Instituto René Rachou IRR, Fundação Oswaldo Cruz - FIOCRUZ/Minas, Avenida Augusto de Lima, Belo Horizonte, MG, 1715, Brazil.
Parasit Vectors. 2019 Jan 25;12(1):60. doi: 10.1186/s13071-019-3305-2.
Glycosylphosphatidylinositol is a surface molecule important for host-parasite interactions. Mannosyltransferase (GPI-14) is an essential enzyme for adding mannose on the glycosylphosphatidyl group. This study attempted to overexpress the GPI-14 gene in Leishmania braziliensis to investigate its role in the antimony-resistance phenotype of this parasite.
GPI-14 mRNA levels determined by quantitative real-time PCR (qRT-PCR) showed an increased expression in clones transfected with GPI-14 compared to its respective wild-type line. In order to investigate the expression profile of the surface carbohydrates of these clones, the intensity of the fluorescence emitted by the parasites after concanavalin-A (a lectin that binds to the terminal regions of α-D-mannosyl and α-D-glucosyl residues) treatment was analyzed. The results showed that the clones transfected with GPI-14 express 2.8-fold more mannose and glucose residues than those of the wild-type parental line, indicating effective GPI-14 overexpression. Antimony susceptibility tests using promastigotes showed that clones overexpressing the GPI-14 enzyme are 2.4- and 10.5-fold more resistant to potassium antimonyl tartrate (Sb) than the parental non-transfected line. Infection analysis using THP-1 macrophages showed that amastigotes from both GPI-14 overexpressing clones were 3-fold more resistant to Sb than the wild-type line.
Our results suggest the involvement of the GPI-14 enzyme in the Sb-resistance phenotype of L. braziliensis.
糖基磷脂酰肌醇是一种对宿主-寄生虫相互作用很重要的表面分子。甘露糖基转移酶（GPI-14）是在糖基磷脂酰基上添加甘露糖的必需酶。本研究试图在巴西利什曼原虫中过表达 GPI-14 基因，以研究其在该寄生虫对锑耐药表型中的作用。
通过定量实时 PCR（qRT-PCR）测定的 GPI-14 mRNA 水平显示，与相应的野生型系相比，转染 GPI-14 的克隆表达增加。为了研究这些克隆表面碳水化合物的表达谱，分析了用伴刀豆球蛋白 A（一种与α-D-甘露糖基和α-D-葡萄糖基残基末端区域结合的凝集素）处理后寄生虫发出的荧光强度。结果表明，转染 GPI-14 的克隆表达的甘露糖和葡萄糖残基比野生型亲本系多 2.8 倍，表明 GPI-14 过表达有效。使用前鞭毛体进行的锑敏感性测试表明，过表达 GPI-14 酶的克隆对酒石酸锑钾（Sb）的耐药性分别比未转染的亲本系高 2.4 倍和 10.5 倍。使用 THP-1 巨噬细胞进行的感染分析表明，来自两个 GPI-14 过表达克隆的无鞭毛体对 Sb 的耐药性比野生型系高 3 倍。
我们的结果表明，GPI-14 酶参与了巴西利什曼原虫对 Sb 的耐药表型。
