Hemoglobin Endocytosis and Intracellular Trafficking: A Novel Way of Heme Acquisition by Leishmania

Abstract

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.

Keywords: Leishmania; Rab GTPase; endocytosis; heme; hemoglobin; traffick.

Figure 1

Schematic representation shows the life cycle of Leishmania and source of hemoglobin.Leishmania has a digenetic life cycle. Transmission to humans occurs through the bite of a female Phlebotomine sandfly. In humans, promastigotes are taken up by the macrophages and transform into amastigotes in the parasitophorous vacuole (PV). Macrophages also ingest senescent RBCs, and lysed RBCs serve as source of hemoglobin for Leishmania. Amastigotes multiply inside the macrophages and eventually lyse cells. Parasites are released and further infect the surrounding macrophages leading to the manifestation of disease. Parasitized cells are ingested by the sandflies during the blood meal. Inside the midgut of the sandfly, amastigote transforms into motile procyclic promastigotes and colonizes their digestive tract. The insect also takes RBCs during the blood meal, which serves as a source of hemoglobin. Finally, they differentiate into infective metacyclic promastigote form and remain in the saliva of the sandfly. Parasites are transmitted to a new vertebrate host during their next blood meal.

Figure 2

Schematic diagram shows the localization and function of different Rab GTPases in mammalian cells. Rab GTPases are present in distinct membranous compartments and regulate transport of cargo between the various compartments. In the endocytic pathway, Rab5 involves trafficking of the cargo from the plasma membrane to the early endosome and localizes on the early endocytic compartment. Rab4 participates in the fast-recycling pathway from the early endosome, while Rab11 and Rab35 regulate the slow recycling pathway. Rab7 helps in the trafficking of cargo to the lysosome from the early endosome, and Rab9 regulates cargo transport from the lysosome to the TGN. In the secretory pathway, Rab1 localizes on the ER Golgi intermediate compartment and regulates anterograde trafficking from ER to Golgi, whereas Rab2 involves trafficking from Golgi to ER in retrograde. Rab6 controls intra-Golgi trafficking. Rab3, Rab27, Rab8, and Rab37 regulate the transport of different secretory vesicles via the exocytic pathway.

Figure 3

Schematic diagram shows the localization and function of different SNAREs in mammalian cells. Similar to Rab GTPases, SNAREs are also localized on selective compartments and provide specificity in vesicle fusion steps. They are subdivided into v-SNARE and t-SNAREs highlighted as VAMP and STX, respectively, in the figure. STX (syntaxin) 1, STX2, STX3, STX4, VAMP5, SNAP-25 and SNAP-23 are localized at the plasma membrane and possibly regulate the fusion of endocytic as well as secretory vesicles. VAMP1, VAMP2 and VAMP3, along with STX7, STX8 and STX13 are found in different endocytic compartments and regulate the different steps in trafficking of endocytic cargo from plasma membrane to lysosomes. STX11, STX5 and SEC28b are located at the Golgi apparatus, whereas STX18, Sec22b, BET1 and Membrin are found at the endoplasmic reticulum, suggesting that traffic between ER and Golgi is regulated by these proteins along with VAMP4 and SNAP29. STX6, STX10, STX11 and STX16, along with VAMP1, VAMP2 and SNAP3, regulate the transport of different secretory vesicles from the Golgi.

Figure 4

Schematic representation demonstrates hemoglobin endocytosis in Leishmania donovani promastigotes. (Left Panel) shows that Alexa-Red labeled Hb bind to the Hb receptor on the flagellar pocket of Leishmania, which traffics the Rab5 positive early endosome by 10 min and finally reaches the lysosome via late endosome in about 45 min. Nucleus is marked with green. (Right Panel) depicts the schematic diagram of Hb trafficking from the flagellar pocket to the lysosomes via early and late endocytic compartments in Rab5b and Rab7 dependent processes in Leishmania promastigotes.

Figure 5

Schematic diagram shows the heme acquisition process in Leishmania. Macrophages engulf senescent RBC and internalize hemoproteins such as transferrin (Tf) and hemoglobin (Hb) by specific receptor-mediated endocytic processes. These hemoproteins and RBCs are transported to the low pH compartment, where iron and hemoglobin are released from their respective hemoprotein. Leishmania residing in such compartment takes in the hemoglobin via their hemoglobin receptor (HbR). Subsequently, internalized Hb is transported to the parasite lysosomes, where it is degraded and releases heme. The released heme is transported to the parasite cytosol by heme transporter LHR1 and FLVCRb, which is finally transferred into mitochondria by mitochondrial heme importer LABCB3 and LMIT1. In addition, Fe³⁺ iron released by the degradation of transferrin in low acidity parasitophorous vacuole (PV) first reduces to Fe²⁺ by LFR1 and is imported by the parasite via its heme transporter LIT1.