Non-Vesicular Lipid Transport Machinery in Leishmania donovani: Functional Implications in Host-Parasite Interaction

Abstract

Eukaryotic cells have distinct membrane-enclosed organelles, each with a unique biochemical signature and specialized function. The unique identity of each organelle is greatly governed by the asymmetric distribution and regulated intracellular movement of two important biomolecules, lipids, and proteins. Non-vesicular lipid transport mediated by lipid-transfer proteins (LTPs) plays essential roles in intra-cellular lipid trafficking and cellular lipid homeostasis, while vesicular transport regulates protein trafficking. A comparative analysis of non-vesicular lipid transport machinery in protists could enhance our understanding of parasitism and basis of eukaryotic evolution. Leishmania donovani, the trypanosomatid parasite, greatly depends on receptor-ligand mediated signalling pathways for cellular differentiation, nutrient uptake, secretion of virulence factors, and pathogenesis. Lipids, despite being important signalling molecules, have intracellular transport mechanisms that are largely unexplored in L. donovani. We have identified a repertoire of sixteen (16) potential lipid transfer protein (LTP) homologs based on a domain-based search on TriTrypDB coupled with bioinformatics analyses, which signifies the presence of well-organized lipid transport machinery in this parasite. We emphasized here their evolutionary uniqueness and conservation and discussed their potential implications for parasite biology with regards to future therapeutic targets against visceral leishmaniasis.

Keywords: Leishmania donovani; drug development; lipid signaling; lipid transfer protein; pathogenesis.

Figure 1

Schematic diagram illustrates the life cycle of L. donovani. L. donovani has two life cycle stages: the sandfly stage (top) and the mammalian stage (bottom). L. donovani proliferate in the midgut as procyclic promastigotes and differentiate into highly infectious metacyclic promastigotes. When a sandfly bites a mammalian host, the L. donovani promastigotes are injected into the dermis of the host. Then they are taken up by phagocytosis, once inside phagolysosomes of macrophages, they transform into amastigotes. Infected macrophages lyse and release amastigotes, which reinvade other macrophages to multiply further. The figure has been modified from Colineau, 2018 [30].

Figure 2

Classification and domain organization of lipid transfer proteins (LTPs) from human, yeast, and L. donovani BPK282A1. Based on domain organization and cellular localization, LTPs are grouped into cytosolic and membrane bound LTPs. Cytosolic LTPs have only lipid transfer domains (LTDs) such as, (A) oxysterol-binding protein (OSBP)-related domain (ORD, binds to sterols and to PtdIns4P), (B) steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain (binds to either sterols, phospholipids, or ceramides), (C) Sec14 domain (bind to PC and PtdIns), and (D) Lipocalin domain (binds to palmitate), all of which can accommodate the hydrophobic moieties of various lipid ligands from aqueous environment of cytoplasm. Membrane bound LTPs possess various combinations of LTDs with other additional membrane-anchored domains/motifs such as pleckstrin-homology (PH) domain, diphenylalanine-in-an-acidic-tract (FFAT) motif, Golgi dynamics (GOLD) domain, Ankyrin repeats, Phox homology (PX) domain, FYVE (Fab-1, YGL023, Vps27, and EEA1) domain etc. and function as membrane contact sites (MCSs). L. donovani has sixteen (16) potential LTP homologs including one START candidate, fourteen Sec14 candidates and one Lipocalin domain containing protein. L. donovani lacks homolog of eukaryotic OSBP-related proteins (ORPs) and other eukaryotic proteins, discussed in Section 3.1.3. TriTrypDB ID of L. donovani BPK282A1 LTP homologs are shown. The e-values for each of the identified LTP homologs in L. donovani BPK282A1 genome are shown after the TriTrypDB ID of each LTP homolog.

Figure 3

Modeling of the 3D structure of representative LTP candidates of L. donovani. The protein sequences of lipid transfer domains (LTDs) of representative homologs from each of the classified groups [i.e., (A) START, (B) Sec14 and (C) Lipocalin] were used for protein homology modelling. The PDB ID of each template and TriTrypDB ID of representative homologs from amastigote form of L. donovani are shown. The structural resolution of the Protein Data Bank structures (2.3 Å for PDB ID: 6ser.1.A, 2.0 Å for PDB ID: 1o6u.3.A and 2.3 Å for PDB ID: 6hhg.1.A) have been provided. The representative homologs (i.e., LdBPK_292840, LdBPK_312110 and LdBPK_281800) from each classified groups shared weak homologies with their counterparts from higher eukaryotes, indicating their potentiality as parasite-specific intervention candidates.

Figure 4

Relative mRNA expression of LTP homologs in L. donovani BPK282A1 amastigotes. Survey of L. donovani BPK282A1 genome has a repertoire of sixteen (16) LTP homologs. The levels of mRNA expression are shown with TPM (transcript per million) value as per TriTrypDB. The levels of mRNA expression of LTP candidates, shown in the figure are obtained from amastigotes form of L. donovani parasite. Two members of L. donovani Sec14 homologs (LdBPK_353610.1 followed by LdBPK_312090.1) show the higher mRNA expression in BPK282A1 strain among all identified LTP candidates, while another two Sec14 homologs (LdBPK_321330.1 and LdBPK_310330.1) exhibits the minimal mRNA expression in BPK282A1 strain among all candidates. The identified START (LdBPK_292840.1) and lipocalin (LdBPK_281800.1) homolog show moderate level of mRNA expression.