Lu S, Suzuki T, Iizuka N, Ohshima S, Yabu Y, Suzuki M, Wen L, Ohta N
Department of Molecular Parasitology, Nagoya City University, Graduate School of Medical Sciences, Nagoya 467-8601, Japan.
Parasitology. 2007 Oct;134(Pt 11):1639-47. doi: 10.1017/S0031182007003046. Epub 2007 Jun 19.
Procyclic forms of Trypanosoma brucei brucei remain and propagate in the midgut of tsetse fly where iron is rich. Additional iron is also required for their growth in in vitro culture. However, little is known about the genes involved in iron metabolism and the mechanism of iron utilization in procyclic-form cells. Therefore, we surveyed the genes involved in iron metabolism in the T. b. brucei genome sequence database. We found a potential homologue of vacuole protein sorting 41 (VPS41), a gene that is required for high-affinity iron transport in Saccharomyces cerevisiae and cloned the full-length gene (TbVPS41). Complementation analysis of TbVPS41 in DeltaScvps41 yeast cells showed that TbVPS41 could partially suppress the inability of DeltaScvps41 yeast cells to grow on low-iron medium, but it could not suppress the fragmented vacuole phenotype. Further RNA interference (RNAi)-mediated gene knock-down in procyclic-form cells resulted in a significant reduction of growth in low-iron medium; however, no change in growth was observed in normal culture medium. Transmission electron microscopy showed that RNAi caused T. b. brucei cells to have larger numbers of small intracellular vesicles, similar to the fragmented vacuoles observed in DeltaScvps41 yeast cells. The present study demonstrates that TbVPS41 plays an important role in the intracellular iron utilization system as well as in the maintenance of normal cellular morphology.
布氏布氏锥虫的前循环型在富含铁的采采蝇中肠中存活并繁殖。它们在体外培养时的生长也需要额外的铁。然而，对于前循环型细胞中铁代谢相关基因以及铁利用机制知之甚少。因此，我们在布氏布氏锥虫基因组序列数据库中调查了参与铁代谢的基因。我们发现了液泡蛋白分选41（VPS41）的一个潜在同源物，该基因是酿酒酵母中高亲和力铁转运所必需的，并克隆了全长基因（TbVPS41）。在DeltaScvps41酵母细胞中对TbVPS41进行互补分析表明，TbVPS41可以部分抑制DeltaScvps41酵母细胞在低铁培养基上生长的缺陷，但不能抑制液泡碎片化的表型。进一步在前循环型细胞中通过RNA干扰（RNAi）介导的基因敲低导致在低铁培养基中生长显著减少；然而，在正常培养基中未观察到生长变化。透射电子显微镜显示，RNAi导致布氏布氏锥虫细胞具有大量小的细胞内囊泡，类似于在DeltaScvps41酵母细胞中观察到的碎片化液泡。本研究表明，TbVPS41在细胞内铁利用系统以及维持正常细胞形态方面发挥着重要作用。
