Fatty Acid Composition and Metabolism in Leishmania Parasite Species: Potential Biomarkers or Drug Targets for Leishmaniasis?

Fatty acids have received growing interest in Leishmania biology with the characterization of the enzymes allowing the complete fatty acid synthesis of this trypanosomatid parasite. This review presents a comparative analysis of the fatty acid profiles of the major classes of lipids and phospholipids in different species of Leishmania with cutaneous or visceral tropism. Specificities relating to the parasite forms, resistance to antileishmanial drugs, and host/parasite interactions are described as well as comparisons with other trypanosomatids. Emphasis is placed on polyunsaturated fatty acids and their metabolic and functional specificities, in particular, their conversion into oxygenated metabolites that are inflammatory mediators able to modulate metacyclogenesis and parasite infectivity. The impact of lipid status on the development of leishmaniasis and the potential of fatty acids as therapeutic targets or candidates for nutritional interventions are discussed.


Introduction
Leishmaniases are a complex of tropical and subtropical diseases caused by unicellular parasites of the genus Leishmania transmitted by a sandfly vector. The localization of parasites either in dermal macrophages or their migration to different tissues within internal macrophages contribute to disease establishment in the host, causing different phenotypes such as cutaneous (CL), mucocutaneous (MCL), and visceral leishmaniasis (VL) [1][2][3]. Twenty Leishmania species are distributed worldwide and transmitted to humans and animals by the sandfly under the different clinical forms mentioned above. Nearly 350 million people are exposed to leishmaniases in more than 90 countries worldwide, mainly in India and Africa. The emergence of leishmaniases is now observed throughout the Maghreb, the Middle East, and South America, with around 2 million new cases reported annually [4]. Canine leishmaniasis is endemic in more than 70 countries in southern Europe, Africa, Asia, and South and Central America and it has also been reported in the United States of America [5]. Dogs constitute a major reservoir for the Leishmania species that also affect humans, and several hundred cases occur annually in humans in the Mediterranean basin (WHO, 2020).
The Leishmania life cycle involves two different stages: the promastigote inside the insect vector and the amastigote in the vertebrate's macrophages. The promastigotes inoculated by the vector are internalized by host macrophages via phagocytosis and undergo a transformation into the amastigote stage within a parasitophorous vacuole of phagolysosomal origin [6]. Differentiation of promastigotes into amastigotes involves complex mechanisms including morphological changes and genomic expressions such as surface molecules that are required for parasite infectivity and survival into macrophages.
Entry of intracellular pathogens and cell differentiation are generally associated with the reorganization of the plasma membrane where lipids, essentially phospholipids, glycolipids, and sterols, are the main components. The host cell's plasma membrane serves as a portal for the entry of intracellular pathogens [7]. There are also intricate relationships between parasites and host cell intracellular organelles through interactions with the parasitophorous vacuole membranes [7][8][9].
During the last decades, advances in biochemical and molecular approaches have both contributed to highlighting the importance of lipids and lipid metabolism in Leishmania biology and the course of macrophage infection. Highly sensitive analytical techniques such as liquid chromatography-mass spectrometry (LC-MS/MS) allow the revealing of lipidomic signatures related to the Leishmania life cycle [10,11] and lipid alterations in drug-resistant Leishmania strains [12,13]. Genomic and transcriptomic approaches have also reported some correlations between lipid-related gene expression and antileishmanial drug resistance [14,15]. To date, fatty acids (FA), phospholipids (PL), and sterols have emerged as biological actors in Leishmania physiology and virulence [12,13].
Recent reviews have emphasized the potential of enzymes involved in lipid and FA synthesis and metabolism to serve as targets for anti-trypanosomal drugs [12,16]. In this review, we will focus on the specificities of Leishmania parasites in terms of FA composition and the production of oxygenated FA derivatives in both promastigote and amastigote forms and in interaction with host cell macrophages. We will open an opinion on the potential of FA to reveal biomarkers for leishmaniasis and to emerge as therapeutic targets or as candidates for nutritional intervention.

Leishmania Species and Leishmaniasis
The diversity of Leishmania species and their wide geographical distribution as well as their sensitivity to treatments illustrate the influence of the environment and the microenvironment, whether of the host or reservoirs, on the development of the disease and species tropism.
The reference method for Leishmania typing is multilocus enzyme electrophoresis (MLEE). The most commonly used is the Montpellier system (MON) which is based on the analysis of 15 enzymes. As an example, L. infantum is characterized by a large enzymatic polymorphism and comprises 30 zymodemes in humans. Some of them are related to VL (MON27, 28, 72, 77, and 187), others only to CL (MON11, 29, 33, 78, and 111), and some zymodemes can cause both VL and CL, such as MON1 and MON24 [17][18][19][20].
Leishmania species cause different clinical forms depending on the localization of the parasite in the host. VL or Kala Azar in the Indian subcontinent is the most serious form and its evolution is fatal in the absence of treatment. It is caused by two species: L. donovani, highly endemic in East Africa and the Indian subcontinent, anthroponotic and affecting all age groups; L. infantum, zoonotic with a canine reservoir, located in countries around the Mediterranean, the Middle East, and South America, pathogenic in children and immunocompromised (HIV) patients [4,21]. CL is the least severe form of the disease. It is caused by different Leishmania species such as L. major and L. tropica in the old world, L. amazonensis, L. guyanensis, L. panamensis, and L. braziliensis in various regions of Central and South America [22,23]. Diffuse CL is a much rarer form linked to immunosuppression and is more difficult to treat. In the old world, it is caused by L. aethiopica, while in the new world, it is caused by L. mexicana and L. amazonensis [24,25]. MCL or "espundia" appears from a few days to several years after CL. It is caused by L. braziliensis, L. panamensis, L. amazonensis, and L. guyanensis [26]. South America is the most important endemic area. Post-kala-azar leishmaniasis (PKDL) is a nonfatal dermatological complication that occurs in some patients with VL [27]. It is still not understood why some species of Leishmania cause cutaneous symptoms while other species, which are fatal for humans and animals, cause visceral symptoms. Even more challenging is the double tropism of L. infantum or the shifting tropism from CL to VL in Leishmania/HIV co-infections [17]. It has been globally proposed that the development of the disease depends mainly on the intrinsic metabolism of the parasite species, although factors in the vectors and the biological status of the host have also been involved [28].
Some differences in FA distribution are also remarkable among individual PL (e.g., phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylinositol PI, and sphingomyelin). In L. donovani promastigotes, LA was the most abundant FA in PE with only trace amounts of higher PUFAs; OA and ALA were major acyl chains in PC and PI, respectively [29,31]. Longer chain PUFAs such as DHA and AA were found in high proportions in diphosphatidylglycerol [29], PC, and PI [30]. Analyses of molecular species showed the predominance in L. donovani of C16 and C18 acyl chains in PC, PE, and PI, as well as DHA-containing species in PC [32]. Wassef et al. [30] showed that the relative distributions of FA in PC, PE, and PI were different between PL isolated from whole cells and those isolated from surface membranes, with a higher ratio of unsaturated to saturated FA in PL surface membranes. In L. mexicana and L. infantum, C19∆ was mainly recovered in plasmenylethanolamine, the dominant class of PE in Leishmania [37][38][39].

Interspecies Differences and Similarities in FA Composition
In addition to L. infantum, L. donovani, and L. major, as summarized above, FA profiles were determined in many other Leishmania species including L. tropica, L. mexicana, L. amazonensis, and L. tarentolae [29][30][31][32][33]35]. Using nonmetric multidimensional scaling analysis, de Azevedo et al. showed that L. infantum/chagasi and L. amazonensis present different FA profiles; however, the method used did not allow the identification of which FA. GCMS analysis indicated that DHA was not detected in these two species, unlike other Leishmania species [33].
To further investigate interspecies specificities, we have compared the FA composition of total lipids from nine Leishmania isolates in the same series of analyses to limit methodology variability. Isolates were of human or canine origin, causing either visceral (L. infantum) or cutaneous (L. tropica, L. major, and L. infantum) leishmaniasis in Tunisia ( Table 1). The data indicate only small differences between the L. infantum and L. tropica strains, regardless of the human/canine origin or the visceral/cutaneous form. These strains all contained about 30% saturated fatty acids (SFA), 20% monounsaturated fatty acids (MUFAs), and 50% polyunsaturated fatty acids (PUFAs), with quite an even distribution between the n-6 and the n-3 series. Within each fatty acid series, the C18 fatty acids were found in the highest proportions, consistent with previous analyses on total lipids or lipid/PL classes of various Leishmania species [29][30][31][32][33]35]. Long-chain n-6 PUFAs were present in a much lower proportion than the long-chain n-3 PUFA, especially AA, which accounted for 1% of total FA compared to 10% for DHA. While the FA profiles of L. infantum and L. tropica are broadly similar, some differences are noticeable in the L. major strains, such as a significantly higher proportion of myristic acid (14:0) and lower proportions of n-3 FA, especially ALA and DHA. A recent study reported the FA composition of L. major strains of different host origins-human or rodents by combining in silico and GC-MS [36]. The authors also pointed out some differences in L. major compared to other Leishmania species, notably the absence of detectable ALA and DPA. From our observations and [36], L. major is the species whose FA composition shows the most differences (high 14:0; low ALA and DHA) compared to other Leishmania species. Of interest, 14:0 and DHA are among biologically active FAs in Leishmania pathogenicity (see paragraph 4) and ALA is an essential precursor for n-3 PUFA including DHA. Moreover, although the FA composition of L. major strains of human and rodent origin is globally conserved, the rodent clones show a lower quantitative abundance of LA, which is correlated with lower infectivity against macrophages [36]. Whether or not specific FAs turn out to be species biomarkers and the differences in FA composition contribute or not to parasite pathogenicity deserves further investigation.  11.2 ± 2.3 11.1 ± 0.8 9.4 ± 0.8 11.2 ± 0.7 7.6 ± 1.5 9.5 ± 1.4 10.0 ± 1.9 4.5 ± 1.0 a 3.9 ± 0.6 a 20:3n-3

Changes in FA Composition Relating to Parasite Differentiation and Drug Resistance/ Sensitivity
Some attempts have also been made to identify specific FAs as biomarkers of biological processes in Leishmania parasites.
In this respect, we showed that the FA composition of L. donovani and L. infantum lipids changed during the differentiation of the promastigote form into the amastigote form inside the host macrophages [35]. The most pronounced changes in amastigotes were the increase in total n-3 FA especially DHA in total PL, and the decrease in total n-6 FAs in all lipid classes analyzed (total PL, TG, and free FA). Noteworthily, there was a remodeling in the distribution of n-6 FA in PL with a significant increase in AA at the expense of its precursors LA and GLA which were conversely reduced. Since the FA composition of amastigotes was close to those of host cells, we have suggested that the remodeling of FAs in amastigote lipids could either depend on FA and/or desaturase/elongase activities available in macrophages or the regulation of FA biosynthesis enzymes in amastigotes.
Changes in FA compositions have been observed in several Leishmania species upon exposure to antileishmanial drugs as well as in resistant Leishmania strains. These changes have been proposed as putative mechanisms for drug toxicity or the development of drug resistance.
It is assumed that the unsaturation level of plasma membrane PL modulates membrane fluidity, which could impact drug membrane interactions and/or drug transport. A diminution of membrane fluidity has been reported in various Leishmania parasites resistant to antileishmanial drugs such as miltefosine [40], amphotericin B [41], and antimony [33,42]. In Rakotomanga et al. [40], the authors described the FA changes in miltefosine-resistant L. donovani strains. They observed a significant decrease in unsaturated alkyl chains in PL of miltefosine-resistant parasites, such as OA, AA, and ALA chains, that could result from reduced desaturation activities. It was proposed that the lower PL unsaturation in miltefosine-resistant parasites would reduce membrane fluidity and impair miltefosine affinity [43]. Decreased levels of unsaturated FAs were also reported in L. donovani strains resistant to amphotericin B compared to sensitive strains, nevertheless, the increase in membrane fluidity was rather attributed to sterol changes [41]. By contrast, FA profiles of L. donovani, L. chagasi, or L. amazonensis strains resistant to antimony revealed an increased level of unsaturation compared to sensitive strains, which was hypothesized to impair antimony transport and therefore antimony sensitivity [44,45]. Altogether, although correlations between unsaturation level and drug resistance have been reported, the data are variable depending on Leishmania strains and/or drugs.
Several studies have pointed out variations in some fatty acids associated with drug resistance. Exposure of L. donovani parasites to antimony induced a marked downregulation of OA and conversely a marked upregulation of vaccenic acid (18:1n-7) and very long-chain FAs including AA and DHA [34]. These very long-chain FAs would first be responsible for antimony-induced killing through the generation of oxidative stress in treated parasites, and secondarily causative of antimony resistance by increasing ergosterol synthesis. The resistance of L. infantum/chagasi and L. amazonensis isolates to antimony was also associated with a marked decrease in OA while AA showed the opposite trend [33,42]. Bouadid et al. [36] showed that the rodent clone of L. major with the lowest amount of LA was the least sensitive to miltefosine.
More extensive lipidomic studies have also highlighted specific changes in lipid species that could turn out to be biomarkers of drug-sensitive vs. drug-resistant Leishmania parasites. These include PE species (C19∆ or 24:0-enriched species) and inositolphosphoceramide (IPC) species in L. infantum or L. donovani strains resistant to miltefosine or amphotericin B [44,45]. Gutierrez Guarnizo et al. [46] have shown that L. tropica strains resistant to antimony exhibited a strong downregulation of PC, especially 16:1, 18:2, or 18:3-containing species, while sensitive strains strongly upregulated TG with long-chain FAs after drug exposure.

FA Acquisition in Leishmania
It is commonly admitted that Leishmania parasites acquire FAs both by de novo synthesis and by the uptake of lipids from their host environment [47].

De Novo Synthesis of FA and PUFA in Leishmania Parasites
The pathways of de novo FA synthesis in Leishmania have been extensively described in recent reviews [16,48] and will only be briefly presented here. It should be noted that comparative studies have been carried out on other trypanosomatids highlighting some specificities for Leishmania. Most of the available data come from genomic and biochemical studies in L. major, T. cruzi, and T. brucei. Several enzymes involved in de novo FA synthesis have been characterized in trypanosomatids and have emerged as potential targets for antileishmanial drugs [16].
Trypanosoma and Leishmania use a mitochondrial type II FA synthesis pathway (FAS II) that mediates the synthesis of caprylate (C8) and palmitate (C16). Three genes coding for at least two of the four enzymes involved in the FAS II pathway have been identified in L. major. Trypanosomatids also use the unconventional elongase (ELO) system to synthesize FAs [47,49]. ELO enzymes are integral membrane proteins of the endoplasmic reticulum that catalyze the extension of acyl chains. Genomes of L. major and T. brucei have genes encoding putative ELO1-3 proteins to synthesize FAs from C4 to C18. ELO1 extends C4 to C10, ELO2 extends C10 to C14, and ELO3 extends C14 to C18. Additional ELOs such as ELO4, ELO5, and ELO6 are involved in the extension of PUFAs [47,49].
Desaturases are enzymes responsible for the synthesis of unsaturated FAs and PUFAs by the insertion of double bonds in the FA carbon skeleton. Several desaturases including ∆4, ∆5, ∆6, ∆9, ∆12, and ∆15 were characterized in L. major; only ∆4, ∆9, and ∆12 desaturases were identified in T. brucei and T. cruzi [50]. Of note, as ∆12 desaturase activity is not detected in mammals, this enzyme turned out to be a potential drug target for novel therapeutics against trypanosomatids.
Cyclopropanated fatty acids (CFAs) are generated from cyclopropane fatty acid synthase (CFAS) that catalyzes the transfer of a methylene group from S-adenosyl methionine to an unsaturated FA. The gene encoding CFAS has been identified in several Leishmania species. Including L. infantum, L. donovani, L. mexicana, and L. braziliensis, but is missing in L. major [37]. CFAS has been characterized in both promastigote and amastigote forms of the parasite in L. infantum and L. mexicana [37].

FA Uptake
Several studies have suggested the presence of FA binding proteins (FABP) and FA transport protein (FATP) that would be involved in the specific uptake of FAs in leishmanial parasites [51]. Orthologues of human FATP have been identified in the genome of L. major [47]. The uptake of free FAs (PA, SA, and OA) and their esterification in glycerolipids has been described in axenic amastigotes from L. mexicana [52]. We recently showed that supplementation of the culture medium of L. infantum promastigotes with AA or DHA led to specific FA enrichment in parasite lipids indicating that promastigotes efficiently uptake exogenous FAs [53]. Specific FA enrichment of promastigote lipids was also observed after supplementation with OA (unpublished observations).
Plasma lipoproteins have been described as an important source of lipids, especially cholesterol for trypanosomatids, through internalization processes [54][55][56]. Low-density lipoprotein (LDL) is likely a source of FAs as it contains large amounts of FAs esterified in TG and cholesterol esters.

Significance of FAs in Parasite/Host Interactions and Parasite Survival in Host Cell
Several studies have investigated the role of some specific FAs in Leishmania parasites at both promastigote and amastigote stages, through the exogenous supply or inhibition of their biosynthesis. Among them, 14:0, some PUFAs and cyclopropanated FAs have emerged as essential or potential actors in Leishmania biology.

Myristic Acid (14:0) and Myristoylation
Myristic acid (14:0) is important for N-myristoylation which consists of the transfer of C14 from myristoyl-CoA onto the N terminal glycine residue of cellular proteins. This transfer is catalyzed by the enzyme N-myristoyltransferase (NMT) which has emerged as a potentially druggable enzyme in Leishmania [57]. NMT has been characterized in L. donovani and L. major and reported for both Leishmania species to be essential for cellular growth, vesicular trafficking, and survival in the mammalian host [57][58][59]. Thirty high-confidence Nmyristoylated proteins have been identified with roles in protein phosphorylation, protein transport, and degradation and Golgi functions in both promastigote and amastigote stages of L. donovani [60].
The essentiality of NMT In Leishmania viability has been demonstrated by both genetic and pharmacological approaches. Double knockout is lethal in L. donovani and L. major promastigotes [57,58]. In vivo studies using the plasmid shuffle method further demonstrated that NMT is also essential for the viability of intracellular amastigotes of L. donovani [61]. Several pharmacological NMT inhibitors have been developed that exert killing activity on Leishmania promastigotes although failing to inhibit axenic or intracellular amastigotes or show low selectivity over human NMT [57,62]. Despite low activity toward L. donovani amastigotes, Corpas-Lopez et al. have shown that the pharmacological inhibition of NMT significantly reduced parasite burden in a mouse VL model, therefore, validating NMT as a pharmacological target in Leishmania [63].
Myristate is also a component of glycosylphosphatidylinositol (GPI) lipid anchors that attach major classes of surface molecules such as promastigote surface protease (or gp123) to the plasma membrane of the Leishmania promastigote. These surface proteins with GPI anchors play a crucial role in Leishmania recognition [59].

Cyclopropanated Fatty Acids
The functions of CFA and CFAS are not totally understood and vary depending on the Leishmania species. Subcellular fractionation studies indicate that the cyclopropanated FA C19∆ mainly locates in both the endoplasmic reticulum and plasma membrane-enriched fractions in L. infantum [37]. CFAS was preferentially detected during the log and early stationary phases of promastigotes in L. infantum and L.mexicana promastigotes and in L. infantum amastigotes upon macrophage infection [37,39]. In L. infantum promastigotes, knockout of the CFAS gene lowered parasite burdens in the spleen and liver during in vivo mice infection [37]. In L. mexicana, CFA modifies the fatty acid chain of plasmenylethanolamine [39]. CFAS plays a key role in the regulation of the cellular shape of L.mexicana, its resistance to acidic environments, and to cell membrane targeting of lipophosphoglycan, but in contrast to L. infantum, is not essential for parasite virulence [39]. Increased content of C19∆ has been reported in resistance to antileishmanial drugs such as amphotericin B and miltefosine supporting its role for the pathogenicity or survival of the parasite [44].

PUFAs: LA, AA and DHA
PUFAs such as LA, AA, and DHA are known to play a key role in maintaining membrane fluidity and regulating inflammatory and oxidative status. Whether they could have an impact on parasite infectivity and survival is, therefore, an interesting but still understudied issue.
Saini and Rai [64] showed that LA supplementation of culture medium during macrophage infection with L. donovani promastigotes decreased parasite load by strength-ening macrophage inflammatory response. LA supplementation also inhibited L. donovani promastigotes from secreting exosomes containing immunomodulatory factors.
Recently, by carrying out AA supplementation on two L. infantum strains, a visceral MON-1, and a cutaneous MON-24, we showed that promastigote lipids became enriched with AA, which correlated with higher infectivity toward J774 macrophages. DHA supplementation induced DHA enrichment of lipids which was also associated with higher infectivity, although specifically for MON-24 and not the MON-1 strain [53]. We proposed that these effects could be mediated through the accumulation in supplemented promastigotes of PUFA-derived oxygenated metabolites exhibiting pro/anti-inflammatory activities (see paragraph 5).
Due to their high level of unsaturation, AA and DHA are very sensitive to peroxidation, thus promoting the production of reactive oxygen species (ROS) [65]. Likewise, exogenously added AA and DHA were shown to promote ROS production in L. donovani promastigotes [34]. Several studies have reported an increase in ROS production in either murine or human macrophages during Leishmania (L. chagasi, L. amazonensis, and L. braziliensis) infection, most likely as a defense mechanism to eliminate parasites by activating inflammatory and immune signaling pathways [66][67][68][69][70]. In our AA and DHA supplementation studies, we found that ROS production induced in macrophages upon L. infantum infection was similar for control and supplemented parasites [53]. However, since parameters other than ROS production (including antioxidant enzyme activity and vitamin E level) contribute to the fine regulation of oxidative status, we cannot exclude that infection with promastigotes supplemented with AA or DHA may actually increase oxidative stress in macrophages. Furthermore, it was recently shown that PUFAs including AA, EPA, and DHA stimulate the formation of lipid bodies (LB) in L. braziliensis and L. infantum procyclic promastigotes [71]. As mentioned below, LB has recently emerged as an important organelle in Leishmania for lipid metabolism and parasite pathogenicity.
Eicosanoids play an important role in Leishmania infection, as parasite infection results in an intense inflammatory response into host cells (macrophages) associated with an increased expression and release of proinflammatory mediators. The balance between lipid mediators, especially leukotriene B4 (LTB4) issued from 5-LOX and prostaglandin E2 (PGE2) issued from PGE2 synthase (PGE2S), determines the macrophage inflammatory response and the parasite survival. The activity of 5-LOX helps macrophages in eliminating parasite infections such as T. cruzi [75,76], L. donovani, L. amazonensis [77], and L. infantum [78]. L. major-infected neutrophils release large amounts of LTB4 during the first hours of infection [79]. LTB4 is involved in NO production and reduces parasite load in different cellular models of L. amazonensis infection [80][81][82][83][84]. With respect to PGE2, its production is induced in macrophages infected with L. infantum and L. donovani [85,86], L. amazonensis, and L. major [86] and benefits parasite survival [87]. Indeed, PGE2 promotes the growth of L. major [88] and L. donovani [77] and reduces the macrophage immune response against L. donovani [86]. In mice infected with L. mexicana and treated with a COX inhibitor, a reduction in lesion size was observed associated with reduced levels of PGE2 in splenocyte supernatants [89]. However, PGE2 was conversely reported to exert antileishmanial activity. It induced L. amazonensis or L. infantum killing in infected macrophages [86,87]. PGE2 released by macrophages under exposure to L.infantum was shown to mediate a proinflammatory response [90]. PGE2S was also highly upregulated in macrophages infected with L. donovani [91].
Besides the production of lipid mediators by infected macrophages, trypanosomatid parasites have also been shown to produce both proinflammatory and proresolving mediators, thereby modulating macrophage responses to parasite infection [71,87,[92][93][94][95] (Figure 1). These parasites possess the same classes of eicosanoids biosynthesis enzymes as mammals, including COX, LOX, and cytochrome P450 (CYP450) as well as parasite-specific enzymes [96]. Many parasites modulate host immune response through PG, but only a few COX activities have been described in trypanosomatids. PGF2α synthase (PGF2S) was identified in T. brucei [97] as well as in T. cruzi modulating the parasite infection [98]. PGF2S expression is increased during metacyclogenesis in L. infantum, L. braziliensis, and L. amazonensis [71]. In L. infantum/chagasi, the production of PGF2S is carried out in the lipid bodies [99]. The overexpression of PGF2S in L. braziliensis increased parasite virulence [100]. The metalloprotease gp63 was identified in L. mexicana as being responsible for COX2 activity in both promastigote and amastigote forms [101]. Several PGs, including PGE2, PGD2, and PGF2α, are produced by trypanosomatid parasites among which PGF2α is the dominant eicosanoid species in L. donovani, L. tropica, L. major, and T. brucei [102,103]. PGF2α produced by L. infantum is important for parasite virulence and increases the parasite burden [99]. proinflammatory and proresolving mediator profiles in noninfectious (procyclic) and infectious (metacyclic) Leishmania promastigotes, revealing that oxygenated metabolites of AA and DHA were increased in the metacyclic form, partially depending on CYP-like enzyme activities. The highest increases were shown for 5-and 8-HETE derived from AA, and 14-and 17-HDoHE derived from DHA. HdoHE are the precursors for resolvins and maresins and the authors further showed that lipid extracts of infectious promastigotes induced the production of RvD2, maresin1, and protectin Dx in host macrophages and their polarization into the M2 phenotype. We recently showed that the L. infantum promastigote was able to convert exogenous AA and DHA into active metabolites, leading to the accumulation of EET, 5-and 8-HETE, and 14-and 17-HDoHE associated with an increase in parasite infectivity [53]. Another recent study quantified the production of eicosanoids in different Leishmania species including L. infantum, L. amazonensis, and L. braziliensis in the presence of exogenous AA. Several compounds were identified, including PGE2, PGD2, PGF2α, and numerous HETEs [71]. An alternative mechanism for the generation of prostaglandin-like compounds is the nonenzymatic generation of molecules called isoprostanes. Increased oxidative stress associated with increased levels of isoprostanes was measured in sera from patients suffering from CL [105]. Specific aldo-keto reductase (AKR) was identified in T. cruzi and L. donovani, converting AA into isoprostanes, especially 8isoPGF2α [106]. The biosynthesis of AA-and DHA-derived active lipids in trypanosomatids. LOX enzymes are in blue, COX enzymes in red, and CYP450 enzymes in green. Arachidonic acid (AA) can be metabolized into thromboxanes and prostaglandins by the COX pathway; into hydroxyeicosatetraenoic acid (HETEs), leukotrienes, oxoeicosatrienoic acids (oxoETES), dihydroxyeicosatetraenoic acid (diHETEs) via the LOX pathway; into HETEs, epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acid (diHETrEs) via CYP450. Docosahexaenoic acid (DHA) can be metabolized into hydroxydocosahexaenoic acids (HDoHEs), D-resolvins (RvD1; RvD2), maresins, protectins via the LOX pathway, and hydroxydocosahexaenoic acid (HDoHEs) via the CYP pathway. 17-hydroperoxydocosahexaenoic acid (17-H(p)DoHE) issued from the COX pathway is the precursor of DHA-derived specialized proresolving mediators.

Current Treatments
The treatment of leishmaniasis in its various forms is limited to a restricted number of molecules, most of which are toxic; their administration is difficult and parasite resistance has developed. Several current therapeutics have been shown to target lipid me-  Figure 1. The biosynthesis of AA-and DHA-derived active lipids in trypanosomatids. LOX enzymes are in blue, COX enzymes in red, and CYP450 enzymes in green. Arachidonic acid (AA) can be metabolized into thromboxanes and prostaglandins by the COX pathway; into hydroxyeicosatetraenoic acid (HETEs), leukotrienes, oxoeicosatrienoic acids (oxoETES), dihydroxyeicosatetraenoic acid (diHETEs) via the LOX pathway; into HETEs, epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acid (diHETrEs) via CYP450. Docosahexaenoic acid (DHA) can be metabolized into hydroxydocosahexaenoic acids (HDoHEs), D-resolvins (RvD1; RvD2), maresins, protectins via the LOX pathway, and hydroxydocosahexaenoic acid (HDoHEs) via the CYP pathway. 17-hydroperoxydocosahexaenoic acid (17-H(p)DoHE) issued from the COX pathway is the precursor of DHA-derived specialized proresolving mediators.
Resolution of inflammation is an active process that promotes the normal function of infected tissues. Among proresolving mediators, resolvin D1 (RvD1) protects inflammatory responses and restores tissue homeostasis [103]. RvD1 administration is beneficial in Leishmania infection in the modulation of the cutaneous manifestation of the disease [87]. During T. cruzi infection, the parasite itself produces proresolving lipids such as RvD1, RvD5, and RvE2 associated with a modulation of host cell responses to infection [92]. RvD1 also favors L. amazonensis infection by promoting intracellular parasite replication in human macrophages [104]. Recently, Paloque and co-workers [94] reported the production of PUFA-derived oxygenated metabolites in L. infantum. They compared proinflammatory and proresolving mediator profiles in noninfectious (procyclic) and infectious (metacyclic) Leishmania promastigotes, revealing that oxygenated metabolites of AA and DHA were increased in the metacyclic form, partially depending on CYP-like enzyme activities. The highest increases were shown for 5-and 8-HETE derived from AA, and 14-and 17-HDoHE derived from DHA. HdoHE are the precursors for resolvins and maresins and the authors further showed that lipid extracts of infectious promastigotes induced the production of RvD2, maresin1, and protectin Dx in host macrophages and their polarization into the M2 phenotype. We recently showed that the L. infantum promastigote was able to convert exogenous AA and DHA into active metabolites, leading to the accumulation of EET, 5and 8-HETE, and 14-and 17-HDoHE associated with an increase in parasite infectivity [53]. Another recent study quantified the production of eicosanoids in different Leishmania species including L. infantum, L. amazonensis, and L. braziliensis in the presence of exogenous AA. Several compounds were identified, including PGE2, PGD2, PGF2α, and numerous HETEs [71].
An alternative mechanism for the generation of prostaglandin-like compounds is the nonenzymatic generation of molecules called isoprostanes. Increased oxidative stress associated with increased levels of isoprostanes was measured in sera from patients suffering from CL [105]. Specific aldo-keto reductase (AKR) was identified in T. cruzi and L. donovani, converting AA into isoprostanes, especially 8isoPGF2α [106].

Current Treatments
The treatment of leishmaniasis in its various forms is limited to a restricted number of molecules, most of which are toxic; their administration is difficult and parasite resistance has developed. Several current therapeutics have been shown to target lipid metabolism, as recently reviewed by Arya et al. [16]. The major treatments and their interaction with lipid metabolism are only briefly described below.
Pentavalent antimonials (SbV) are the first-line drugs used to treat visceral, cutaneous, and mucocutaneous leishmaniasis. The mechanism of action of SbV and its active trivalent form (SbIII) is not completely established but it may interfere with oxidative status and fatty acid oxidation [52,107]. Antimonials are administered intralesionally or parenterally and produce severe side effects, such as cardiotoxicity, nephrotoxicity, and hepatotoxicity. These compounds have developed serious resistance in certain endemic areas, such as in northern Bihar in India [108]. In the new world, mainly in Brazil, the effectiveness of these drugs is over 90% while in Bihar, India, and Nepal, the treatment failure is around 60%.
Amphotericin B, the second treatment against leishmaniasis, is a polyene antibiotic isolated from Streptomyces nodosus first used as an antifungal in the treatment of systemic mycoses. It is currently used by parenteral administration against severe forms including VL or forms resistant to antimonials [109]. Amphotericin B acts on both promastigotes and amastigotes by targeting ergosterol in the parasite surface membrane and increasing permeability [22]. It also stimulates the phagocytic capacities of macrophages. In the new world, mainly Brazil, Amphotericin B has been used successfully to treat VL in special conditions, including older patients, children, transplant recipients, and patients with comorbidities such as diabetes and HIV infection, but it presents strong renal and hematological toxicity. Liposomal amphotericin B is significantly less nephrotoxic while remaining very effective and is at present the treatment of choice for immunocompetent patients in the Mediterranean region and the preferred drug for HIV/visceral leishmaniasis co-infection. The main obstacle to its widespread use, especially in the world's poorest countries, is its high cost.
Miltefosine is an alkyl-lysophospholipid originally developed for the treatment of cancer that exerts antileishmanial activity by inhibiting the biosynthesis of Leishmania phospholipids and sterols [110]. It is the first oral drug available for the treatment of VL and CL and shows 95% efficacy against moderate VL in the Indian subcontinent [111]. However, its use is limited due to gastrointestinal, hepatic, renal toxicity, and teratogenic effects. Moreover, the efficiency of miltefosine is rather low in the new world, which may be due to natural resistance in the patients in this region.
Imidazoles and triazoles (ketoconazole, fluconazole, and itraconazole) are antifungal drugs mainly used against CL in the new world. They both act as inhibitors of lanosterol 14α-demethylase in Leishmania parasites [112]. Triazoles are metabolized more slowly, interfere less with the synthesis of human sterols, and are, therefore, less toxic than imidazoles [113].

Lipid Status as Prognostic/Diagnostic Biomarker for Leishmaniasis
Although not yet fully understood, nutrition may have a strong influence on the course and severity of leishmaniases. With respect to lipid supply, it was shown that in children with VL and suffering from undernutrition, the development of the disease is favored by a low-fat mass and the disease itself leads to a loss of fat mass [114]. It is now admitted that lipids and lipoproteins play an important role in host defense as well as in the infectivity of trypanosomatids.
Host cholesterol is widely recognized as a key lipid player in Leishmania infection [115]. It has been demonstrated that host cell cholesterol is mandatory for the binding, internalization, and development of Leishmania into macrophages [7,116] and required for the biogenesis of the parasitophorous vacuole [9,117]. Despite the lack of cholesterol synthesis enzymes in the parasite Leishmania, traces of cholesterol have been detected in promastigotes likely reflecting uptake from their environment. An increase in cholesterol content at the expense of ergosterol, the major sterol in Leishmania parasites, has been reported in L. infantum promastigotes during metacyclogenesis [14]. We previously showed a strong remodeling of sterols during the intramacrophagic transformation of the L. donovani and L. infantum promastigotes into the amastigote stage, with a marked enrichment in cholesterol and loss of ergosterol [35]. Altogether these observations suggest that macrophage cholesterol is a helping player in Leishmania infection.
Increased synthesis of TG and formation of LB were reported in macrophages upon infection with L. major, L. infantum, or L. donovani [118][119][120], and LB of macrophage origin were recovered in the parasitophorous vacuole [118,121]. The formation of LB was also observed in metacyclic L. infantum and L. braziliensis promastigotes and these organelles were recovered in the parasitophorous vacuole after macrophage infection. Regardless of macrophage or parasite origin, LB have recently emerged as important modulators of Leishmania pathogenicity [95,99,119].
With respect to the clinic, it was reported that VL patients had lower LDL, HDL, and total cholesterol levels compared to controls [122,123]. The reduction in serum cholesterol correlates with a high parasite load [124,125]. In mouse models (mice fed a high-cholesterol diet or Apo E-deficient mice), it was demonstrated that high circulating cholesterol levels exert protective effects against Leishmania infection [126]. In normocholesterolemic conditions, L. donovani infection induces the cholesterol depletion of macrophage membranes, which disorganizes lipid rafts and impairs host cell defense. It was shown that cholesterol replenishment via systemic liposomal cholesterol administration offers protection in hamsters infected with L. donovani [114,127]. Liposomal cholesterol delivery would help the host to outwit the Leishmania parasite by maintaining a high membrane cholesterol level and activating macrophage immune function [128].
Hypertriglyceridemia has been proposed as a prognostic/diagnostic marker in VL. Patients with VL exhibit increased TG levels at diagnosis that return to normal after VL resolution [122]. Serum TG levels were found to be significantly higher in VL than in control subjects and correlate with the severity of the disease [129,130].
Both hypocholesterolemia and hypertriglyceridemia were measured in pediatric VL patients [131]. Low HDL and elevated TG levels in patients with a mutation of lipoprotein lipase and PPAR alpha genes have been proposed as risk factors for the development of VL [131]. Feeding mice a high-sugar/high-fat diet was shown to increase parasite burden in both the liver and spleen after infection with L. infantum/chagasi [132]. High TG and low HDL levels were also measured in dogs infected with L. infantum [133,134].
It is noteworthy that the levels of cholesterol and triglycerides that impact Leishmania infectivity can both be modulated by FAs. Hypothetic mechanisms to explore would be that FAs may increase cholesterol esterification, therefore, reducing free cholesterol and limiting host cell defense. In addition, FAs may modulate TG content through synthesis/esterification/hydrolysis pathways, therefore, interfering with the formation of lipid bodies.

Conclusions and Perspectives
So far, mainly pharmacological approaches to target specific lipid enzymatic pathways in Leishmania parasites, especially involved in PL and sterol biosynthesis and myristoylation, have been used for the development of antileishmanial drugs. As concluding perspectives, we would like to highlight the potential of FAs and PUFAs to modulate parasite or host cell lipid status and consequently parasite/host cell interactions and parasite pathogenicity.
From our data and data from others cited in this review, saturated FA myristate as well as several PUFAs including LA, AA for the n-6 series, and ALA and DHA for the n-3 series appear as FAs of highest interest. Not only do these FAs exhibit changes associated with infection stages, drug sensitivity, or resistance but they are also involved in Leishmania metabolism and/or infectivity.
Developing nutritional strategies may thus be worthwhile. Interestingly, Saini et al. [135] reported that serum LA levels were decreased in patients with VL. Macrophage supplementation with LA, either preventively or postinfection, reduced the parasite load in infected macrophages. They suggest that the regular consumption of LA-rich oils in endemic regions may be a valuable strategy to control leishmaniases. In another model of parasite/host cell interaction (i.e., the freshwater crustacean Daphnia magna and its parasite Pasteuria ramosa), Scholtz et al. [136] showed that a PUFA-enriched diet or specific AA and EPA supplementation significantly reduced the likelihood of infection.
These PUFAs, especially LA, AA, and DHA, have been described as precursors of oxylipins in several Leishmania species and infected macrophages. Among them, PGE2, PGF2α, HETE, LTB4, proresolving mediators, and their precursors HDoHE, have been proposed as being involved in Leishmania infection. Only a few studies on the oxygen metabolism of PUFAs have been published to date and the characterization of the enzymes has not yet been carried out. Furthermore, the biological activities of specific PUFA metabolites issued from parasites are not well described and deserve further investigation. These lipid mediators certainly open a new and promising way to better understand the role of PUFAs in host cell/parasite interactions and bring them to the fore in therapeutic strategies.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.