Leishmania species are causative agents of human leishmaniasis, affecting 12 million people annually. Drugs available for leishmaniasis are toxic, and no vaccine is available. Thus, the major thrust is to identify new therapeutic targets. Leishmania is an auxotroph for heme and must acquire heme from the host for its survival. Thus, the major focus has been to understand the heme acquisition process by the parasites in the last few decades. It is conceivable that the parasite is possibly obtaining heme from host hemoprotein, as free heme is not available in the host. Current understanding indicates that Leishmania internalizes hemoglobin (Hb) through a specific receptor by a clathrin-mediated endocytic process and targets it to the parasite lysosomes via the Rab5 and Rab7 regulated endocytic pathway, where it is degraded to generate intracellular heme that is used by the parasite. Subsequently, intra-lysosomal heme is initially transported to the cytosol and is finally delivered to the mitochondria via different heme transporters. Studies using different null mutant parasites showed that these receptors and transporters are essential for the survival of the parasite. Thus, the heme acquisition process in Leishmania may be exploited for the development of novel therapeutics.

Lipids stored in lipid-bodies (LBs) in host cells are potential sources of fatty acids for pathogens. However, the mechanism of recruitment of LBs from the host cells by pathogens to acquire fatty acids is not known. Here, we have found that Leishmania specifically upregulates the expression of host Rab18 and its GEF, TRAPPC9 by downregulating the expression of miR-1914-3p by reducing the level of Dicer in macrophages via their metalloprotease gp63. Our results also show that miR-1914-3p negatively regulates the expression of Rab18 and its GEF in cells. Subsequently, Leishmania containing parasitophorous vacuoles (Ld-PVs) recruit and retain host Rab18 and TRAPPC9. Leishmania infection also induces LB biogenesis in host cells and recruits LBs on Ld-PVs and acquires FLC₁₂-labeled fatty acids from LBs. Moreover, overexpression of miR-1914-3p in macrophages significantly inhibits the recruitment of LBs and thereby suppresses the multiplication of parasites in macrophages as parasites are unable to acquire fatty acids. These results demonstrate a novel mechanism how Leishmania acquire fatty acids from LBs for their growth in macrophages.

Leishmania internalize hemoglobin (Hb) via a specific receptor (HbR) for their survival. To identify the Hb‐binding domain of HbR, we cloned and expressed several truncated proteins of HbR and determined their ability to bind Hb. Our findings reveal that 90% of Hb‐binding activity is retained in HbR^41–80 in comparison with HbR^1–471. We synthesized a 40 amino acid peptide (SSEKMKQLTMYMIHEMVEGLEGRPSTVRMLPSFVYTSDPA) corresponding to HbR^41–80 and found that it specifically binds Hb. Subsequently, we found that the HbR^41–80 peptide completely blocks Hb uptake in both promastigote and amastigote forms of Leishmania and, thereby, inhibits the growth of the parasite. These results demonstrate that HbR^41–80 is the Hb‐binding domain of HbR, which might be used as a potential therapeutic agent to inhibit the growth of Leishmania.

In clathrin-mediated endocytosis, the cytoplasmic domain of the receptor binds to the AP2 adaptor, which recruits clathrin to mediate endocytosis. The classical AP2 adaptor in Leishmania has not yet been characterized. Here, we identified ATP synthase epsilon (LdATPSε) subunit as a novel adaptor in Leishmania using yeast two-hybrid screening. Subsequently, we cloned and expressed LdATPSε from Leishmania and showed that LdATPSε colocalizes with LdClathrin and the hemoglobin receptor in Leishmania. We found that LdATPSε directly binds to a cargo-binding motif, ‘YLAP’, in the cytoplasmic domain of the high-affinity hemoglobin receptor, whereas it interacts with the LdClathrin terminal domain via a clathrin-binding motif, ‘LSELD’. Consequently, we showed that mutated clathrin binding box LdATPSεL133A/L136A/D137A does not bind to clathrin and fails to localize in the flagellar pocket, and its overexpression completely blocks hemoglobin internalization in Leishmania. LdATPSε−/− parasites are not viable, indicating the essential function of LdATPSε. However, hemoglobin internalization in LdATPSε+/− parasites is significantly blocked, and LdATPSε+/− parasites fail to grow in macrophages as the parasites are unable to internalize hemoglobin. Our results demonstrate that LdATPSε is a novel adaptor for clathrin in hemoglobin endocytosis in Leishmania.