Host-lipidome as a potential target of protozoan parasites

Microbes and Infection 15 (2013) 649e660 www.elsevier.com/locate/micinf

Review

Host-lipidome as a potential target of protozoan parasites Abdur Rub a,*, Mohd Arish a, Syed Akhtar Husain a, Niyaz Ahmed b, Yusuf Akhter c a

Infection and Immunity Lab, Department of Biotechnology, Jamia Millia Islamia (A Central University), New Delhi 110025, India b Department of Biotechnology and Bioinformatics, University of Hyderabad, Prof C.R. Rao Road, Hyderabad 500046, India c School of Life Sciences, Central University of Himachal Pradesh, Post Box 21, Dharamshala, Kangra 176215, H.P., India Received 24 January 2013; accepted 18 June 2013 Available online 27 June 2013

Abstract Host-lipidome caters parasite interaction by acting as first line of recognition, attachment on the cell surface, intracellular trafficking, and survival of the parasite inside the host cell. Here, we summarize how protozoan parasites exploit host-lipidome by suppressing, augmenting, engulfing, remodeling and metabolizing lipids to achieve successful parasitism inside the host. ! 2013 Institut Pasteur . Published by Elsevier Masson SAS. All rights reserved. Keywords: Host-lipidome; Protozoans; Bioactive lipids; Membrane; Enzymes

1. Introduction Protozoan parasites are among most dreadful pathogens that entail billions of fatal outcomes worldwide. Among them protozoan such as Toxoplasma, Leishmania, Plasmodium and Trypanosoma cruzi are intracellular parasites having multistage life cycle in more than one host. These protozoans are among deadliest disease causing pathogen in humans claiming millions of death annually. Protozoan such as Cryptosporidium, Entamoeba and Giardia parasitize the gastrointestinal tract, causing diarrhea, which can be fatal if left untreated. Trichomonas is one of the sexually transmitted disease causing organism that adheres to the vaginal epithelial cells causing vaginitis and inflammation. Few decades ago exploitation of host lipidome by protozoan parasites was not so explored but due to the recent advancement in the field, this fact is not much obscured. Increasing evidences demonstrated the exploitation of host lipidome by protozoan parasites. These protozoan parasites exploit host cellular lipidome which begins with the initial interaction with the host plasma membrane [1e3], till the intracellular survival and proliferation of the parasites, by not * Corresponding author. Tel.: þ91 11 26981717. E-mail addresses: [email protected], [email protected] (A. Rub).

only synthesizing enzymes targeting host lipids but also taking advantage of host’s lipid metabolizing enzymes [4]. As these parasites lacks or have incomplete de novo lipid metabolic machinery, these protozoan parasites selectively scavenge host lipids to meet their lipids requirement [5]. These different varieties of lipid precursors are either directly utilized or metabolized to complex lipids [6]. Additionally, these parasites also up-regulates or down-regulates the synthesis of various bioactive lipid mediators, which modulates the host function in the favor of parasites [7e9]. Thus, host lipidome can be considered as potential target for protozoan parasites as majority of immune evasion and survival strategies deployed by these parasites revolve around the manipulation of host lipid and lipid-derived metabolites. Hence, in this review we provide an overview on recent paradigms of the modulation of cellular functions by protozoan parasites through exploiting host lipidome for a successful parasitism inside host cell. 2. Role of host lipidome in the entry of protozoan parasites Although a variety of strategies appear to have been developed by protozoan parasites to invade host cells, increasing evidences suggested that most of these pathogens

1286-4579/$ - see front matter ! 2013 Institut Pasteur . Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.micinf.2013.06.006

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may have a common dependence on host lipidome for invasion of host cells. Protozoan parasites with an intracellular life cycle stage have developed several strategies to manipulate host lipidome for entry and immune system evasion. In most of the cases these parasites take advantage of lipid rafts which are enriched in cholesterol and sphingolipids, and have been thought to act as a platform through which parasites gain entry to host cells [10]. However in some of the cases these parasites either synthesize enzymes that may target host lipids or take advantage of host’s lipids metabolizing enzymes, which act on plasma membrane of the host to release arachidonic acids and other bioactive lipid mediators which may trigger signal transduction events in favor of the parasite. During invasion T. cruzi elicits signals which invoke the recruitment of host-cell lysosomes to the cytosolic face of the plasma membrane for fusion at the site of parasite internalization [11]. Ca2þ dependent exocytosis of recruited lysosomes results in the extracellular release of acid sphingomyelinases (aSMase) from lysosomes, that induces formation of ceramide enriched endocytic vesicles that can facilitate trypomastigotes entry into host cells (Fig. 1). Impaired lysosomal recruitment and exocytosis events reduces invasion by T. cruzi trypomastigotes as it was observed with methyl-beta cyclodextrin (bMCD) treated cardiomyocytes that depletes cholesterol from membrane and disrupts raft organization thus deregulating lysosomal exocytosis eventually leading to reduction in parasite load [12]. Host membrane cholesterol also plays important role in efficient attachment and parasite internalization as several studies demonstrated that cholesterol depletion by bMCD had a more significant inhibitory effect on the invasion

of most of the protozoan parasites [13e16]. Furthermore, the reduction in the ability of the parasite to infect host cells can be reversed upon replenishment of cell membrane cholesterol [12,14]. Cholesterol depletion from host cells prevents Plasmodium falciparum and Plasmodium yoelii entry through the CD81-dependent pathway as host cholesterol mediates the localization of CD81 into tetraspanin-enriched microdomains that facilitates Plasmodium entry [17]. Apart from entry, host cholesterol depletion also plays an important role in immune evasion strategy as Leishmania major depletes membrane cholesterol that induces alteration of CD40 signaling toward IL-10 production, which exacerbates Leishmania infection [18]. Another immune evasion strategy was discussed in case of Plasmodium during liver stage egress, where it was demonstrated that host cell death initiated by Plasmodium results in disintegration of host cell plasma membrane releasing merosomes, containing merozoites. Intact host cell membrane around merosomes allows Plasmodium to mask itself from the host immune system before establishing blood stage infection [19]. Upon entry most of the intracellular protozoan parasites is surrounded by parasite vacuolar membrane (PVM), which is derived from host cell plasma membrane. PVM formation in Toxoplasma gondii is contributed by rhoptries, cholesterolenriched parasite apical organelles, which is discharged at the time of cell entry. However, rhoptry cholesterol is not essential for entry process and in contrast, host plasma membrane cholesterol is incorporated into the forming PVM during invasion, through a caveolae-independent mechanism [20]. Additionally, ceramide generation during early

Fig. 1. Targeting host lipidome by Trypanosoma: During invasion T. cruzi elicits signals, which invoke the recruitment of host cell lysosomes to the cytosolic face of the plasma membrane for the fusion at the site of parasite internalization. Ca2þ dependent exocytosis of recruited lysosomes results in the extracellular release of ASM from lysosomes, that induces the formation of ceramide enriched endocytic vesicles that can facilitate trypomastigotes entry into the host cells. Membrane bound PLA activity of Trypanosoma significantly modified the host cell lipid profile with generation of lipid secondary messengers such as DAG and IP3 which is ultimately required for increase in Ca2þ concentration in infected cells.

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parasitophorous vacuoles formation suggested that during T. cruzi invasion, ceramide plays an important role in the plasma membrane deformation and entry process of trypomastigotes [1]. Role of ceramide generation is also recently demonstrated during Leishmania donovani internalization [2]. L. donovani promastigotes induces host aSMase activation that hydrolyzes host sphingomyelin, thereby generating ceramide-enriched membrane platforms, which are then used for the parasite internalization [2] (Fig. 2). Similar observations were earlier documented in the case of Cryptosporidium parvum entry in host epithelial cells, where C. parvum activates host aSMase to generate ceramide resulting in the recruitment of sphingolipid microdomain thereby facilitating parasite entry [21]. Protozoan parasites entry inside host cell is also followed by host membrane remodeling as it was observed during Plasmodium invasion. Plasmodium entry inside mammalian erythrocytes is followed by remodeling of phospholipids on the cytoplasmic face of the malarial vacuole. During endovacuolation of parasite phosphatidylinositol (4, 5) bisphosphate (PIP2) is excluded from the vacuole, while phosphatidylserine (PS) is detected in newly formed PVM (Fig. 3). Loss of PIP2 from the vacuole may be seen as critical as for establishment of successful erythrocytic infection [22]. Lastly, it have been also observed that some of these intracellular protozoan parasites secretes phospholipase that creates a pore in the host cell membrane, which fuses the parasite and the host membranes, or may alter the host membrane fluidity which could facilitate parasite invagination. C. parvumderived soluble phospholipase A2 (PLA2) activity has been demonstrated during parasite-host cell interactions that result in parasite invasion and intracellular development in host enterocytes [23]. Furthermore, it was reported that Plasmodium berghei sporozoites secretes phospholipase that wounds

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the cell membrane and allows the access of sporozoites through cells and enables the infection [24]. These protozoan parasites not only secrete phospholipase but also take advantage of host derived phospholipase, for example, during the T. gondii penetration both parasite and the host cell phospholipases are involved in release of arachidonic acid from host cell membrane phospholipids thereby altering host membrane fluidity and facilitating the invasion of the host cells by parasites [3].

3. Host lipid uptake by protozoan parasites Lipid metabolism has been extensively studied in protozoa parasites. Several evidences demonstrated that most of the protozoan parasites either lacks or have incomplete de novo lipid synthesis. However, some of these parasites can synthesize complex lipids by salvaging precursors from the host cell. These parasites scavenge host lipids for survival, proliferation, membrane biogenesis and for the energy requirements of the parasite. Low-density lipoprotein (LDL) is the major carrier of plasma cholesterol in humans and the main source of cholesterol for protozoa [25,26]. Human LDL is a type of lipoprotein composed of a core of triglycerides and cholesteryl esters and a shell of polar phospholipids, cholesterol and apolipoproteins [27]. It has been observed that cholesterol acquisition by protozoan parasites is mainly occurred through receptor mediated uptake of host LDL. However, there are also few reports regarding non receptor mediated endocytosis and enzymatic uptake of host lipids by these parasites. Thus, protozoan parasites scavenge lipids from their host environment not only by via receptor mediated but also through nonreceptor endocytosis and by enzymatic uptake.

Fig. 2. Manipulation of host lipidome by Leishmania: Leishmania utilizes sphingolipids and cholesterol rich membrane micro-domain for entry. Leishmania infection induces host ASM activation that hydrolyzes host sphingomyelin, generating ceramide-enriched membrane platforms, which are then used for the parasite internalization. Ceramide generation is involved in the down-regulation of protein kinase C, AP-1, NF-kB activity and NO generation, thereby facilitating the intracellular survival of Leishmania. De novo synthesis of ceramide results in the cholesterol depletion from the membrane.

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Fig. 3. Host lipid remodeling by Plasmodium: Plasmodium entry inside mammalian erythrocytes is followed by remodeling of phospholipid on the cytoplasmic face of the malarial vacuole. During endo-vacuolation of parasite phosphatidylserine (PS) is detected in newly formed PVM. P. falciparum membrane bound neutral sphingomyelinases activity hydrolyzes host sphingomyelin to produce ceramide that might regulates the progression of the cell cycle of the parasite.

Toxoplasma synthesizes most of their lipids, mostly phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI), from scavenged host cell precursors. However, presence of some of the unknown lipids, suggested the co-existence of both de novo pathway and salvage pathway in this parasite [28]. Previous studies have shown that variety of host lipids are exchange across the PVM from the host, including fatty acids, phospholipids and cholesterol, some of which are further metabolized by the parasite to ensure its survival and proliferation [6,29]. Acquisition of LDL-derived cholesterol from the host cell by Toxoplasma occurs via host LDL receptormediated endocytosis from endo-lysosomes that favors growth of the parasite [26,30] (Fig. 4). However, T. gondii growth in LDL receptor knockout (LDLr"/") mice was similar to LDLRþ/þ when mice were subjected to hypercholesterolemic diet, this data suggested that the presence of alternative receptors in cholesterol uptake which facilitates parasite growth and survival [30]. Later it was demonstrated that host P-glycoprotein, a member of the ABC transporter superfamily, is required for the transport of host cholesterol to the parasite vacuole [31] (Fig. 4). Moreover, another protein, sterol carrier protein-2 (SCP-2) was characterized in T. gondii, where it was observed that this protein plays multiple roles in uptake and metabolism of host cholesterol, fatty acid, and phospholipids [32]. Leishmania lack de novo mechanism for cholesterol synthesis and recently it was demonstrated that promastigote, amastigote and the infective metacyclic forms of Leishmania amazonensis are able to internalize human LDL as a source of cholesterol from culture medium, which involves the participation of detergent-resistant membrane lipid microdomains

[33]. Recent microarray analysis indicates that Leishmania infection in bone marrow derived macrophages of mouse perturbed the transcription of genes implicated in lipid metabolism and enhanced the expression of scavenger receptors involved in the uptake of LDL [34]. Trichomonads are unable to synthesize fatty acids de novo hence they actively uptake phospholipids, triacylglycerol and fatty acids from the culture medium which are then utilized for sphingolipids synthesis and phospholipids acylation [35]. Similar to T. gondii, Trichomonas vaginalis is also dependence on cholesterol derived from the host LDL. T. vaginalis possess surface receptors that have specificity for human HDL and LDL. Binding and uptake of host LDL by T. vaginalis is required for the growth and assembly of new membranes [36,37]. Plasmodium cannot synthesize fatty acids and cholesterol de novo hence must obtain these and other lipid components from the host cell [25]. Plasmodium parasites efficiently incorporated exogenously acquired choline and ethanolamine and metabolize them into PC and PE, respectively, via de novo Kennedy pathway [38]. However, Plasmodium also actively internalizes fatty acids and phospholipids such as PC, PE, and PS from erythrocyte membrane and human serum that is necessary for growth and survival of the parasite [39,40]. Plasmodium parasites, during intra-hepatic life cycle, are not just capable to internalize cholesterol from LDL but also from other alternative sterol sources from hepatocytes cytosol to maintain pathogenesis [25]. Giardia lamblia trophozoites are also unable to synthesize fatty acids and cholesterol de novo therefore it was suggested that this gut parasite exogenously obtains fatty acids and cholesterol from its environment. Investigators have found that these protozoans are capable of uptake of exogenous PC, PI, sphingomyelin, cholesterol,

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Fig. 4. Exploitation of host lipidome by Toxoplasma: During the T. gondii penetration both parasite and the host cell phospholipase are involved in releasing of arachidonic acid (AA) from host cell membrane phospholipids thereby altering host membrane fluidity and facilitating the invasion of the host cells by parasite. After parasite entry, the growth of the parasite is dependent on the acquisition of LDL-derived cholesterol from the host cell occurred via host LDL receptormediated endocytosis from endo-lysosomes. Host P-glycoprotein is required for the transport of host cholesterol to the parasite vacuole, which can be, utilize for cholesterol ester (CE) synthesis by ACAT activities of T. gondii. PIP2 conversion to IP3 and DAG by phospholipase C activity of Toxoplasma is ultimately required for increase in Ca2þ concentration during parasite egress.

ceramide and fatty acids from the intestinal milieu [41]. Ceramides and sphingolipids that are taken up by nonendocytic and clatherin dependent endocytic pathways are utilized for cyst wall biosynthesis during encystations process by Giardia [42]. It was further demonstrated that encysting trophozoites enhanced the expression of the gspt genes encoding the gardial serine palmitoyltransferase (gSPT) enzymes that is required for ceramide endocytosis [43]. T. cruzi depends on exogenous lipids in all developmental stages which is derived from host that plays critical roles in growth and infection [44]. Lysophosphatidylcholine (LPC) uptake is occurred through a pathway consisting of three enzymes, phospholipase A1, acyl-CoA ligase, and LPC: acylCoA acyltransferase, all of which are associated with trypanosomal plasma membrane. Acquisition of lysophospholipids (LPL) from the plasma or tissue fluid is not only important as a source of fatty acids and choline, but also contributes as energy source for the parasite [45]. There are also various reports regarding parasite-derived enzymes that utilize scavenged host lipid precursors for the synthesis of complex lipids, which can be used for membrane biogenesis. In Plasmodium there are various enzymes that are important for synthesis of phospholipids from precursor that are scavenged from the host cell, which are ultimately used for parasite’s membrane biogenesis [46e48]. In particular, phosphoethanolamine methyltransferase (PMT) and glycerol3-phosphate acyltransferases (GPT) are required for membrane biogenesis of this parasite by utilizing host lipid precursors [47,48].

4. Host lipidome as a target for parasite derived enzymes Several important milestones have been achieved in identifying factors that are critical to parasite’s virulence and the establishment of disease. Among the most widely studied of these factors are parasite-derived lipids metabolizing enzymes that targets host lipidome [Table 1]. In many of the cases, parasites engage synthesis of surface and secreted lipid metabolizing enzymes that degrade phospholipids present in host membrane thereby facilitating invasion of host cell. However, some of the enzymes are also involved in intracellular trafficking and triggering specific signaling pathways, both in the parasite and in the host cell, that are critical for establishment of pathogenesis. There have also been reports of some enzymes that take part in the membrane biogenesis of these protozoan parasites, utilizing host lipid precursors. Parasite-derived lipid metabolizing enzymes thus can play a variety of roles in invasion, survival, establishment and exacerbation of disease. 4.1. Phospholipases Phospholipases are group of enzymes that could also cleave cell membrane and intracellular phospholipids releasing a variety of products such as phosphoinositides, phosphatidic acid and secondary messenger such as LPL, free fatty acids (FFA), and diacylglycerols (DAG) [49]. Phospholipases are classified as A1, A2, C, and D, depending on the site of hydrolysis. Phospholipases A1 (PLA1) and PLA2 specifically

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Table 1 Parasite derived enzymes targeting host lipidome. Protozoan parasites

Type of enzymes

Functions

Ref.

Trypanosoma

Phospholipase

Differentiation of parasites and generation of lipid secondary messengers in host cells Ceramide production in host cell Invasion and egress of parasite Lipid droplet biogenesis in parasite Sphingolipid synthesis in parasite Host sphingolipid remodeling Growth and survival of parasite Survival and replication of parasite Ceramide production in host cell Invasion in host cell Membrane biogenesis of parasite Membrane biogenesis of parasite Invasion and intracellular development of parasite Virulence of parasite PGE2 production in host cell AA production in host cell and cytolytic activity Virulence of parasite Ceramide endocytosis by parasite Ceramide generation in parasite

[51,52]

Toxoplasma

Leishmania

Plasmodium

Cryptosporidium Entameoba Trichomonas Giardia

Ceramide synthase Phospholipase ACAT IPC synthase IPC synthase LmDAT Sphingomyelinase Sphingomyelinases Phospholipase PMT GPT Phospholipase Phospholipase COX-like enzyme Phospholipase Phospholipase gSPT Sphingomyelinase

hydrolyzes acyl group from phospholipids at sn-1 and sn-2 positions respectively, releasing FFA and LPL [49]. PLA1 and PLA2 activities have been linked to invasion in various protozoan pathogens [49], as these pathogens require enzymatic penetration and membrane disruption processes that often occur during host cell invasion. PLA thus create pore in the host cell membrane, fuse the parasite and host membranes, or alter host membrane fluidity, which could facilitate parasite entry. PLA1, PLA2 and phospholipase C (PLC) activity has been observed in membranes of T. cruzi epimastigotes [50]. Phospholipase activity that has been associated with the membranes of amastigotes and trypomastigotes is implicated in the differentiation of T. cruzi [51]. Moreover, membranebound activity of PLA1 has been demonstrated in the infective amastigotes and trypomastigotes stages of T. cruzi, which was remarkably higher with respect to the non-infective epimastigotes [52]. Additionally, it was also observed that during infection, PLA1 significantly modified the host cell lipid profile with generation of lipid secondary messengers such as DAG, FFA and LPC activating PKC signaling pathway [52]. Later, Phosphoinositide specific phospholipase C (TcPI-PLC) gene encoding a PI-PLC was characterized and it was demonstrated that TcPI-PLC gene was developmentally regulated during the differentiation of trypomastigotes into amastigotes [53], where it has been found to localize to the outer surface of the plasma membrane of amastigotes [54]. TcPI-PLC enzyme is released from the parasite during its differentiation to reach the host plasma membrane that catalyzes the hydrolysis of PIP2 to generate the second messenger inositol 1,4,5-trisphosphate (IP3) [53] (Fig. 1). Previously it was demonstrated that T. gondii invasion triggered self calcium dependent PLA which increase permeability and fluidity of host cell membrane that facilitates parasite entry [55] (Fig. 4). Moreover presence of putative PIPLC has also been suggested in this protozoan parasite which

[104] [55e57] [4] [77] [74] [67] [82] [80] [24] [47] [48] [21] [63] [99] [61,60] [62,64] [43] [43]

is required for the conversion of PIP2 to IP3 and DAG [56]. IP3 then binds to the IP3 receptor (IP3-R), present on the endoplasmic reticulum which is ultimately required for increase in Ca2þ concentration during parasite egress [57]. Another class of phospholipase, phospholipase D (PLD) is present in the parasitic protozoan L. donovani which is calcium and magnesium dependent and its activity is increased in response to acute osmotic stress [58]. However its role in the pathogenesis of Leishmania has not been claimed yet. Since no other phospholipase activity has been documented in Leishmania, therefore substantial research is needed in this direction. Presence of surface associated phospholipase was investigated in P. berghei sporozoites, where it was demonstrated that this phospholipase hydrolyzes host PC present in host cell membranes that allows access of sporozoites through cells and enables Plasmodium infection in the mammalian hosts [24]. Previously, it was observed that lysocholinephospholipids, PLC and sphingomyelinase (SMase) activities are associated with the protein encoded in the PfNSM gene of Plasmodium flaciparum that degrade host-derived lysophosphatidylcholine to supply the parasite with phosphocholine for their efficient intra-erythrocytic growth [59]. A soluble PLA2 activity has been demonstrated in C. parvum during parasite-host cell interactions that results in parasite invasion and intracellular development in host enterocytes [23]. In T. vaginalis, a lytic factor was purified that was responsible for the host cell destruction and in vitro analysis showed that this lytic factor possesses PLA2 activity. This PLA2 activity was suggested to hydrolyze the host phospholipids and releases arachidonic acid, which is then, converted to prostanoids and leukotrienes by cyclooxygenases (COX) and lipoxygenases leading to inflammation during infection [60]. Later PLA1 and PLA2 activity was also observed in the sub cellular fraction of T. vaginalis that was suggested for the hemolytic and cytolytic activity of this protozoan parasite [61]. Similarly intestinal

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parasites such as Giardia and Entamoeba histolytica also found to have PLA activity [62e64] that plays an important role in the virulence of these intestinal parasites. 4.2. Acyltransferase Acyltransferase is a type of transferase enzyme that catalyzes the transfer of an acyl group from one substance to another. Acyltransferase are involved in the fatty acid remodeling of membrane phospholipids and the metabolism of bioactive lipids in mammalian cells [65]. Increasing bodies of evidence suggested the role of this class of enzymes in the survival and proliferation of various pathogens especially those with intracellular life cycle. Phospholipid precursors and fatty acids that are scavenged from the human host are required for the membrane biogenesis of Plasmodium during asexual life cycle, initiated by glycerol3-phosphate acyltransferases [48]. In another study, homologous gene of the family membrane-bound O-acyltransferase family has been discovered in the P. falciparum genome and is suggested that it may be responsible for the production of triacylglycerols, using free fatty acids of host as substrate [66]. Leishmania expresses two acyltransferase, dihydroxyacetone phosphate acyltransferase (LmDAT) and glycerol-3-phosphate acyltransferase (LmGAT), which is required for the biosynthesis of its cellular glycerolipids using lipid precursor dihydroxyacetone phosphate and glycerol-3-phosphate respectively. LmDAT is localized in the peroxisome, important for growth, survival and essential for virulence whereas LmGAT is important for triacylglycerol synthesis but not essential for virulence [67,68]. Replication of T. gondii in its parasitophorous vacuole is dependent on the conversion of host cell acquired cholesterol [29] for lipid droplet biogenesis. Host cell derived acylCoA:cholesterol acyltransferase (ACAT) are thought to plays an essential role in the intracellular proliferation of T. gondii [69]. However, not only host derived but also endogenous ACAT activities of T. gondii are involved in the parasite’s cholesterol-ester synthesis and lipid droplet biogenesis [4]. Conversion of free cholesterol to cholesterol-ester is seen as critical step as accumulation of free cholesterol is toxic to parasite development [4]. The parasite expresses two isoforms of ACAT that differ from mammalian ACAT in their substrate affinity, specificity, and mechanism of regulation. Due to these reasons, this enzyme could be targeted for efficient antitoxoplasmosis drug therapy. 4.3. Sphingolipid synthase Sphingolipids are essential structural components of plasma membranes and found ubiquitously among pathogenic protozoan [70]. The primary complex sphingolipid in trypanosomatids is inositol phosphorylceramide (IPC) which is synthesized by IPC synthase that is detectable in the pathogenic stages of all the Leishmania and Trypanosoma sps. [70]. IPC synthase catalyzes the transfer of an inositol phosphate group from phosphatidylinositol to the 1-hydroxyl group of

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ceramide releasing DAG as a by-product [71]. However major sphingolipid in Plasmodium is sphingomyelin that is synthesized by the activity of endogenous sphingomyelin synthase (SMS) utilizing host lipid precursors [70]. During intra-macrophage life cycle amastigotes of Leishmania expresses active IPC synthase that remodels acquired host sphingolipid into IPC [72]. Indeed it has been shown that L. donovani stimulates host macrophages to up-regulate the production of ceramide, a precursor of IPC and a substrate of IPC synthase [73]. Later an enzyme, LmIPCS was isolated in L. major using bioinformatics and functional genetic approaches and it was demonstrated that this enzyme possess IPC synthase activity [71]. Recently, active IPC synthase was also reported in Leishmania mexicana that synthesizes the primary complex sphingolipid IPC utilizing host sphingolipid [74]. IPC synthase activity has also been characterized in T. cruzi [75] where it has been suggested that IPC synthesis is important for infectivity and transformation of the plasma membrane, a necessary step in the differentiation of trypomastigotes to amastigotes [51]. T. gondii has been demonstrated to synthesize complex sphingolipids de novo [76], the identification of the enzymes responsible for this have remained ambiguous until recently, SL synthase was identified in T. gondii that demonstrated IPC synthase activity [77]. Further investigation is necessary to validate whether de novo synthesis of IPC in T. gondii required host precursor or not. Plasmodium infected erythrocytes contains high activity of SMS while uninfected erythrocytes contain no detectable SMS activity, hence it was suggested that this SMS could be of parasitic origin [78]. Interestingly, SMS was found to be actively present in both the intra-erythrocytic parasite and in extracellular merozoites stages. However, in the intracellular ring and trophozoite stages the parasite exports a fraction of the activity beyond the parasite plasma membrane [79]. Further it was observed that P. falciparum membrane bound neutral sphingomyelinases (nSMase) activity hydrolyze host sphingomyelin to produce ceramide, which could be a source of the re-synthesis of sphingomylein by a plasmodial SMS [80]. 4.4. Sphingomyelinase Sphingomyelinase (SMase) is a principal enzyme catalyzing the hydrolysis of SM to ceramide and phosphocholine. There are five main types of SMases; the acidic Zn2þ-dependent and independent, the neutral Mg2þ-dependent and independent and lastly the secreted alkaline SMase [81]. Various reports are documented regarding the activation of host SMase upon infection that causes ceramide-mediated entry of intracellular pathogens [1,2]. However increasing evidence demonstrated that apart from utilizing host SMase, these pathogens also secretes endogenous SMase to release ceramide on the cell surface, that is required for the invasion process [80]. The presence of a single Mg2þ dependent, membrane bound nSMase activity in P. falciparum was reported. It was suggested that nSMase may be secreted to the erythrocyte membrane to hydrolyze sphingomyelin for the production of

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ceramide that might modulate the progression of the cell cycle of the parasite [80]. Another class of SMase that is expressed by P. falciparum functions as a sphingomyelin/ lysocholinephospholipid-phospholipase C and has been shown to be required for the infection of erythrocytes with P. falciparum. Inhibition of this enzyme prevented intraerythrocytic proliferation [59], suggesting that this enzyme might alter the composition of host lipid rafts to permit infection. There are also various observations regarding the presence of endogenous SMase activity in another intracellular protozoan parasite Leishmania. L. major promastigotes possess a potent SMase activity, which is dependent on the Inositol phospho-sphingolipid phospholipase C-Like (ISCL) protein. Although, ISCL can also degrade IPC, its activity with sphingomyelin is 10e20-fold greater than that of IPC [82]. Additionally, L. amazonensis amastigotes express SMase that is essential for amastigote survival and replication in the mammalian host [82]. Degradation of host sphingomyelin into ceramide is a necessary step in the formation of IPC and acidic tolerance that could promote parasite entry, survival and proliferation. These findings strongly implicate that endogenous SMase may play a pivotal role in establishing infection in Leishmania in the mammalian host. Recent studies showed that intestinal parasites such as Giardia and Entamoeba genome contain putative SMase encoding genes. G. lamblia possess endogenous SMase activity that is encoded by gsmaseB and gsmase3b whose transcription were elevated during encystation. Therefore it was suggested that both cytoplasmic and secreted SMases are involved in degradation of sphingomyelin from the dietary components in intestine for the generation of excess ceramide that is further required for encystation process [43]. Soluble and membrane associated neutral SMase-C activity was in vitro identified and characterized in E. histolytica trophozoites. It was further postulated that this soluble and membrane associated SMase activity might be essential for the virulence and sphingolipid metabolism in this protozoan parasite [83]. 5. Manipulation of host lipid-derived signaling mediators by protozoan parasites Apart from just exploiting, scavenging and remodeling of host lipidome, these intracellular parasites also control the release of bioactive lipid molecules, which modulates the course of infection. Several bioactive lipid mediators are released during the course of infection and results in exacerbation of the disease. In contrast, some of the bioactive lipid mediators play an important role in the host defense during the infection hence these parasite down-regulates their synthesis in order to survive and proliferate. 5.1. Eicosanoids Eicosanoids are produced by several parasitic organisms which are considered as potent regulators of host immune

responses [84]. T. gondii tachyzoites markedly alters the release of eicosanoids, in particular 5-1ipoxygenase arachidonic acid, by human mononuclear phagocytes. Release of bioactive lipids such as thromboxane and leukotrienes LTB4 is important for toxoplasmacidal activity and provide host defense [85,86], hence T. gondii tachyzoites deregulates 51ipoxygenase pathway in human monocytes and suppress activation of this pathway in monocyte derived macrophages which is important for the survival of these pathogens [9]. Thromboxane TXA2 and TXB2 levels are elevated in mice infected with T. cruzi [87]. In particular, TXA2 are released during all life stages of T. cruzi and is considered as an important modulator of survival and disease progression in host [87]. In vivo study in T. gondii infected mice showed that T. gondii induced Lipoxin LXA4 production that suppresses the IL-12 production by dendritic cells. Hence it was suggested that LXA4 plays a major host protective role in preventing parasite-induced inflammation and mortality [88]. Intestinal protozoan parasite E. histolytica infection in macrophages results in alteration of arachidonic acid metabolism leading to eicosanoids formation through cyclooxygenase (COX) and lipoxygenase (LOX) pathways [89]. 5.2. Prostaglandins Prostaglandins (PG) are class of bioactive lipid mediators that are produced by arachidonic acid metabolism through the action of COX-1, COX-2 and PG synthase. It is a well-known fact that several protozoan parasites increase the amount and synthesis of prostaglandins in the host cell during infection [90e92]. Earlier studies on murine splenic mononuclear cells and peritoneal macrophages infected with L. donovani have shown increased COX and LOX activities which results in more prostaglandin E2 (PGE2) and other arachidonic acid metabolite production [93]. It has been also reported that Leishmania infection initiated in vivo PGE2 production that it may favor Leishmania persistence and progression [94]. Initial contact of L. donovani with the host cell may induce a rapid activation of the PKC pathway, thus increasing COX-2 activity and consequently up-regulating PGE2 release [90]. T. cruzi induces an anti-inflammatory response through activation of prostaglandins, specifically, PGE2 [91]. Similarly, T. gondii also induces PGE2 biosynthesis in RAW264.7 macrophages by regulation of arachidonic acid production and induction of COX-2 expression by a PKC-dependent pathway [8]. P. falciparum infected RBC produces significant amount of PG D2, E2, and F2a that is suggested to modulates the host defense mechanism by lowering the host TNF-a production [95], that limits malarial parasitemia but also exacerbate pathogenesis at high concentrations. Intriguingly, the suppression of COX-2 derived PGE production in peripheral blood mononuclear cells by malarial pigment, hemozoin, was demonstrated that resulted in overproduction of TNF-a, leading to the development of malarial anemia [7]. Intestinal parasite E. histolytica is known to produce and secrete PGE2. Earlier studies have shown that E. histolytica stimulates host cells such as macrophages, colonic epithelial

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cells and polymorphonuclear cells to produce high levels of PGE2 that can modulate macrophage functions by cytokine production and also alters tight junction permeability colonic epithelial cells by a PGE2 mechanism [92,96e98]. This increment in PGE2 production was due to increase of COX-2 mRNA expression [92,96]. PGE2 results in the production of IL-8, which supports acute inflammation associated with intestinal amebiasis [97]. COX-like enzyme in E. histolytica was isolated and characterized that was responsible for the biosynthesis of PGE2 utilizing exogenous arachidonic acid substrates [99]. These observations suggested that E. histolytica is not only enhancing the expression of prostaglandins but also synthesizes the bioactive lipid mediator that plays an important role in modulating host immune response during infection. 5.3. Ceramide Ceramide is a family of lipid molecules that are produced in cells either by the de novo synthesis or by hydrolysis of complex sphingolipids. L. donovani induced immunesuppression and modulation of host cell signaling that is also mediated by ceramide generation. Different studies conducted have proved that ceramide is involved in the downregulation of protein kinase C, dephosphorylation of Akt and suppression of ERK activation, AP-1, NF-kB activity and NO generation, thereby facilitating the intracellular survival of L. donovani [73,100,101]. Recently, biphasic generation of ceramide during Leishmania infection is also discussed. During the first phase ceramide is generated from activation of acid sphingomyelinase which aids in parasite internalization and during second phase ceramide is generated by de novo synthesis which results in the depletion of cholesterol from the membrane leading to impairment of antigen presentation to the T cells [2]. Role of ceramide generation also has studied in Giardia, where it has been suggested that excess ceramide generation is necessary for the encystations process. This intestinal protozoa lacks de novo synthesis of ceramide hence it is selectively up-taken from the intestinal milieu [42]. However, it also possesses enzymes those are required for the generation of excess ceramide by expression of SMases that hydrolyze host sphingomyelin into ceramide [43]. Similarly, P. falciparum lacks de novo synthesis of ceramide, therefore it has been suggested that plasmodial nSMase activity hydrolyzes sphingomyelin present in erythrocyte membrane to produce ceramide. This ceramide production has been suggested for the re-synthesis of sphingomyelin or to modulate the progression of the cell cycle of the parasite [80]. Earlier studies have demonstrated the presence of free ceramide in all stages of T. cruzi [102]. Later it was demonstrated that ceramide containing glycolipids from T. cruzi synergizes with the host cytokine IFN-g to induce macrophage apoptosis that release viable parasites. This pro-apoptotic activity of T. cruzi derived by ceramide was suggested as virulence mechanism to spread disease [103]. Ceramide generation is also required for the differentiation and pathogenesis of T. cruzi and membrane associated enzymes such as

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PLA1, PLA2, inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and phospholipase C of T. cruzi amastigotes and trypomastigotes stages are involved in releasing ceramide [51]. Recently, T. cruzi ceramide synthase (TcCERS1) gene was identified that putatively encodes ceramide synthase activity where it was suggested that this enzymes might be involved in ceramide synthesis using exogenous sources of sphingolipids from host cell [104]. 6. Conclusion It is quite clear from the above thesis that the protozoan parasites sabotage the host cellular functions by exploiting host lipidome in various ways. These protozoans not just directly acquire host lipids as energy source but also manipulate it for their invasion, intra-cellular survival, proliferation and subsequent progression of pathogenesis. Therefore, host lipidome can be considered as a key player in the hosteparasite interaction that plays important roles in shaping the nature and severity of a parasitic infection. Since the current state of art in this area is still evolving, concurrent efforts to mechanistically unravel the lipidome exploitation pathways operating at the base of human parasitic infections caused by protozoa. There is a need for comprehensive transcriptomic, functional genomics and metabolic approaches to explore novel gene functions entailing lipidmetabolizing enzymes in the genome of protozoan parasites, as most of such putative enzymes/functions have not been identified and characterized in detail. Moreover, the metabolic machineries for the synthesis of phospholipids and fatty acids in these parasites have generated significant interest due to their importance in survival, growth and proliferation during various stages of a parasitic life cycle. Recent observations concerning the metabolism of host lipidome by these parasites via unusual lipid metabolizing enzymes that are not present in mammalian host come with a notion that these metabolic enzymes may form potential drug targets. With the advancements made during the last few years in drug designing, it would be possible to target these metabolic enzymes for the development of novel therapeutic strategies. Acknowledgments The authors are grateful to Mr. Atahar Husein for helping in designing of figures. M. A. is financially supported by UGC Govt. of India. References [1] M.C. Fernandes, M. Cortez, A.R. Flannery, C. Tam, R.A. Mortara, N.W. Andrews, Trypanosoma cruzi subverts the sphingomyelinasemediated plasma membrane repair pathway for cell invasion, J. Exp. Med. 208 (2011) 909e921. [2] S. Majumder, R. Dey, S. Bhattacharjee, A. Rub, G. Gupta, S. Bhattacharyya Majumdar, B. Saha, S. Majumdar, Leishmaniainduced biphasic ceramide generation in macrophages is crucial for uptake and survival of the parasite, J. Infect. Dis. 205 (2012) 1607e1616.

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