Host-lipidome as a potential target of protozoan parasites

Microbes and Infection 15 (2013) 649e660www.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, IndiabDepartment of Biotechnology and Bioinformatics, University of Hyderabad, Prof C.R. Rao Road, Hyderabad 500046, Indiac 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, andsurvival 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 pathogensthat entail billions of fatal outcomes worldwide. Among themprotozoan such as Toxoplasma, Leishmania, Plasmodium andTrypanosoma cruzi are intracellular parasites having multi-stage life cycle in more than one host. These protozoans areamong deadliest disease causing pathogen in humans claimingmillions of death annually. Protozoan such as Cryptospo-ridium, Entamoeba and Giardia parasitize the gastrointestinaltract, causing diarrhea, which can be fatal if left untreated.Trichomonas is one of the sexually transmitted disease causingorganism that adheres to the vaginal epithelial cells causingvaginitis and inflammation.

Few decades ago exploitation of host lipidome by proto-zoan parasites was not so explored but due to the recentadvancement in the field, this fact is not much obscured.Increasing evidences demonstrated the exploitation of hostlipidome by protozoan parasites. These protozoan parasitesexploit host cellular lipidome which begins with the initialinteraction with the host plasma membrane [1e3], till theintracellular survival and proliferation of the parasites, by not

* Corresponding author. Tel.: þ91 11 26981717.

E-mail addresses: [email protected], [email protected] (A. Rub).

1286-4579/$ - see front matter � 2013 Institut Pasteur . Published by Elsevier M

http://dx.doi.org/10.1016/j.micinf.2013.06.006

only synthesizing enzymes targeting host lipids but also takingadvantage of host’s lipid metabolizing enzymes [4]. As theseparasites lacks or have incomplete de novo lipid metabolicmachinery, these protozoan parasites selectively scavenge hostlipids to meet their lipids requirement [5]. These differentvarieties of lipid precursors are either directly utilized ormetabolized to complex lipids [6]. Additionally, these para-sites also up-regulates or down-regulates the synthesis ofvarious bioactive lipid mediators, which modulates the hostfunction in the favor of parasites [7e9].

Thus, host lipidome can be considered as potential targetfor protozoan parasites as majority of immune evasion andsurvival strategies deployed by these parasites revolve aroundthe manipulation of host lipid and lipid-derived metabolites.Hence, in this review we provide an overview on recent par-adigms of the modulation of cellular functions by protozoanparasites through exploiting host lipidome for a successfulparasitism inside host cell.

2. Role of host lipidome in the entry of protozoanparasites

Although a variety of strategies appear to have beendeveloped by protozoan parasites to invade host cells,increasing evidences suggested that most of these pathogens

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650 A. Rub et al. / Microbes and Infection 15 (2013) 649e660

may have a common dependence on host lipidome for invasionof host cells. Protozoan parasites with an intracellular lifecycle stage have developed several strategies to manipulatehost lipidome for entry and immune system evasion. In mostof the cases these parasites take advantage of lipid rafts whichare enriched in cholesterol and sphingolipids, and have beenthought to act as a platform through which parasites gain entryto host cells [10]. However in some of the cases these parasiteseither synthesize enzymes that may target host lipids or takeadvantage of host’s lipids metabolizing enzymes, which act onplasma membrane of the host to release arachidonic acids andother bioactive lipid mediators which may trigger signaltransduction events in favor of the parasite.

During invasion T. cruzi elicits signals which invoke therecruitment of host-cell lysosomes to the cytosolic face of theplasma membrane for fusion at the site of parasite internali-zation [11]. Ca2þ dependent exocytosis of recruited lysosomesresults in the extracellular release of acid sphingomyelinases(aSMase) from lysosomes, that induces formation of ceramideenriched endocytic vesicles that can facilitate trypomastigotesentry into host cells (Fig. 1). Impaired lysosomal recruitmentand exocytosis events reduces invasion by T. cruzi trypomas-tigotes as it was observed with methyl-beta cyclodextrin (b-MCD) treated cardiomyocytes that depletes cholesterol frommembrane and disrupts raft organization thus deregulatinglysosomal exocytosis eventually leading to reduction in para-site load [12]. Host membrane cholesterol also plays importantrole in efficient attachment and parasite internalization asseveral studies demonstrated that cholesterol depletion by b-MCD had a more significant inhibitory effect on the invasion

Fig. 1. Targeting host lipidome by Trypanosoma: During invasion T. cruzi elicits sig

of the plasma membrane for the fusion at the site of parasite internalization. Ca2þ de

ASM from lysosomes, that induces the formation of ceramide enriched endocytic v

bound PLA activity of Trypanosoma significantly modified the host cell lipid profile

ultimately required for increase in Ca2þ concentration in infected cells.

of most of the protozoan parasites [13e16]. Furthermore, thereduction in the ability of the parasite to infect host cells canbe reversed upon replenishment of cell membrane cholesterol[12,14]. Cholesterol depletion from host cells prevents Plas-modium falciparum and Plasmodium yoelii entry through theCD81-dependent pathway as host cholesterol mediates thelocalization of CD81 into tetraspanin-enriched microdomainsthat facilitates Plasmodium entry [17]. Apart from entry, hostcholesterol depletion also plays an important role in immuneevasion strategy as Leishmania major depletes membranecholesterol that induces alteration of CD40 signaling towardIL-10 production, which exacerbates Leishmania infection[18]. Another immune evasion strategy was discussed in caseof Plasmodium during liver stage egress, where it wasdemonstrated that host cell death initiated by Plasmodiumresults in disintegration of host cell plasma membranereleasing merosomes, containing merozoites. Intact host cellmembrane around merosomes allows Plasmodium to maskitself from the host immune system before establishing bloodstage infection [19].

Upon entry most of the intracellular protozoan parasites issurrounded by parasite vacuolar membrane (PVM), which isderived from host cell plasma membrane. PVM formation inToxoplasma gondii is contributed by rhoptries, cholesterol-enriched parasite apical organelles, which is discharged atthe time of cell entry. However, rhoptry cholesterol is notessential for entry process and in contrast, host plasmamembrane cholesterol is incorporated into the forming PVMduring invasion, through a caveolae-independent mechanism[20]. Additionally, ceramide generation during early

nals, which invoke the recruitment of host cell lysosomes to the cytosolic face

pendent exocytosis of recruited lysosomes results in the extracellular release of

esicles that can facilitate trypomastigotes entry into the host cells. Membrane

with generation of lipid secondary messengers such as DAG and IP3 which is

651A. Rub et al. / Microbes and Infection 15 (2013) 649e660

parasitophorous vacuoles formation suggested that during T.cruzi invasion, ceramide plays an important role in the plasmamembrane deformation and entry process of trypomastigotes[1]. Role of ceramide generation is also recently demonstratedduring Leishmania donovani internalization [2]. L. donovanipromastigotes induces host aSMase activation that hydrolyzeshost sphingomyelin, thereby generating ceramide-enrichedmembrane platforms, which are then used for the parasiteinternalization [2] (Fig. 2). Similar observations were earlierdocumented in the case of Cryptosporidium parvum entry inhost epithelial cells, where C. parvum activates host aSMase togenerate ceramide resulting in the recruitment of sphingolipidmicrodomain thereby facilitating parasite entry [21].

Protozoan parasites entry inside host cell is also followedby host membrane remodeling as it was observed duringPlasmodium invasion. Plasmodium entry inside mammalianerythrocytes is followed by remodeling of phospholipids onthe cytoplasmic face of the malarial vacuole. During endo-vacuolation of parasite phosphatidylinositol (4, 5) bisphos-phate (PIP2) is excluded from the vacuole, while phosphati-dylserine (PS) is detected in newly formed PVM (Fig. 3). Lossof PIP2 from the vacuole may be seen as critical as forestablishment of successful erythrocytic infection [22]. Lastly,it have been also observed that some of these intracellularprotozoan parasites secretes phospholipase that creates a porein the host cell membrane, which fuses the parasite and thehost membranes, or may alter the host membrane fluiditywhich could facilitate parasite invagination. C. parvum-derived soluble phospholipase A2 (PLA2) activity has beendemonstrated during parasite-host cell interactions that resultin parasite invasion and intracellular development in hostenterocytes [23]. Furthermore, it was reported that Plasmo-dium berghei sporozoites secretes phospholipase that wounds

Fig. 2. Manipulation of host lipidome by Leishmania: Leishmania utilizes sphin

infection induces host ASM activation that hydrolyzes host sphingomyelin, generatin

internalization. Ceramide generation is involved in the down-regulation of protein

intracellular survival of Leishmania. De novo synthesis of ceramide results in the

the cell membrane and allows the access of sporozoitesthrough cells and enables the infection [24]. These protozoanparasites not only secrete phospholipase but also take advan-tage of host derived phospholipase, for example, during the T.gondii penetration both parasite and the host cell phospholi-pases are involved in release of arachidonic acid from host cellmembrane phospholipids thereby altering host membranefluidity and facilitating the invasion of the host cells by par-asites [3].

3. Host lipid uptake by protozoan parasites

Lipid metabolism has been extensively studied in protozoaparasites. Several evidences demonstrated that most of theprotozoan parasites either lacks or have incomplete de novolipid synthesis. However, some of these parasites can synthe-size 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 theparasite. Low-density lipoprotein (LDL) is the major carrier ofplasma cholesterol in humans and the main source ofcholesterol for protozoa [25,26]. Human LDL is a type of li-poprotein composed of a core of triglycerides and cholesterylesters and a shell of polar phospholipids, cholesterol andapolipoproteins [27]. It has been observed that cholesterolacquisition by protozoan parasites is mainly occurred throughreceptor mediated uptake of host LDL. However, there are alsofew reports regarding non receptor mediated endocytosis andenzymatic uptake of host lipids by these parasites. Thus,protozoan parasites scavenge lipids from their host environ-ment not only by via receptor mediated but also through non-receptor endocytosis and by enzymatic uptake.

golipids and cholesterol rich membrane micro-domain for entry. Leishmania

g ceramide-enriched membrane platforms, which are then used for the parasite

kinase C, AP-1, NF-kB activity and NO generation, thereby facilitating the

cholesterol depletion from the membrane.

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 phos-phatidylcholine (PC), phosphatidylethanolamine (PE), phos-phatidylserine (PS), and phosphatidylinositol (PI), fromscavenged host cell precursors. However, presence of some ofthe unknown lipids, suggested the co-existence of both denovo pathway and salvage pathway in this parasite [28]. Pre-vious studies have shown that variety of host lipids are ex-change across the PVM from the host, including fatty acids,phospholipids and cholesterol, some of which are furthermetabolized by the parasite to ensure its survival and prolif-eration [6,29]. Acquisition of LDL-derived cholesterol fromthe host cell by Toxoplasma occurs via host LDL receptor-mediated endocytosis from endo-lysosomes that favorsgrowth of the parasite [26,30] (Fig. 4). However, T. gondiigrowth in LDL receptor knockout (LDLr�/�) mice was similarto LDLRþ/þ when mice were subjected to hypercholesterol-emic diet, this data suggested that the presence of alternativereceptors in cholesterol uptake which facilitates parasitegrowth and survival [30]. Later it was demonstrated that hostP-glycoprotein, a member of the ABC transporter super-family, is required for the transport of host cholesterol to theparasite 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 inuptake and metabolism of host cholesterol, fatty acid, andphospholipids [32].

Leishmania lack de novo mechanism for cholesterol syn-thesis and recently it was demonstrated that promastigote,amastigote and the infective metacyclic forms of Leishmaniaamazonensis are able to internalize human LDL as a source ofcholesterol from culture medium, which involves the partici-pation of detergent-resistant membrane lipid microdomains

[33]. Recent microarray analysis indicates that Leishmaniainfection in bone marrow derived macrophages of mouseperturbed the transcription of genes implicated in lipid meta-bolism and enhanced the expression of scavenger receptorsinvolved in the uptake of LDL [34]. Trichomonads are unableto synthesize fatty acids de novo hence they actively uptakephospholipids, triacylglycerol and fatty acids from the culturemedium which are then utilized for sphingolipids synthesisand phospholipids acylation [35]. Similar to T. gondii, Tri-chomonas vaginalis is also dependence on cholesterol derivedfrom the host LDL. T. vaginalis possess surface receptors thathave specificity for human HDL and LDL. Binding and uptakeof host LDL by T. vaginalis is required for the growth andassembly of new membranes [36,37].

Plasmodium cannot synthesize fatty acids and cholesterolde novo hence must obtain these and other lipid componentsfrom the host cell [25]. Plasmodium parasites efficientlyincorporated exogenously acquired choline and ethanolamineand metabolize them into PC and PE, respectively, via de novoKennedy pathway [38]. However, Plasmodium also activelyinternalizes fatty acids and phospholipids such as PC, PE, andPS from erythrocyte membrane and human serum that isnecessary for growth and survival of the parasite [39,40].Plasmodium parasites, during intra-hepatic life cycle, are notjust capable to internalize cholesterol from LDL but also fromother alternative sterol sources from hepatocytes cytosol tomaintain pathogenesis [25]. Giardia lamblia trophozoites arealso unable to synthesize fatty acids and cholesterol de novotherefore it was suggested that this gut parasite exogenouslyobtains fatty acids and cholesterol from its environment. In-vestigators have found that these protozoans are capable ofuptake of exogenous PC, PI, sphingomyelin, cholesterol,

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 receptor-

mediated 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 non-endocytic and clatherin dependent endocytic pathways areutilized for cyst wall biosynthesis during encystations processby Giardia [42]. It was further demonstrated that encystingtrophozoites enhanced the expression of the gspt genesencoding the gardial serine palmitoyltransferase (gSPT) en-zymes that is required for ceramide endocytosis [43].

T. cruzi depends on exogenous lipids in all developmentalstages which is derived from host that plays critical roles ingrowth and infection [44]. Lysophosphatidylcholine (LPC)uptake is occurred through a pathway consisting of three en-zymes, phospholipase A1, acyl-CoA ligase, and LPC: acyl-CoA acyltransferase, all of which are associated withtrypanosomal plasma membrane. Acquisition of lysophos-pholipids (LPL) from the plasma or tissue fluid is not onlyimportant as a source of fatty acids and choline, but alsocontributes as energy source for the parasite [45]. There arealso various reports regarding parasite-derived enzymes thatutilize scavenged host lipid precursors for the synthesis ofcomplex lipids, which can be used for membrane biogenesis.In Plasmodium there are various enzymes that are importantfor synthesis of phospholipids from precursor that are scav-enged from the host cell, which are ultimately used for para-site’s membrane biogenesis [46e48]. In particular,phosphoethanolamine methyltransferase (PMT) and glycerol-3-phosphate acyltransferases (GPT) are required for mem-brane biogenesis of this parasite by utilizing host lipid pre-cursors [47,48].

4. Host lipidome as a target for parasite derived enzymes

Several important milestones have been achieved in iden-tifying factors that are critical to parasite’s virulence and theestablishment of disease. Among the most widely studied ofthese factors are parasite-derived lipids metabolizing enzymesthat targets host lipidome [Table 1]. In many of the cases,parasites engage synthesis of surface and secreted lipidmetabolizing enzymes that degrade phospholipids present inhost membrane thereby facilitating invasion of host cell.However, some of the enzymes are also involved in intracel-lular trafficking and triggering specific signaling pathways,both in the parasite and in the host cell, that are critical forestablishment of pathogenesis. There have also been reports ofsome enzymes that take part in the membrane biogenesis ofthese protozoan parasites, utilizing host lipid precursors.Parasite-derived lipid metabolizing enzymes thus can play avariety of roles in invasion, survival, establishment andexacerbation of disease.

4.1. Phospholipases

Phospholipases are group of enzymes that could also cleavecell membrane and intracellular phospholipids releasing avariety of products such as phosphoinositides, phosphatidicacid and secondary messenger such as LPL, free fatty acids(FFA), and diacylglycerols (DAG) [49]. Phospholipases areclassified as A1, A2, C, and D, depending on the site of hy-drolysis. Phospholipases A1 (PLA1) and PLA2 specifically

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

[51,52]

Ceramide synthase Ceramide production in host cell [104]

Toxoplasma Phospholipase Invasion and egress of parasite [55e57]

ACAT Lipid droplet biogenesis in parasite [4]

IPC synthase Sphingolipid synthesis in parasite [77]

Leishmania IPC synthase Host sphingolipid remodeling [74]

LmDAT Growth and survival of parasite [67]

Sphingomyelinase Survival and replication of parasite [82]

Plasmodium Sphingomyelinases Ceramide production in host cell [80]

Phospholipase Invasion in host cell [24]

PMT Membrane biogenesis of parasite [47]

GPT Membrane biogenesis of parasite [48]

Cryptosporidium Phospholipase Invasion and intracellular development of parasite [21]

Entameoba Phospholipase Virulence of parasite [63]

COX-like enzyme PGE2 production in host cell [99]

Trichomonas Phospholipase AA production in host cell and cytolytic activity [61,60]

Giardia Phospholipase Virulence of parasite [62,64]

gSPT Ceramide endocytosis by parasite [43]

Sphingomyelinase Ceramide generation in parasite [43]


hydrolyzes acyl group from phospholipids at sn-1 and sn-2positions respectively, releasing FFA and LPL [49]. PLA1 andPLA2 activities have been linked to invasion in various pro-tozoan pathogens [49], as these pathogens require enzymaticpenetration and membrane disruption processes that oftenoccur during host cell invasion. PLA thus create pore in thehost cell membrane, fuse the parasite and host membranes, oralter host membrane fluidity, which could facilitate parasiteentry. PLA1, PLA2 and phospholipase C (PLC) activity hasbeen observed in membranes of T. cruzi epimastigotes [50].Phospholipase activity that has been associated with themembranes of amastigotes and trypomastigotes is implicatedin the differentiation of T. cruzi [51]. Moreover, membrane-bound activity of PLA1 has been demonstrated in the infec-tive amastigotes and trypomastigotes stages of T. cruzi, whichwas remarkably higher with respect to the non-infective epi-mastigotes [52]. Additionally, it was also observed that duringinfection, PLA1 significantly modified the host cell lipidprofile with generation of lipid secondary messengers such asDAG, FFA and LPC activating PKC signaling pathway [52].Later, Phosphoinositide specific phospholipase C (TcPI-PLC)gene encoding a PI-PLC was characterized and it wasdemonstrated that TcPI-PLC gene was developmentallyregulated during the differentiation of trypomastigotes intoamastigotes [53], where it has been found to localize to theouter surface of the plasma membrane of amastigotes [54].TcPI-PLC enzyme is released from the parasite during itsdifferentiation to reach the host plasma membrane that cata-lyzes the hydrolysis of PIP2 to generate the second messengerinositol 1,4,5-trisphosphate (IP3) [53] (Fig. 1).

Previously it was demonstrated that T. gondii invasiontriggered self calcium dependent PLA which increase perme-ability and fluidity of host cell membrane that facilitatesparasite entry [55] (Fig. 4). Moreover presence of putative PI-PLC has also been suggested in this protozoan parasite which

is required for the conversion of PIP2 to IP3 and DAG [56]. IP3then binds to the IP3 receptor (IP3-R), present on the endo-plasmic reticulum which is ultimately required for increase inCa2þ concentration during parasite egress [57]. Another classof phospholipase, phospholipase D (PLD) is present in theparasitic protozoan L. donovani which is calcium and mag-nesium dependent and its activity is increased in response toacute osmotic stress [58]. However its role in the pathogenesisof Leishmania has not been claimed yet. Since no otherphospholipase activity has been documented in Leishmania,therefore substantial research is needed in this direction.

Presence of surface associated phospholipase was investi-gated in P. berghei sporozoites, where it was demonstrated thatthis phospholipase hydrolyzes host PC present in host cellmembranes that allows access of sporozoites through cells andenables Plasmodium infection in the mammalian hosts [24].Previously, it was observed that lysocholinephospholipids,PLC and sphingomyelinase (SMase) activities are associatedwith the protein encoded in the PfNSM gene of Plasmodiumflaciparum that degrade host-derived lysophosphatidylcholineto supply the parasite with phosphocholine for their efficientintra-erythrocytic growth [59]. A soluble PLA2 activity hasbeen demonstrated in C. parvum during parasite-host cell in-teractions that results in parasite invasion and intracellulardevelopment in host enterocytes [23]. In T. vaginalis, a lyticfactor was purified that was responsible for the host celldestruction and in vitro analysis showed that this lytic factorpossesses PLA2 activity. This PLA2 activity was suggested tohydrolyze the host phospholipids and releases arachidonicacid, which is then, converted to prostanoids and leukotrienesby cyclooxygenases (COX) and lipoxygenases leading toinflammation during infection [60]. Later PLA1 and PLA2activity was also observed in the sub cellular fraction of T.vaginalis that was suggested for the hemolytic and cytolyticactivity of this protozoan parasite [61]. Similarly intestinal


parasites such as Giardia and Entamoeba histolytica alsofound to have PLA activity [62e64] that plays an importantrole in the virulence of these intestinal parasites.

4.2. Acyltransferase

Acyltransferase is a type of transferase enzyme that cata-lyzes the transfer of an acyl group from one substance toanother. Acyltransferase are involved in the fatty acidremodeling of membrane phospholipids and the metabolism ofbioactive lipids in mammalian cells [65]. Increasing bodies ofevidence suggested the role of this class of enzymes in thesurvival and proliferation of various pathogens especiallythose with intracellular life cycle.

Phospholipid precursors and fatty acids that are scavengedfrom the human host are required for the membrane biogenesisof Plasmodium during asexual life cycle, initiated by glycerol-3-phosphate acyltransferases [48]. In another study, homolo-gous gene of the family membrane-bound O-acyltransferasefamily has been discovered in the P. falciparum genome and issuggested that it may be responsible for the production oftriacylglycerols, using free fatty acids of host as substrate [66].Leishmania expresses two acyltransferase, dihydroxyacetonephosphate acyltransferase (LmDAT) and glycerol-3-phosphateacyltransferase (LmGAT), which is required for the biosyn-thesis of its cellular glycerolipids using lipid precursor dihy-droxyacetone phosphate and glycerol-3-phosphaterespectively. LmDAT is localized in the peroxisome, importantfor growth, survival and essential for virulence whereasLmGAT is important for triacylglycerol synthesis but notessential for virulence [67,68].

Replication of T. gondii in its parasitophorous vacuole isdependent on the conversion of host cell acquired cholesterol[29] for lipid droplet biogenesis. Host cell derived acyl-CoA:cholesterol acyltransferase (ACAT) are thought to playsan essential role in the intracellular proliferation of T. gondii[69]. However, not only host derived but also endogenousACAT activities of T. gondii are involved in the parasite’scholesterol-ester synthesis and lipid droplet biogenesis [4].Conversion of free cholesterol to cholesterol-ester is seen ascritical step as accumulation of free cholesterol is toxic toparasite development [4]. The parasite expresses two isoformsof ACAT that differ from mammalian ACAT in their substrateaffinity, specificity, and mechanism of regulation. Due to thesereasons, this enzyme could be targeted for efficient anti-toxoplasmosis drug therapy.

4.3. Sphingolipid synthase

Sphingolipids are essential structural components ofplasma membranes and found ubiquitously among pathogenicprotozoan [70]. The primary complex sphingolipid in trypa-nosomatids is inositol phosphorylceramide (IPC) which issynthesized by IPC synthase that is detectable in the patho-genic stages of all the Leishmania and Trypanosoma sps. [70].IPC synthase catalyzes the transfer of an inositol phosphategroup from phosphatidylinositol to the 1-hydroxyl group of

ceramide releasing DAG as a by-product [71]. However majorsphingolipid in Plasmodium is sphingomyelin that is synthe-sized by the activity of endogenous sphingomyelin synthase(SMS) utilizing host lipid precursors [70].

During intra-macrophage life cycle amastigotes of Leish-mania expresses active IPC synthase that remodels acquiredhost sphingolipid into IPC [72]. Indeed it has been shown thatL. donovani stimulates host macrophages to up-regulate theproduction of ceramide, a precursor of IPC and a substrate ofIPC synthase [73]. Later an enzyme, LmIPCS was isolated inL. major using bioinformatics and functional genetic ap-proaches and it was demonstrated that this enzyme possessIPC synthase activity [71]. Recently, active IPC synthase wasalso reported in Leishmania mexicana that synthesizes theprimary 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 isimportant for infectivity and transformation of the plasmamembrane, a necessary step in the differentiation of trypo-mastigotes to amastigotes [51]. T. gondii has been demon-strated to synthesize complex sphingolipids de novo [76], theidentification of the enzymes responsible for this haveremained ambiguous until recently, SL synthase was identifiedin T. gondii that demonstrated IPC synthase activity [77].Further investigation is necessary to validate whether de novosynthesis of IPC in T. gondii required host precursor or not.

Plasmodium infected erythrocytes contains high activity ofSMS while uninfected erythrocytes contain no detectable SMSactivity, hence it was suggested that this SMS could be ofparasitic origin [78]. Interestingly, SMS was found to beactively present in both the intra-erythrocytic parasite and inextracellular merozoites stages. However, in the intracellularring and trophozoite stages the parasite exports a fraction ofthe activity beyond the parasite plasma membrane [79].Further it was observed that P. falciparum membrane boundneutral sphingomyelinases (nSMase) activity hydrolyze hostsphingomyelin to produce ceramide, which could be a sourceof the re-synthesis of sphingomylein by a plasmodial SMS[80].

4.4. Sphingomyelinase

Sphingomyelinase (SMase) is a principal enzyme catalyzingthe hydrolysis of SM to ceramide and phosphocholine. Thereare five main types of SMases; the acidic Zn2þ-dependent andindependent, the neutral Mg2þ-dependent and independent andlastly the secreted alkaline SMase [81]. Various reports aredocumented regarding the activation of host SMase uponinfection that causes ceramide-mediated entry of intracellularpathogens [1,2]. However increasing evidence demonstratedthat apart from utilizing host SMase, these pathogens also se-cretes endogenous SMase to release ceramide on the cell sur-face, that is required for the invasion process [80].

The presence of a single Mg2þ dependent, membranebound nSMase activity in P. falciparum was reported. It wassuggested that nSMase may be secreted to the erythrocytemembrane to hydrolyze sphingomyelin for the production of


ceramide that might modulate the progression of the cell cycleof the parasite [80]. Another class of SMase that is expressedby P. falciparum functions as a sphingomyelin/lysocholinephospholipid-phospholipase C and has beenshown to be required for the infection of erythrocytes with P.falciparum. Inhibition of this enzyme prevented intra-erythrocytic proliferation [59], suggesting that this enzymemight alter the composition of host lipid rafts to permitinfection.

There are also various observations regarding the presenceof endogenous SMase activity in another intracellular proto-zoan parasite Leishmania. L. major promastigotes possess apotent SMase activity, which is dependent on the Inositolphospho-sphingolipid phospholipase C-Like (ISCL) protein.Although, ISCL can also degrade IPC, its activity withsphingomyelin is 10e20-fold greater than that of IPC [82].Additionally, L. amazonensis amastigotes express SMase thatis essential for amastigote survival and replication in themammalian host [82]. Degradation of host sphingomyelin intoceramide is a necessary step in the formation of IPC and acidictolerance that could promote parasite entry, survival and pro-liferation. These findings strongly implicate that endogenousSMase may play a pivotal role in establishing infection inLeishmania in the mammalian host.

Recent studies showed that intestinal parasites such asGiardia and Entamoeba genome contain putative SMaseencoding genes. G. lamblia possess endogenous SMase ac-tivity that is encoded by gsmaseB and gsmase3b whose tran-scription were elevated during encystation. Therefore it wassuggested that both cytoplasmic and secreted SMases areinvolved in degradation of sphingomyelin from the dietarycomponents in intestine for the generation of excess ceramidethat is further required for encystation process [43]. Solubleand membrane associated neutral SMase-C activity wasin vitro identified and characterized in E. histolytica tropho-zoites. It was further postulated that this soluble and mem-brane associated SMase activity might be essential for thevirulence and sphingolipid metabolism in this protozoanparasite [83].

5. Manipulation of host lipid-derived signaling mediatorsby protozoan parasites

Apart from just exploiting, scavenging and remodeling ofhost lipidome, these intracellular parasites also control therelease of bioactive lipid molecules, which modulates thecourse of infection. Several bioactive lipid mediators arereleased during the course of infection and results in exacer-bation of the disease. In contrast, some of the bioactive lipidmediators play an important role in the host defense during theinfection hence these parasite down-regulates their synthesisin order to survive and proliferate.

5.1. Eicosanoids

Eicosanoids are produced by several parasitic organismswhich are considered as potent regulators of host immune

responses [84]. T. gondii tachyzoites markedly alters therelease of eicosanoids, in particular 5-1ipoxygenase arach-idonic acid, by human mononuclear phagocytes. Release ofbioactive lipids such as thromboxane and leukotrienes LTB4 isimportant for toxoplasmacidal activity and provide host de-fense [85,86], hence T. gondii tachyzoites deregulates 5-1ipoxygenase pathway in human monocytes and suppressactivation of this pathway in monocyte derived macrophageswhich is important for the survival of these pathogens [9].Thromboxane TXA2 and TXB2 levels are elevated in miceinfected with T. cruzi [87]. In particular, TXA2 are releasedduring all life stages of T. cruzi and is considered as animportant modulator of survival and disease progression inhost [87]. In vivo study in T. gondii infected mice showed thatT. gondii induced Lipoxin LXA4 production that suppressesthe IL-12 production by dendritic cells. Hence it was sug-gested that LXA4 plays a major host protective role in pre-venting parasite-induced inflammation and mortality [88].Intestinal protozoan parasite E. histolytica infection in mac-rophages results in alteration of arachidonic acid metabolismleading to eicosanoids formation through cyclooxygenase(COX) and lipoxygenase (LOX) pathways [89].

5.2. Prostaglandins

Prostaglandins (PG) are class of bioactive lipid mediatorsthat are produced by arachidonic acid metabolism through theaction of COX-1, COX-2 and PG synthase. It is a well-knownfact that several protozoan parasites increase the amount andsynthesis of prostaglandins in the host cell during infection[90e92]. Earlier studies on murine splenic mononuclear cellsand peritoneal macrophages infected with L. donovani haveshown increased COX and LOX activities which results inmore prostaglandin E2 (PGE2) and other arachidonic acidmetabolite production [93]. It has been also reported thatLeishmania infection initiated in vivo PGE2 production that itmay favor Leishmania persistence and progression [94]. Initialcontact of L. donovani with the host cell may induce a rapidactivation of the PKC pathway, thus increasing COX-2 activityand consequently up-regulating PGE2 release [90]. T. cruziinduces an anti-inflammatory response through activation ofprostaglandins, specifically, PGE2 [91]. Similarly, T. gondiialso induces PGE2 biosynthesis in RAW264.7 macrophagesby regulation of arachidonic acid production and induction ofCOX-2 expression by a PKC-dependent pathway [8]. P. fal-ciparum infected RBC produces significant amount of PG D2,E2, and F2a that is suggested to modulates the host defensemechanism by lowering the host TNF-a production [95], thatlimits malarial parasitemia but also exacerbate pathogenesis athigh concentrations. Intriguingly, the suppression of COX-2derived PGE production in peripheral blood mononuclearcells by malarial pigment, hemozoin, was demonstrated thatresulted in overproduction of TNF-a, leading to the develop-ment of malarial anemia [7].

Intestinal parasite E. histolytica is known to produce andsecrete PGE2. Earlier studies have shown that E. histolyticastimulates host cells such as macrophages, colonic epithelial


cells and polymorphonuclear cells to produce high levels ofPGE2 that can modulate macrophage functions by cytokineproduction and also alters tight junction permeability colonicepithelial cells by a PGE2 mechanism [92,96e98]. Thisincrement in PGE2 production was due to increase of COX-2mRNA expression [92,96]. PGE2 results in the production ofIL-8, which supports acute inflammation associated with in-testinal amebiasis [97]. COX-like enzyme in E. histolytica wasisolated and characterized that was responsible for thebiosynthesis of PGE2 utilizing exogenous arachidonic acidsubstrates [99]. These observations suggested that E. histo-lytica is not only enhancing the expression of prostaglandinsbut also synthesizes the bioactive lipid mediator that plays animportant role in modulating host immune response duringinfection.

5.3. Ceramide

Ceramide is a family of lipid molecules that are producedin cells either by the de novo synthesis or by hydrolysis ofcomplex sphingolipids. L. donovani induced immune-suppression and modulation of host cell signaling that isalso mediated by ceramide generation. Different studies con-ducted have proved that ceramide is involved in the down-regulation of protein kinase C, dephosphorylation of Akt andsuppression of ERK activation, AP-1, NF-kB activity and NOgeneration, thereby facilitating the intracellular survival of L.donovani [73,100,101]. Recently, biphasic generation of cer-amide during Leishmania infection is also discussed. Duringthe first phase ceramide is generated from activation of acidsphingomyelinase which aids in parasite internalization andduring second phase ceramide is generated by de novo syn-thesis which results in the depletion of cholesterol from themembrane leading to impairment of antigen presentation tothe T cells [2]. Role of ceramide generation also has studied inGiardia, where it has been suggested that excess ceramidegeneration is necessary for the encystations process. This in-testinal protozoa lacks de novo synthesis of ceramide hence itis selectively up-taken from the intestinal milieu [42]. How-ever, it also possesses enzymes those are required for thegeneration of excess ceramide by expression of SMases thathydrolyze host sphingomyelin into ceramide [43]. Similarly, P.falciparum lacks de novo synthesis of ceramide, therefore ithas been suggested that plasmodial nSMase activity hydro-lyzes sphingomyelin present in erythrocyte membrane toproduce ceramide. This ceramide production has been sug-gested for the re-synthesis of sphingomyelin or to modulatethe progression of the cell cycle of the parasite [80].

Earlier studies have demonstrated the presence of freeceramide in all stages of T. cruzi [102]. Later it was demon-strated that ceramide containing glycolipids from T. cruzisynergizes with the host cytokine IFN-g to induce macrophageapoptosis that release viable parasites. This pro-apoptotic ac-tivity of T. cruzi derived by ceramide was suggested as viru-lence mechanism to spread disease [103]. Ceramidegeneration is also required for the differentiation and patho-genesis of T. cruzi and membrane associated enzymes such as

PLA1, PLA2, inositolphosphoceramide-fatty acid hydrolase,acyltransferase, and phospholipase C of T. cruzi amastigotesand trypomastigotes stages are involved in releasing ceramide[51]. Recently, T. cruzi ceramide synthase (TcCERS1) genewas identified that putatively encodes ceramide synthase ac-tivity where it was suggested that this enzymes might beinvolved in ceramide synthesis using exogenous sources ofsphingolipids from host cell [104].

6. Conclusion

It is quite clear from the above thesis that the protozoanparasites sabotage the host cellular functions by exploitinghost lipidome in various ways. These protozoans not justdirectly acquire host lipids as energy source but also manip-ulate it for their invasion, intra-cellular survival, proliferationand subsequent progression of pathogenesis. Therefore, hostlipidome can be considered as a key player in thehosteparasite interaction that plays important roles in shapingthe 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 lipidomeexploitation pathways operating at the base of human parasiticinfections caused by protozoa. There is a need for compre-hensive transcriptomic, functional genomics and metabolicapproaches to explore novel gene functions entailing lipid-metabolizing enzymes in the genome of protozoan parasites,as most of such putative enzymes/functions have not beenidentified and characterized in detail. Moreover, the metabolicmachineries for the synthesis of phospholipids and fatty acidsin these parasites have generated significant interest due totheir importance in survival, growth and proliferation duringvarious stages of a parasitic life cycle. Recent observationsconcerning the metabolism of host lipidome by these parasitesvia unusual lipid metabolizing enzymes that are not present inmammalian host come with a notion that these metabolicenzymes may form potential drug targets. With the advance-ments made during the last few years in drug designing, itwould be possible to target these metabolic enzymes for thedevelopment of novel therapeutic strategies.

Acknowledgments

The authors are grateful to Mr. Atahar Husein for helping indesigning of figures. M. A. is financially supported by UGCGovt. of India.

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Host-lipidome as a potential target of protozoan parasites

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