Changes in Lipid and Fatty Acid Composition During Intramacrophagic Transformation of Leishmania donovani Complex Promastigotes into Amastigotes

Lipids (2017) 52:433–441 DOI 10.1007/s11745-017-4233-6 ORIGINAL ARTICLE Changes in Lipid and Fatty Acid Composition During Intramacrophagic Transformation of Leishmania donovani Complex Promastigotes into Amastigotes Hana Bouazizi‑Ben Messaoud1,2,3 · Marion Guichard1,2 · Philippe Lawton1,2 · Isabelle Delton3 · Samira Azzouz‑Maache1,2 Received: 3 June 2016 / Accepted: 10 January 2017 / Published online: 4 February 2017 © The Author(s) 2017. This article is published with open access at Springerlink.com Abstract Leishmania sp., are trypanosomatid parasites that are phagocytized by human and animal macrophages. Transformation from the vector promastigote stage to the intracellular amastigote host cell stage is mandatory, since development in the host depends on the internalization of the parasite. We identified and analyzed the lipids involved in the promastigote to amastigote transformation process in the Leishmania donovani complex. Four lipid classes, phospholipids, free fatty acids, triglycerides and sterols were studied. The derivatization method of Bligh and Dyer was used to establish the fatty acid composition in each stage of the parasite. To stay within the context of Leishmania infection, we used amastigotes extracted from macrophages after experimental in vitro infection. The purification process was checked by electronic microscopy, the absence of major contamination by host-cell debris and a correct purification yield validated our experimental model. Our results show that free fatty acids and cholesterol increased, whereas triglycerides and ergosterol decreased during the transition between promastigotes to amastigotes. With respect to phospholipid classes, we found increased proportion of sphingomyelin and phosphatidylserine and lowered proportion of phosphatidylinositol * Samira Azzouz‑Maache samira.azzouz‑maache@univ‑lyon1.fr 1 Institut de recherche pour le développement (IRD), UMR InterTryp IRD/CIRAD, campus international de Baillarguet, Montpellier, France 2 Department of Parasitology and Medical Mycology, Lyon University, Lyon, France 3 Inserm U1060 CarMeN Laboratory, INSA-Lyon, Villeurbanne, France and lysophosphatidylethanolamine. Regarding fatty acid composition, a significant increase of n-7 fatty acids was observed in amastigotes. Overall, the total n-6 fatty acids were decreased in PL. Several of the changes were also observed in TG and free fatty acids. Particularly, n-7 fatty acids and 20:4n-6 were highly increased, whereas n-9 fatty acid and n-6 precursors decreased. Keywords Leishmania donovani complex · Amastigotes · Lipid variations · Cholesterol · Fatty acids · Phospholipids Abbreviations Ama Amastigote BSTFA Bis(trimethylsilyl) trifluoroacetamide FA Fatty acid FAME Fatty acid methyl esters CerPCho Sphingomyelin C Total cholesterol ERG Ergosterol GC Gas chromatography HILIC Hydrophilic interaction liquid chromatography LPtdCho Lysophosphatidylcholine LPtdEtn Lysophosphatidylethanolamine MbCD Methyl-b-cyclodextrin PBS Phosphate-buffered saline PL Phospholipids PLFA Phospholipid fatty acid Pro Promastigote PtdCho Phosphatidylcholine PtdEtn Phosphatidylethanolamine PtdIns Phosphatidylinositol PtdSer Phosphatidylserine SEM Standard error of the mean TG Triglycerides VL Visceral leishmaniasis 13 434 Introduction Leishmania donovani and L. infantum are the causative agents of visceral leishmaniasis (VL) in humans and of canine leishmaniasis in dogs. Leishmania is an intracellular pathogen, whose establishment depends on its successful internalization and multiplication inside macrophages, its mammalian host cell. The Leishmania life cycle is divided into two phases, each of them involving a different stage, the promastigote stage in the insect vector and the amastigote stage inside the host’s macrophages. The promastigotes inoculated during the blood meal of the hematophagous sandfly are phagocytosed by endocytosis and undergo a transformation into amastigotes within a parasitophorous vacuole of phagolysosomal origin. This process is a vital step in the Leishmania life cycle and could be pivotal in the research for new treatments against Leishmania. To date, studies have essentially focused on the role of surface glycosylated residues and proteins in host-parasite interactions. Recent interest has grown for the role of lipids in Trypanosomatids and especially in Trypanosoma cruzi, T. brucei and Leishmania spp. [1]. Lipid metabolism is of paramount importance for parasites, and especially for intracellular parasites, which rely on a complex system of uptake and synthesis mechanisms to satisfy their lipid needs. The parameters of this system change dramatically as the parasite transits through the various stages of its life cycle. Within host cells, intracellular pathogens often develop in specialized vacuoles and the flow of lipids between host and pathogen-controlled membranous compartments is pivotal to the pathogen’s ultimate success [2–4]. Intracellular pathogens have evolved sophisticated mechanisms to manipulate and tap into the lipid metabolism of their host cells. These include interference with vesicular and non-vesicular cellular lipid trafficking in viral [5], bacterial [6] and protozoal [7] pathogens. According to Zhang and Beverley [8], phospholipids (PL) and sphingolipids are both abundant and critical to virulence and viability in Leishmania. The success of miltefosine as an orally available antileishmanial drug is an important validation of lipid metabolism as a drug target. Moreover, it has been previously reported that macrophage cholesterol is important for parasite internalization. The entry of intracellular parasites and particularly of Leishmania, involves interaction with the plasma membrane of host cells. A number of previous studies have demonstrated the requirement of membrane cholesterol in host-pathogen interactions [9, 10]. Cholesterol (C) is an important component of higher eukaryotic cellular membranes and plays a crucial role in the function and the organization of membrane proteins and receptors 13 Lipids (2017) 52:433–441 [11, 12], some of which being necessary for parasite entry [4]. Our goal was to analyze the variations in major lipid classes during the metamorphosis from the promastigote to the amastigote stage, in terms of quantities and fatty acid composition. Biological membranes are composed of fatty acids (FA) and phospholipids, which are present in constant proportions, but when exposed to some stress conditions, such as pathogens or drugs, these proportions might change [13]. The identification of these specific variations could be used as biomarkers to study virulence or resistance to treatments currently used against leishmaniasis. Materials and Methods Promastigote Culture The strains used in this study were L. donovani and L. infantum that both belong to the Leishmania donovani complex. Leishmania donovani (LCR-133) was provided from the Leishmania Referen (...truncated)