Balcazar Darío E, Vanrell María Cristina, Romano Patricia S, Pereira Claudio A, Goldbaum Fernando A, Bonomi Hernán R, Carrillo Carolina
Laboratorio de Parasitología Molecular y Bioquímica, Instituto de Ciencia y Tecnología Dr. Cesar Milstein, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Saladillo, (C1440FFX) Ciudad Autónoma de Buenos Aires, Argentina.
Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM), Universidad Nacional de Cuyo-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Casilla de correo 56, Ciudad de Mendoza, Argentina.
PLoS Negl Trop Dis. 2017 Apr 13;11(4):e0005513. doi: 10.1371/journal.pntd.0005513. eCollection 2017 Apr.
Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown.
METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems.
CONCLUSIONS/SIGNIFICANCE: The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT/SLC52 and RibJ present, make the riboflavin transporter -and its downstream metabolism- a potential trypanocidal drug target.
锥虫寄生虫是一个重大的健康问题，影响着全球数亿人，其临床治疗方法部分有效且/或毒性很大。它们导致严重的人类和植物疾病，包括克氏锥虫（恰加斯病）、布氏锥虫（昏睡病）、利什曼原虫属（利什曼病）和植生滴虫属（植物寄生虫）。动物和锥虫都缺乏生物合成核黄素（维生素B2）的途径，而核黄素是黄素单核苷酸（FMN）和黄素腺嘌呤二核苷酸（FAD）辅因子的重要前体。后生动物通过RFVT/SLC52转运蛋白从饮食中获取核黄素，而锥虫中的核黄素转运机制仍然未知。
方法/主要发现：在这里，我们使用放射性标记的核黄素转运试验表明，克氏锥虫、布氏锥虫、墨西哥利什曼原虫（利什曼原虫属）、束状短膜虫和植生滴虫Jma中核黄素以高亲和力被摄取。维生素通过可饱和的载体介导过程被吸收。核黄素类似物玫瑰红菌素、发光黄素和发光色素以及辅因子衍生物FMN和FAD会发生有效的竞争性摄取。此外，在克氏锥虫中评估的重要生物学过程（即增殖、循环体形成和无鞭毛体复制）取决于核黄素的可用性。此外，发现核黄素竞争性类似物会干扰寄生虫在核黄素依赖性过程中的生理功能。通过生物信息学分析，我们在锥虫中鉴定出一个新的核黄素转运蛋白家族（RibJ）。分别来自克氏锥虫和布氏锥虫的两个RibJ成员，即TcRibJ和TbRibJ，使用同源和/或异源表达系统进行了功能表征。
结论/意义：RibJ家族是原生生物中发现的首个核黄素转运蛋白，也是迄今为止已知的第三个真核生物家族。核黄素对锥虫的必要性，以及RFVT/SLC52和RibJ存在的结构/生化差异，使核黄素转运蛋白及其下游代谢成为潜在的抗锥虫药物靶点。
