前鞭毛体中，用于基因表达的体外研究。然后，对感染转基因利什曼原虫和野生型菌株的BALB/c小鼠以及病变的数量和大小进行体内研究。
研究表明，在第3天至第20天，突变型利什曼原虫中NT4基因的相对表达低于对照组。感染野生型硕大利什曼原虫菌株的巨噬细胞中无鞭毛体的百分比和数量高于感染突变型寄生虫的巨噬细胞。感染转基因利什曼原虫的BALB/c小鼠的病变数量和大小低于感染野生型菌株的BALB/c小鼠。
研究结果表明，使用反义RNA可降低硕大利什曼原虫中NT4基因的表达。此外，需要进一步研究以确定使用反义RNA是否可被视为利什曼病的一种新治疗方法。
