Rastogi Ruchir, Kapoor Anjali, Verma Jitender Kumar, Ansari Irshad, Sood Chandni, Kumar Kamal, Mukhopadhyay Amitabha
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Kusuma School of Biological Sciences, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India; National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Biochim Biophys Acta Mol Cell Res. 2021 Jan;1868(1):118868. doi: 10.1016/j.bbamcr.2020.118868. Epub 2020 Oct 2.
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 LdRab5bLeishmania survival in macrophage indicating that LdRab5b function is essential for the acquisition of heme from internalized Hb for the survival of Leishmania.
此前，我们发现Rab5a和Rab5b分别对利什曼原虫的液相内吞作用和受体介导的内吞作用进行差异性调控。为明确证明Rab5b在利什曼原虫血红蛋白内吞作用中的作用，我们通过依次用潮霉素和新霉素抗性基因盒替换LdRab5b的两个拷贝，构建了Rab5b基因缺失的突变体寄生虫。使用LdRab5b特异性引物对基因组DNA进行qPCR分析，证实了LdRab5b基因缺失的突变体寄生虫。LdRab5b细胞表现出严重的生长缺陷，表明LdRab5b在寄生虫中具有重要功能。为了表征Rab5b在寄生虫血红蛋白内吞作用中的作用，通过在生长培养基中外源添加血红素挽救了LdRab5b细胞。我们的结果表明，LdRab5b细胞的尺寸相对较小。超微结构分析显示，与对照细胞相比，这些细胞中存在相对扩大的鞭毛袋和更大的细胞内囊泡。Rab5b基因缺失的突变体寄生虫的前鞭毛体和无鞭毛体均无法内化血红蛋白，但不同标记物的液相内吞作用未受影响。然而，在LdRab5b细胞中互补LdRab5b:WT（LdRab5b:pRab5b:WT）挽救了这些细胞中的血红蛋白内化。有趣的是，与对照细胞相比，LdRab5b细胞表面的血红蛋白受体明显减少，表明血红蛋白受体转运受阻。最后，我们表明LdRab5b寄生虫可以感染巨噬细胞，但与对照细胞相比，感染96小时后无法存活。然而，在生长培养基中添加血红素显著挽救了LdRab5b利什曼原虫在巨噬细胞中的存活，表明LdRab5b功能对于利什曼原虫从内化的血红蛋白中获取血红素以维持生存至关重要。
