Previously, we showed that Rab5a and Rab5b differentially regulate fluid-phase and receptor-mediated endocytosis in Leishmania, respectively. To unequivocally demonstrate the role of Rab5b in hemoglobin endocytosis in Leishmania, we generated null-mutants of Rab5b parasites by sequentially replacing both copies of LdRab5b with the hygromycin and neomycin resistance gene cassettes. LdRab5b^−/− null-mutant parasite was confirmed by qPCR analysis of genomic DNA using LdRab5b specific primers. LdRab5b^−/− cells showed severe growth defect indicating essential function of LdRab5b in parasite. To characterize the role of Rab5b in Hb endocytosis in parasites, LdRab5b^−/− cells were rescued by exogenous addition of hemin in growth medium. Our results showed that LdRab5b^−/− cells are relatively smaller in size. Ultrastructural analysis revealed the presence of relatively enlarged flagellar pocket and bigger intracellular vesicles in these cells in comparison to control cells. Both promastigotes and amastigotes of Rab5b null-mutant parasites were unable to internalize Hb but fluid phase endocytosis of different markers was not affected. However, complementation of LdRab5b:WT in LdRab5b^−/− cells (LdRab5b^−/−:pRab5b:WT) rescued Hb internalization in these cells. Interestingly, LdRab5b^−/− cells showed significantly less Hb-receptor on cell surface in comparison to control cells indicating a block in HbR trafficking. Finally, we showed that LdRab5b^−/− parasites can infect the macrophages but are unable to survive after 96 h of infection in comparison to control cells. However, supplementation of hemin in the growth medium significantly rescued LdRab5b^−/− Leishmania survival in macrophage indicating that LdRab5b function is essential for the acquisition of heme from internalized Hb for the survival of Leishmania.

以前，我们显示Rab5a和Rab5b分别调节利什曼原虫的液相和受体介导的内吞作用。为了明确证明Rab5b在利什曼原虫的血红蛋白内吞作用中的作用，我们通过用潮霉素和新霉素抗性基因盒顺序替换LdRab5b的两个拷贝，从而生成Rab5b寄生虫的无效突变体。通过使用LdRab5b特异性引物的基因组DNA的qPCR分析，证实了LdRab5b ^-/-空突变寄生虫。LdRab5b ^-/-细胞显示出严重的生长缺陷，表明LdRab5b在寄生虫中具有基本功能。为了表征Rab5b在寄生虫Hb内吞中的作用，LdRab5b ^-/-通过在生长培养基中外源添加血红素拯救细胞。我们的结果表明，LdRab5b ^-/-细胞的大小相对较小。超微结构分析显示，与对照细胞相比，这些细胞中存在相对较大的鞭毛袋和较大的细胞内囊泡。Rab5b无效突变寄生虫的前鞭毛体和变形体均不能内化Hb，但不同标记物的液相内吞作用不受影响。然而，LdRab5b ^-/-细胞中LdRab5b：WT的互补（LdRab5b ^-/-：pRab5b：WT）拯救了这些细胞中的Hb内在化。有趣的是，LdRab5b ^-/-与对照细胞相比，细胞在细胞表面显示出明显更少的Hb受体，表明HbR转运受到阻滞。最后，我们表明，与对照细胞相比，LdRab5b ^-/-寄生虫可以感染巨噬细胞，但在感染96小时后无法存活。然而，在生长培养基中补充血红素可显着拯救巨噬细胞中的LdRab5b ^-/- 利什曼原虫存活，表明LdRab5b的功能对于从内在的Hb获取血红素对于利什曼原虫的存活至关重要。