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Mina JG and Denny PW (2018) Everybody needs sphingolipids right Mining for new drug targets inprotozoan sphingolipid biosynthesis Parasitology 145 (2) pp 134-147

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httpsdoiorg101017S0031182017001081

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Everybody needs sphingolipids right Mining for new drugtargets in protozoan sphingolipid biosynthesis

JOHN G M MINA and P W DENNY

Department of Biosciences Lower Mountjoy Stockton Road Durham DH1 3LE UK

(Received 10 April 2017 revised 15 May 2017 accepted 18 May 2017)

SUMMARY

Sphingolipids (SLs) are an integral part of all eukaryotic cellular membranes In addition they have indispensable functions assignallingmolecules controlling a myriad of cellular events Disruption of either the de novo synthesis or the degradation path-ways has been shown to have detrimental effects The earlier identification of selective inhibitors of fungal SL biosynthesispromised potent broad-spectrum anti-fungal agents which later encouraged testing some of those agents against protozoanparasites In this review we focus on the key enzymes of the SL de novo biosynthetic pathway in protozoan parasites of theApicomplexa and Kinetoplastidae outlining the divergence and interconnection between host and pathogen metabolismThe druggability of the SL biosynthesis is considered alongside recent technology advances that will enable the dissectionand analyses of this pathway in the parasitic protozoa The future impact of these advances for the development of newtherapeutics for both globally threatening and neglected infectious diseases is potentially profound

Key words sphingolipids ceramide drug targets protozoan parasites apicomplexa kinetoplastidae

INTRODUCTION

Protozoan parasites and the global burden of theirdiseases

Protozoa (kingdom Protista) are single-cell organismsthat can be free-living or parasitic in nature (Baron1996) Out of more than 50 000 protozoan speciesthat have been described to-date relatively few havebeen identified as major contributors to the globalburden of human diseases (Kuris 2012) and animalagriculture (Dubey 1977) The protozoa represent19 of all human parasites (83 out of 437 species to-date) and are associated with 30 of parasite-inducedhuman morbidity-mortality (Kuris 2012)Of the four groups of infectious protozoa (CDC

2017) the Mastigophora (flagellates) and Sporozoacontain theKinetoplastidae andApicomplexa respect-ively It is to these two phyla that belong many ofthe causative agents of disease Mastigophora ndash theinsect vector-borne kinetoplastids Trypanosomabrucei (Human African Trypanosomiasis HAT)Leishmania spp (leishmaniasis cutaneous andvisceral)and Trypanosoma cruzi (American trypanosomiasisChagasrsquo disease) Sporozoa ndash the apicomplexanToxoplasma gondii (toxoplasmosis) Cryptosporidiumspp (cryptosporidiosis) andEimeria spp (coccidiosisinpoultry andcattle)Theileria spp (EastCoastFeverin cattle) andPlasmodium spp includingPlasmodiumfalciparum the causative agent of severe malaria andone of the lsquoBig Threersquo global infectious diseases

alongside HIV and tuberculosis (Torgerson ampMacpherson 2011)Historically the diseases caused by some of these

parasites have been classified as Neglected TropicalDiseases (NTDs) or Neglected Zoonotic Diseases(King 2011) and were associated with the classicalmodel of the lsquopoverty traprsquo covering tropical andsub-tropical regions in Africa Latin America andthe Indian subcontinent (Kuris 2012) Howeverwith global changes in climate and human demo-graphics and associated practices the classicalmodels do not promise safe boundaries that mightcontain andor stop the further global spread ofmany of these parasitic diseases (Colwell et al 2011)The problems associated with these pathogens arefurther aggravated by the lack of effective vaccines(Dumonteil 2007 Innes et al 2011 McAllister2014 BlackampMansfield 2016) and the paucity of reli-able drugs (Zofou et al 2014) in addition to thedifficulties of vector or reservoir control (Colwellet al 2011) Therefore there is a recognized need tofind new therapeutic targets in these causative agentsin order to develop effective treatment regimens toavoid potentially catastrophic outbreaks both interms of human health and economic impactThis review presents sphingolipid (SL) biosyn-

thesis and ceramide (CER) homoeostasis as a poten-tial gold mine of tractable drug targets for theseprotozoan parasites

State-of-the-art treatment of apicomplexan andkinetoplastid diseases

In general available treatments for the diseasescaused by the Kinetoplastidae and Apicomplexa

Corresponding author Department of BiosciencesLower Mountjoy Stockton Road Durham DH1 3LEUK E-mail jgmminadurhamacuk

1SPECIAL ISSUE REVIEW

Parasitology of 14 copy Cambridge University Press 2017 This is an Open Access article distributed under the terms of theCreative Commons Attribution licence (httpcreativecommonsorglicensesby40) which permits unrestricted re-use distributionand reproduction in any medium provided the original work is properly citeddoi101017S0031182017001081

httpswwwcambridgeorgcoreterms httpsdoiorg101017S0031182017001081Downloaded from httpswwwcambridgeorgcore Durham University Library on 28 Jun 2017 at 091514 subject to the Cambridge Core terms of use available at

are outdated (if not historic) with relatively fewexamples that were introduced recently toxic andrequire a long treatment regimen and thereforeclose monitoring of patientsThe kinetoplastid pathogens in focus here all

cause NTDs and as such there are significant pro-blems with the available drug regimens

Leishmania spp The treatment of leishmaniasisoften requires a long course of intravenous pentavalentantimony drugs (eg Glucantime and Pentostam)aminosidine (paromomycin) or liposomal amphoter-icin B (Croft amp Coombs 2003 Center for FoodSecurity and Public Health 2004 WHO 2004Kedzierski et al 2009) The most recent additionwas the orally available miltefosine (Sunder et al2002 Verma amp Dey 2004) originally developed asanti-neoplastic agent Despite its teratogenic effects(Sunder et al 2002) due to the lack of othereffective medications it has been registered and isnow used in India Colombia Guatemala andGermany (Soto amp Berman 2006) Other regimens oftreatment include Pentamidine (Bray et al 2003)allopurinol dapsone fluconazole itraconazole andketoconazole However to-date all available che-motherapeutic agents suffer from being toxic(Chappuis et al2007) or inaccessible both geographic-ally and financially in endemic areas where publichealth is under-resourced poor and underdevelopedAdditionally the lack of effective vaccines (deOliveira et al 2009) and the alarming emergence ofresistance to these drugs (Croft et al 2006) combinedwith the short-lived prevention resulting fromapplying measures such as vector and reservoirhost control (WHO 2004 Figueiredo et al 2012)demand an intensive search for alternative anti-leishmanials to enable effective treatment andcontrol

Trypanosoma brucei Another compelling exampleof the shortcomings of available treatments is HAT(Mina et al 2009 Buckner et al 2012) wherethere is a lack of effective vaccines (Black ampMansfield 2016) and treatment depends on the stageof the disease Whilst in the first stage the drugsused are less toxic easier to administer and moreeffective treatment in the second stage requiresdrugs that can cross the blood-brain barrier specifi-cally the arsenates (Gibaud amp Jaouen 2010) makingthem considerably more toxic and complex to admin-ister (Babokhov et al 2013) Currently four drugs areregistered for HAT treatment and are provided free ofcharge to endemic countries through a WHO privatepartnership with Sanofi-Aventis (Pentamidine melar-soprol and eflornithine) and Bayer AG (suramin)(Schmidt et al 2012) Unfortunately all of themexhibit a broad range of adverse effects Moreovertreatment regimens are usually highly restrictiveparticularly in the second stage of the disease

requiring hospital-based IV treatment with continu-ous monitoring

Trypanosoma cruzi Despite their toxic side-effects nifurtimox and benznidazole are the onlylicenced drugs available for treatment of Chagasrsquodisease (Carabarin-Lima et al 2013 Bermudezet al 2016) with the latter being the first choicedue to its lower side effects Also benznidazole hasbeen implemented in the treatment of womenbefore pregnancy in order to preventreduce verticaltransmission (Carabarin-Lima et al 2013 Murciaet al 2013) Due to the lack alternatives effortshave been directed towards implementing differenttreatment regimens in order to reduce toxicity egintermittent administration schedules combinationtherapy and re-purposing of commercial drugs(Bermudez et al 2016)

Management of apicomplexan infections is also chal-lenging and faces many of the same shortcomingsencountered in the treatment of kinetoplastidinfections

Toxoplasma gondii Treatment regimens for toxo-plasmosis patients have essentially remained thesame since the 1950s (Eyles amp Coleman 1953)They largely depend on the repurposing of antibacter-ials (sulfadiazine spiramycin and clindamycin) andantimalarials (pyrimethamine and atovaquone)(Opremcak et al 1992 Andrews et al 2014 Antczaket al 2016) in combination therapies that target para-site folic acid synthesis protein synthesis or oxidativephosphorylation (Greif et al 2001 Antczak et al2016) Most of these chemotherapeutics are notreadily bioavailable at the site of infection (egunable to cross the blood-brain barrier) cannot beadministered by patients with hypersensitivity to sul-phonamides have suspected teratogenic properties(Montoya amp Remington 2008 Paquet amp Yudin2013) are threatened by the emergence of resistance(Sims 2009) or require adjuvant therapies (folinicacid supplement) to minimize toxic side effects (for adetailed review see Antczak et al 2016)Toxoplasmosis is a representative of the urgent needfor new antiprotozoal targets In addition to the factthatT gondii is estimated to infect 2ndash3 billion peopleworldwide (Welti et al 2007) its treatment iscomplicated due to two main factors (a) the parasiteundergoes a complex life cycle with two predomin-ant forms in the human host namely tachyzoites(proliferative form) and bradyzoites (encysted formchronic toxoplasmosis) (b) bradyzoite burden iswidespread but usually asymptomatic although ithas been associated with psychiatric disorders(Webster et al 2013) However in immunocom-promised individuals encysted T gondii transforminto proliferative tachyzoite forms causing symp-tomatic disease toxoplasmic encephalitis As such

2John G M Mina and P W Denny


T gondii is an opportunistic parasite Notably allthe above-mentioned drugs act only against thetachyzoite stage with no notable effect againstencysted bradyzoites (Antczak et al 2016) Recentdata from our laboratory (Alqaisi et al 2017) andothers (Sonda et al 2005) have shown that theAureobasidin A and analogous depsipeptidesknown to target yeast SL biosynthesis (Wuts et al2015) exhibit activity against bradyzoite T gondiiThis class of compounds may offer a potential treat-ment for chronic toxoplasmosis and perhaps somepsychiatric disorders although the mechanism ofaction is not via inhibition of parasite SL biosyn-thesis and is yet to be elucidated (Alqaisi et al 2017)

Plasmodium falciparum Falciparum malariaremains one of the lsquoBig Threersquo most prevalent anddeadly infectious diseases across tropical and sub-tropical regions with an estimated 154ndash289 millioncases in 2010 (212 million cases in 2015) and 660000 (429 000 in 2015) associated deaths althoughthe actual numbers might be even higher (Biamonteet al 2013 WHO 2016)Similar to T gondii Plasmodium parasite undergoesa complex life cycle with different stages in differentorgans of the host rendering treatment challengingsporozoites and schizonts in the liver and mero-zoites trophozoites and gametocytes in the blood(Dechy-Cabaret amp Benoit-Vical 2012) Artemisinin-based combination therapies (ACTs) are the standardfor treating malaria cases with typical partner drugsincluding lumefantrine and piperaquine egCoartemtrade (Novartis) and Eurartesimtrade (Sigma-Tau) (Biamonte et al 2013) Other regimens includethe use of parenteral artesunate (severe malaria)(Dondorp et al 2010a) primaquine (liver and trans-mission gametocyte stages) (Dondorp 2013)mefloquine and sulfadoxinepyrimethamine in com-bination (effective as single dose antimalarial drug)(Biamonte et al 2013) and atovaquoneproguanilMalaronetrade (GlaxoSmith Kline) as a prophylactictreatmentHowever although combination therapies have

now been adopted resistance against many existingantimalarials has been observed since the 1950s(Bishop 1951 Hallinan 1953 Sandosham et al1964) and remains a severe threat (Rieckmann ampCheng 2002 Chinappi et al 2010 Dondorp et al2010b Newton et al 2016 Parija 2016 Menard ampDondorp 2017 Zhou et al 2017) This bleak viewof the future of available anti-malarial chemothera-peutics makes it imperative to invest more effortsin identifying new potent chemotypes that willoffer both efficacy and safety

Cryptosporidium spp Like T gondii Cryptospo-ridium parvum and Cryptosporidium hominis usuallycause a self-limiting disease in healthy individualsbut represent a manifest problem in immuno-

compromised patients particularly those withAIDS where infection leads to acute and protractedlife-threatening gastroenteritis (Chen et al 2002)More recent data have led to a radical reassessmentof the impact of cryptosporidiosis with the numberof Cryptosporidium-attributable diarrhoea episodesestimated at gt7middot5 million in children aged lt24months in sub-Saharan Africa and South Asia whereinfection is estimated to contribute to gt250 000 infantdeaths per year (Sow et al 2016) Current treatmentof cryptosporidiosis relies on a single FDA-approveddrug nitazoxanide which has limited efficacy inthose most at risk More recently the repurposing ofantimalarials eg quinolones and allopurinols hasbeen proposed (Gamo et al 2010 Chellan et al 2017)The distinctive metabolic features of this parasitefrom other apicomplexan organisms eg noplastid-derived apicoplast and the absence of thecitrate cycle and cytochrome-based respiratorychain (Ryan amp Hijjawi 2015) confer several limita-tions for the identification of targets necessary for thedevelopment of anticryptosporidial drugs Howeverthe core metabolic pathways eg energy metabolismand lipid synthesis are still present and exhibit highlevel of divergence from the mammalian host thuspresenting an opportunity to identify new drugtargets that promise effective and selective treatment(Chellan et al 2017)

The biological significance of SLs

SLs are a class of lipids that are ubiquitous ineukaryotic cell membranes particularly the plasmamembrane as well as in some prokaryotic organismsand viruses (Merrill amp Sandhoff 2002) Since theirearliest characterization by Thudichum (1884) theyhave been a subject of controversy Initially they hadbeen considered of structural importance onlyhowever over the last couple of decades severalreports have revealed their indispensability to a pleth-ora of functions including but not limited to theformation of structural domains polarized cellulartrafficking signal transduction cell growth differen-tiation and apoptosis (Huwiler et al 2000 Ohanian ampOhanian 2001 Cuvillier 2002 Pettus et al 2002Buccoliero amp Futerman 2003)SLs consist structurally of a sphingoid base back-

bone eg sphingosine (SPH) that can beN-acylatedto form CER To the latter a variety of head groupscharged neutral phosphorylated andor glycosy-lated can be attached to form complex SLs egsphingomyelin (SM) as the primary complex mam-malian SL and inositol phosphorylceramide (IPC)in fungi plants and numerous protozoa (Fig 1)These molecules have both polar and non-polarregions giving rise to their amphipathic characterwhich accounts for their tendency to aggregate intomembranous structures yet retaining the interfacialability to interact with various partners eg

3Druggability of the protozoan sphingolipid biosynthesis


involvement of glycosphingolipids (GSLs) in cellularrecognition complexes cell adhesion and the regula-tion of cell growth (Gurr et al 2002)Furthermore the diversity of their chemical struc-tures allows for distinctive roles within cellularmetabolism eg the signalling functions of SPHand CER vs sphingosine-1-phosphate (S1P) and cer-amide-1-phosphate (C1P) (Merrill amp Sandhoff2002 Metzler 2003)

SLs as indispensable structural components

The unique structural features of SLs (the free3-hydroxy group the amide functionality and theC4ndashC5 trans double bond) affect their biophysicalproperties rendering these molecules different fromtheir glycerolipid counterparts ie SM vs phosphat-idylcholine (PC) (Boggs 1980 1987 Talbott et al2000 Ramstedt amp Slotte 2002) Such interfacialdifferences give complex SLs such as SM theunique ability to form both intra- and intermolecular

hydrogen bonds (Bruzik 1988) that are fine-tunedby the trans double bond (Ramstedt amp Slotte2002) This ability is reflected in the tendency ofSLs to cluster rather than behave like typicallsquofluidrsquo membrane lipids Naturally occurring SLsundergo the Lβ (gel phase) to Lα (lamellar phase)transition near the physiological temperature of37 degC in contrast this transition for naturallyoccurring glycerolipids is near or below 0 degCAdditionally the long saturated alkyl chains of SLsallow them to pack tightly with sterols stabilizedby hydrogen bonding (Ramstedt amp Slotte 2002)to form laterally compact hydrophobic micro-domains commonly known as lsquolipid raftsrsquo(Futerman amp Hannun 2004) Similar results havebeen reported with the fungalplant counterpart ofSM IPC where it was shown that IPC was able toform sterol containing ordered domains in modelsystems (Bjoumlrkbom et al 2010) These membranemicro-domains can readily segregate from the moredisordered and expanded domains of unsaturated

Fig 1 Illustration of the predominant species of complex sphingolipid in organisms from different evolutionary cladesEPC in Drosophila SM in mammals and IPC in Leishmania and T cruzi (as representatives of protozoan parasites) andin fungi and plants IPC is absent from Mammalian cells but essential for many pathogenic organisms (red box)Glycosylated sphingolipids are also ubiquitous across different species Backbone chain length is commonly C18 derivedfrom palmitoyl-CoA Mammals M Fungi and Plants FP Leishmania spp L Trypanosoma cruzi Tc Trypanosoma bruceiTb Toxoplasma gondii Tg and Plasmodium falciparum Pf Denotes developmental regulation EPC ethanolaminephosphorylceramide IPC inositol phosphorylceramide SM sphingomyelin



acyl chains of glycerolipids (Merrill amp Sandhoff2002) They have been proposed to function in adiverse array of processes from polarised traffickingof lipid modified proteins (Brown amp London1998) and the stabilization of other types of bio-logical structures such as lamellar bodies to theassembly and activation of signal transduction com-plexes (Brown amp London 2000 Magee et al 2002Pierce 2002 Vance amp Vance 2002 Hannun ampObeid 2008) They have also been involved inthe formation of detergent-insoluble gel-phasedomains (Ramstedt amp Slotte 2002) via the extensivehydrogen-bonding network in the head groups ofGSLs that have been implicated during the forma-tion of lsquocaveolaersquo and surface recognition (Merrillamp Sandhoff 2002)

SLs as indispensable signalling agents

SLs can also function as bioactive signalling mole-cules due to their biophysical properties eg thelow pKa (7ndash8) of SPH allows it to remain partiallyuncharged at physiological pH retaining the abilityto move across membranes (Merrill amp Sandhoff2002) Likewise CER a neutral species is able tofreely flip flop across membranes (Hannun ampObeid 2008) Many studies have produced evi-dence of such signalling functions eg SPHexerts pleiotropic effects on protein kinases CERmediates many cell-stress responses including theregulation of apoptosis (Georgopapadakou 2000)and S1P has crucial roles in cell survival cellmigration and inflammation (Hannun amp Obeid2008)

SL metabolism and the rationale for druggability

The indispensability of SLs for a myriad of cellularprocesses and functions ranging from structuralintegrity to signalling events makes it is unsurpris-ing that the SL biosynthesis is highly conserved inall eukaryotes where it is alongside its proposed reg-ulators (Holthuis et al 2006) an essential pathway(Heung et al 2006 Sutterwala et al 2007) Thishas lead the pathway to be considered vital for proto-zoan pathogenesis and therefore a drug target egSM synthase activity in Plasmodium (Heung et al2006) In order to characterise the druggability ofprotozoan SL biosynthesis the mammalian pathwayas the most studied system will be used as the refer-ence model in the following discussionsSL metabolism constitutes a highly complex

network involving critical intersections with variousother pathways particularly glycerolipid biosynthesis(Holthuis amp Menon 2014) CER represents thecorner stone for both biosynthesis and catabolismmodulating cell fate (Hannun amp Obeid 2008)Dysregulation of either SL biosynthesis or catabol-ism could result in cell death eg of protozoan

parasites (Yatsu 1971 Brady 1978 Chen et al 1999Merrill amp Sandhoff 2002) however here our focuswill be on the former pathwayConsidering the central position of CER the

druggability of SL metabolism revolves around dys-regulation of lsquoCeramide Homeostasisrsquo (Young et al2012) which in turn leads to ripple effects perturbingthe balance between the pro-apoptotic CER and themitogenic diacylglycerol (DAG) consequentlydetermining cell fate (Fig 2) ndash a mechanism thathas been associated with resistance to anti-cancertreatments (Seacutegui et al 2006) and has been reportedin protozoan parasites eg Plasmodium (Pankova-Kholmyansky et al 2003 Labaied et al 2004)The characterisation of several key enzymesinvolved in SL de novo biosynthesis has revealeddivergence between mammalian and protozoanspecies Thus attention has been given to theexploitation of the SL biosynthetic pathway (para-site andor host) for new drug targets or regimens(Sugimoto et al 2004 Zhang et al 2005 Dennyet al 2006 Tanaka et al 2007 Pruett et al 2008Mina et al 2009 Tatematsu et al 2011 Younget al 2012)

SL METABOLISM

The key steps in de novo biosynthesis

SL de novo biosynthesis can be simplified into threekey steps a gate-keeper and two cell fate modulatorsteps The former comprises the up-stream rate-lim-iting step of the condensation of acyl-CoA and L-serine in the endoplasmic reticulum (ER) viaserine palmitoyltransferase (SPT) to produce dihy-drosphingosine The latter comprises first the for-mation of CER in the ER by the action ofceramide synthase (CerS) and then the formationof complex SLs in the Golgi These products varydepending on the species and are formed underthe catalysis of what could be generically termedSL synthases SM synthase in mammals and IPCsynthase in fungi plants and protozoa It is worthmentioning that another Golgi localized metabolicpathway results in the formation of glycosylatedCER species and also contributes to the regulationCER levels (Holthuis amp Menon 2014) (Fig 2)

Protozoan parasites vs host differences ampopportunities

The cross-species differences encountered in thefirst SPT-catalysed step are mostly minor interms of the chemical structure of the productmainly due to the chain length of the acyl-CoA uti-lised in the reaction eg myristoyl-CoA (inLeishmania spp amongst other odd sphingoid baselengths (Hsu et al 2007)) and palmitoyl-CoA withthe latter more predominant across the Eukaryota



(in mammals Plasmodium and T brucei) (Richmondet al 2010 Botteacute et al 2013) Further differencesmay be apparent with respect to the catalysingenzyme SPT (vide infra) However clear divergenceis observed in the second and the third steps bothof which represent a cell-fate modulator processCerSs exhibit differential preferences for the chainlength of the acyl-CoA substrate (Park et al 2014)and its hydroxylation pattern (Layre amp Moody2013) with 6 isoforms present in humans suggestinga different role for each CER species produced(Levy amp Futerman 2010 Figueiredo et al 2012)To-date one or maximum two genes encodingCerS function have been identified in protozoanparasite species (Koeller amp Heise 2011) Howevermost interesting is the variation in the complex SLformed in the Golgi reflecting significant differencesin the active site of the SL synthases catalysing thetransfer reaction The divergence of the protozoalcomplex SL synthases and the synthetic productswith respect to the mammalian host may provideopportunities to design selective inhibitorsPreviously this step has been validated as a promising

drug target in fungi using aureobasidin A (AbA)(Fig 2) (Denny et al 2006)

Serine palmitoyl transferase (SPT)

SPTs are members of the pyridoxal 5prime-phosphate(PLP)-dependent (Sandmeier et al 1994) α-oxoa-mine synthase family and share a conserved motif(T[FL][GTS]K[SAG][FLV]G) around the PLP-binding lysine (in bold) (Young et al 2012) SPTcatalyses the first rate-limiting step in the de novobiosynthesis of SLs (Weiss amp Stoffel 1997 Hojjatiet al 2005) (Fig 2) a reaction involving the decar-boxylative Claisen-like condensation of serine andan acyl-CoA (Lowther et al 2012) to yield thesphingoid base backbone 3-ketodihydrosphingosine(3-KDS) (Hanada 2003 Raman et al 2009Lowther et al 2012) Therefore SPT representsthe lsquoGatekeeperrsquo of the SL biosynthetic pathwayAll eukaryotic SPTs studied to date are ER-resi-

dent and membrane bound with a heterodimericprotein core consisting of two subunits sharingsim20 identity LCB1 and LCB2 sim53 and sim63

Fig 2 Schematic representation of de novo sphingolipid metabolism Three key steps are highlighted (1) SPTevolutionary divergent in T gondii (2) CerS fewer isoforms in protozoan parasite (cf 6 isoforms in mammals) SLSwhile predominantly synthesising SM in mammals and to a lesser extent EPC orthologues in protozoan parasites(Leishmania spp T brucei T cruzi andT gondii) can synthesise IPC an activity that is absent frommammalian cells andthe target of the highly specific fungal inhibitors shown The scheme also illustrates the differential cellular effects ofceramide vsDAG (diacylglycerol) Accumulation of ceramide elicits an apoptotic response while increasing concentrationsof DAG promotes cell growth CerS ceramide synthase GluCerS glucosylceramide synthase SLS sphingolipidsynthase SPT serine palmitoyltransferase PC phosphatidylcholine PE phosphatidylethanolamine PIphosphatidylinositol SM sphingomyelin EPC ethanolamine phosphorylceramide and IPC inositolphosphorylceramide



kDa respectively (Hanada 2003 Denny et al 2004Han et al 2004 Chen et al 2006) The latter con-tains the canonical PLP cofactor binding site whilethe former has been suggested to be important forcomplex stability (Lowther et al 2012) In contrastthe orthologous SPT from sphingomonad bacteria isa soluble 45 kDa homodimer (Ikushiro et al 2001)SPT activity in apicomplexan parasites has beendetected and was proposed as a potential drugtarget (Gerold amp Schwarz 2001 Bisanz et al 2006Coppens 2013) however the enzyme(s) responsiblehave yet to be further characterized (Mina et al2017) In contrast kinetoplastid parasites havebeen shown to possess a heterodimeric SPT similarto the mammalian orthologue (Denny et al 2004)Inhibiting SPT activity (eg using myriocinFig 2) results in various effects in different speciesMammalian cells exhibited a loss of viability witha partial loss of SPT function resulting in a rare SLmetabolic disease Hereditary Sensory Neuropathytype I (HSN1) (Hanada 2003) In contrastSaccharomyces cerevisiae were found to be relativelytolerant (Nagiec et al 1994) and Leishmania majorlacking LCB2 were viable but unable to differentiateinto infective metacyclic forms (Zhang et al 2003)However T brucei procyclic forms in which SPTexpression was reduced were non-viable (Fridberget al 2008)The SPT catalysed reaction product 3KDS is sub-

sequently reduced by 3-ketosphinganine reductase toform sphinganine (dihydrosphingosine) Subsequentminor metabolic differences are encountered acrossdifferent species mainly concerning the order of thehydroxylation (in fungi and higher plants) and acyl-ation to produce CERs (Sugimoto et al 2004)

Ceramide synthase

In all eukaryotic systems studied to date CerSs areER-resident integral membrane proteins catalysingthe N-acetylation of dihydrosphingosine to producedihydroceramide which is then oxidized to formCER the simplest SL species and a key bioactivemolecule in numerous cellular pathways (Lahari ampFuterman 2007)Mammalian CerSs are orthologues of longevity-

assurance genes LAG1p and LAC1p identified inyeast (Guillas et al 2001) The eukaryotes studiedto date have been found to encode at least twoCerSs with humans expressing six ndash each generatingCER with a defined acyl chain length (C18 to C26)(Pewzner-Jung et al 2006 Levy amp Futerman2010) Whilst little is known regarding structure-function relationships or regulation of CerS theubiquitous Lag1 motif has been shown to be import-ant for functionality (Spassieva et al 2006) likelyforming part of the active siteExperimental evidence (from our laboratory and

others) has previously indicated the presence of

CerS activity in Leishmania spp (Zhang et al2003 Denny et al 2004 2006) and in T cruzi(De Lederkremer et al 2011) More recentlyLAG1 orthologues have been identified and func-tionally and molecularly characterized in the latterparasite (Figueiredo et al 2012) Other results indir-ectly suggest the presence of such activity in Tbrucei (Patnaik et al 1993 Richmond et al 2010Smith amp Buumltikofer 2010) Similarly CerS activityin the Apicomplexa has been inferred (Welti et al2007 Zhang et al 2010 Pratt et al 2013) butremains unexploredOnce formed in the ER CER is transported by

CER transfer protein CERT in mammals(Kumagai et al 2005 Kudo et al 2010 Rao et al2014) to the Golgi apparatus where the synthesisof complex SLs occurs (Ohanian amp Ohanian 2001Bromley et al 2003 Bartke amp Hannun 2009 Pataet al 2010) ER CER concentration is kept undertight control as accumulation of CER here hasbeen shown to result in induction of the mitochon-drial apoptotic pathway (Vacaru et al 2009Tafesse et al 2014) via an unknown mechanism(Bockelmann et al 2015)

Sphingolipid synthase

In the Golgi CER can be phosphorylated by CERkinase (Rovina et al 2009) glycosylated by glucosylor galactosyl CerS (Raas-Rothschild et al 2004) oracquire a variety of neutral or charged head groupsunder the catalysis of what could be called generic-ally SLSs to form various complex phosphosphin-golipids Phylogenetic analyses have identified atleast 4 clades of SLS (Huitema et al 2004 Dennyet al 2006)In mammals CER is a substrate for the SLS SM

synthase to produce SM (Huitema et al 2004)Whilst in fungi and higher plants phytoceramide isutilized by a different SLS IPC synthase toproduce IPC as the principal phosphosphingolipid(Nagiec et al 1997 Wang et al 2008) This land-scape is significantly divergent when it comes toprotozoaIn the kinetoplastid Leishmania spp and T cruzi

CER acquires a phosphorylinositol head group fromphosphatidylinositol (PI) to produce IPC via IPCsynthase (Zhang et al 2005 Denny et al 2006Mina et al 2010) although there are some reportsof SM in T cruzi (Quintildeones et al 2004) (Fig 2)Whilst Leishmania encodes a single copy IPC syn-thase T cruzi has two highly related copies(Denny et al 2006) Further divergence and pos-sible redundancy is encountered in T Bruceiwhich harbours 4 genes that encode SLSs (Dennyet al 2006 Sutterwala et al 2008) This enzymeportfolio results in a diverse profile of the complexSL species (SM IPC and ethanolamine phosophor-ylceramide [EPC]) which are developmentally



regulated during the life cycle of the parasite(Sutterwala et al 2008)In apicomplexan parasites previous reports have

indicated the presence of glycosyl-ceramide andSM in P falciparum and T gondii as summarizedin Zhang et al (2010) However other findingsreported the presence of EPC in T gondii (Weltiet al 2007) and more recently IPC (Pratt et al2013) The latter study also characterized T gondiiSLS as demonstrating IPC synthase activity invitro (Pratt et al 2013)The divergence of SLS function with respect to

the host seen in both kinetoplastid and apicom-plexan protozoan parasites in intriguing andperhaps indicated them as a tractable drug targetIn support of this hypothesis ceramide-analogueswith anti-Plasmodium activity have already beenidentified (Labaied et al 2004)In general SLSs are Golgi-resident transmem-

brane proteins presumed to have 6 transmembranedomains with the active site facing the Golgilumen (Holthuis et al 2006 Sutterwala et al2008) Those orthologues identified in kinetoplastidsdemonstrated two conserved regions(CGDX3SGHT amp HYTXDVX3YX6FX2YH)with respect to the animal SM synthases (Huitemaet al 2004 Denny et al 2006) These regionscontain the so-called the catalytic triad (twoHistidines and one Aspartate residues) that mediatesa nucleophilic attack on lipid phosphate ester duringthe transferasehydrolase activity (Mina et al 2010)Apicomplexan orthologues form a separate evolu-tionary clade yet retain the catalytic triad (Dennyet al 2006 Pratt et al 2013) as does the fungalorthologue AUR1p (Heidler amp Radding 2000)Further evidence for the essentiality of these resi-dues was provided when mutation of the active his-tidine of the triad was shown to deactivate fungalIPC synthase and mammalian SM synthase-relatedactivity (Levine et al 2000 Vacaru et al 2009)Furthermore recently it has been shown that sub-strate selectivity and so the diversity of SLS activ-ity may depend on key residues close to thetransferase active residues or on a luminal loop ofthe protein (Sevova et al 2010 Kol et al 2017)In the Eukaryota SLSrsquos occupy a central position

at the intersection of glycerolipids (PIPCPE andDAG) and SLs ([phyto]ceramide and IPCSMEPC) Accordingly these enzymes act as regulatorsof a delicate balance between pro-apoptotic CERand pro-mitogenic DAG (Holthuis et al 2006)The most significant previous example of SL bio-

synthesis inhibition as a drug target was reported infungi Aureobasidin A (AbA) a depsipeptide wasfirst reported by Ikai et al (1991) and soon after itsantifungal properties were highlighted (Takesakoet al 1993) The target gene was further character-ized (Hashidaokado et al 1995) revealing its identityto be the IPC synthase (AUR1p) AbA is a specific

and potent (low nanomolar) inhibitor of the fungalIPC synthase This ushered in a new era in thesearch for anti-fungal chemotherapeutics position-ing IPC synthase as a promising broad spectrumanti-fungal drug target (Sugimoto et al 2004)Other specific inhibitors were later added to thearsenal of fungal IPC synthase inhibitors khafrefun-gin (Mandala et al 1997) rustmicin (Harris et al1998 Mandala et al 1998) and others (Ohnukiet al 2009) Unfortunately further development ofthese inhibitors stalled either due to physical prop-erties eg aureobasidin A is very sparingly solublein water (Georgopapadakou 2000 Sugimoto et al2004) or because their highly complex chemicalstructures rendered chemical synthesis challengingwith the few synthetic efforts reported resulting incompounds with either reduced or no activity(Sugimoto et al 2004 Aeed et al 2009) Howeverrecent works have highlighted that semi-syntheticstrategies may overcome these barriers (Wuts et al2015)Perhaps reflecting the evolutionary divergence of

these enzymes the protozoan IPC synthase ortholo-gues from Leishmania major and T gondii are notsusceptible to AbA inhibition (Denny et al 2006Pratt et al 2013) Some studies have reported theinhibitory effects of AbA and analogues againstT gondii in culture (Sonda et al 2005 Alqaisiet al 2017) however this is not associated withinhibition of SL biosynthesis Despite this theprotozoan SLSrsquos remain tractable drug targets withno functional equivalent in mammalian cellsSurprisingly at least one SLS isoform fromT brucei was acutely sensitive to AbA treatment(Mina et al 2009) although these findings stirredsome controversy due in part to the redundancyof T brucei SLSs (4 isoforms) compared with thesingle copy found for example in L major andT gondii (Sutterwala et al 2008)

THE ENIGMATIC NATURE OF SL DRUGGABILITY

Difficulties in pinpointing SL functionality

Investigation and deciphering of the functions ofeach specific SL species remains challenging This isdue to the complexity in SL metabolic interconnec-tions their varied biophysical properties (neutral orcharged) chain length variation the hydrophobicnature of the involved enzymes and the presence ofmultiple pathways that can operate in parallel(Hannun amp Obeid 2008) The interaction withother cellular metabolic pathways (eg glycerolipidmetabolism) introduces another layer of complexityOverall the signalling effectrole of an individual

SL could be defined on spatial-temporal basis withat least five parameters (a) subcellular localisation(b) regulation (c) chain length specificity (d) kineticsof trafficking and (e) mechanism of action For



example phosphorylation of 1ndash3 cytosolic SPHmay double the levels of S1P that acts on Gprotein-coupled receptor (GPCR) to elicit aspecific response in a particular cellular locality forcertain period of time (Hannun amp Obeid 2008)Such signalling events can be described as a functionof cytosolic S1P that is regulated by S1P Kinasewith the signal caused through the interactionof S1P with a GPCR The elucidation of suchcomplex systems remains challenging and a compre-hensive discussion of the issue is beyond the scope ofthis review However an additional layer of signifi-cant complexity in terms of the pathogenic protozoaarises when considering the SL signalling network inthe case of obligate intracellular parasites where hostSL biosynthesis and its interaction with parasite denovo synthesis must be taken into account

Parasitendashhost SL interplay

The intimate parasitendashhost interaction in termsof SL metabolism has been well documented Lmajor pathogenic amastigotes isolated frommamma-lian hosts showed normal IPC levels (Zhang et al2005) despite lacking LCB2 a functional SPT andthe ability to synthesis CER de novo Alterations inhost macrophage cell SL biosynthesis upon infec-tion may compensate for this deficiency (Ghoshet al 2001 2002) These studies suggest acomplex and multifaceted interplay between hostand parasite SL metabolism comprising nutritionalfactors and signalling pathways that could modulateparasite survival andor host defence (Zhang et al2010) Similar observations have been reported inthe apicomplexan parasites (Romano et al 2013)This highlights the striking potential of host andparasite SL modulation as an anti-protozoal targetas is similarly proposed for pathogenic fungi(Zhang et al 2010 Ramakrishnan et al 2013)

PERSPECTIVE

Classically dissecting the role and locale of criticalenzymatic steps in SL biosynthesis and assessingthe effect on the parasite fitness and virulencecould turn into an overwhelmingly challenging taskaggravated by the complexity of the metabolicpathway itself the ability of the parasite to salvage(Coppens 2013) hijack and remodel host SL anddevelopmental regulation during the parasitic lifecycle which adds another layer of intricacy render-ing the deconvolution of any observed effectsdifficult to interpret Fortunately many of thoseproblems can be now overcome with advances intechnology High resolution localization studies inprotozoan parasites can benefit greatly from newmicroscopic techniques such as Airy-scan (Huff2015) super-resolution microscopy (Florentinoet al 2014) and upcoming technologies eg phase-

modulation nanoscopy (Pal 2015 Ward amp Pal2017) which can elucidate spatial arrangement ofproteins of interest within the parasite to revealpotential interaction partners and shed light onmechanistic features Similarly new advances inchemical probes and SL analogues in particularsuch as bifunctional lipid technology (Haberkant ampHolthuis 2014) coupled with high throughputproteomic (Ramaprasad et al 2015) could identifydifferent interaction partners that would help mapthe biosynthetic pathway and its critical interactionsThe effects of these probes on the parasite (andhost) cell can now be comprehensively evaluatedby monitoring the transcriptome proteome meta-bolomics (Watson 2010) and lipidome (Marechalet al 2011) Such studies could reveal multiplewindows of opportunity to exploit as potentialdrug targets The targets identified in this waycan now be rapidly genetically validated in theparasitic protozoa by applying modern geneediting technologies such as CRISPRCas9(Sugi et al 2016) Compared with the classicalmethodologies this tool enables fast and efficientapplication for single gene (Serpeloni et al 2016)and systematic genome-wide knockout generation(Sidik et al 2016) Additionally the developmentof novel orthogonal approach for conditionalknockout strategies eg tetracycline-induced genedisruption Tet-system (Meissner et al 2002)rapamycin-induced Cre recombinase-assisted geneexcision (Andenmatten et al 2013 Collins et al2013 Jimenez-Ruiz et al 2014) has allowedtesting of essential gene functionality in Leishmaniaspp (Duncan et al 2016) and T gondii (Pieperhoffet al 2015)Aside from the increase ability to robustly validate

targets such as SL biosynthesis global collaborationbetween academia and pharmaceutical partners isexpediting the process of drug discovery of newanti-protozoal drugs For example within thesphere of targeting SL biosynthesis in the protozoawe have managed several projects with industrialpartners MRCT and Tres Cantos Open LabFoundation (httpswwwopenlabfoundationorgan initiative of GlaxoSmithKline) in the pursuit ofidentifying new compound scaffolds active againstthe Leishmania spp IPC synthase utilising yeast(Norcliffe et al 2014) as a vehicle for drug discovery(Denny amp Steel 2015) The generated results andtechniques could readily be translated to otherdisease targets Other global initiatives includeOpen Innovation Drug Discovery Eli Lilly whichis focused on cancer cardiovascular disease endo-crine disorders neuroscience and tuberculosis TheCenters for Therapeutic Innovation facilitatesPfizer and academic researchers to work together inorder to develop new biologics programs andWIPO ReSearch provide participant researcherswith access to patents and expertise related to drug



discovery for 19 NTDs malaria and tuberculosis(Sheridan 2011)Finally SL biosynthesis represents a gold mine

for new drug targets alongside at least two axes denovo synthesis and salvage and remodelling Onone hand the protozoan de novo SL biosyntheticpathway comprises three key steps and consideringtheir divergence compared with the mammalianhost identifying specific inhibitors for those couldopen an opportunity for anti-protozoal drugs withsynergistic effects and lower incidences of resistanceOn the other hand the nature of obligate intracellu-lar parasites dictates that further efforts should bedirected towards the catabolicsalvage pathwaywhere parasitendashhost dependencies could be exploitedin order to identify additional key steps or hostenzymes where inhibitors would exert further syn-ergism with the de novo inhibitorsTo summarize the landscape of anti-protozoan

drug discovery requires immediate attention withthe re-evaluation of knowledge gained the applica-tion of recent technologies and the support of coor-dinated global discovery efforts The multifacetedeffects of SLs as a dynamic matrix of interaction(spatial and temporal) and function makes SL bio-synthesis highly alluring for drug interventionafter all everybody needs SLs right

ACKNOWLEDGMENTS

We thank Dr Ehmke Pohl for helpful discussions

FINANCIAL SUPPORT

JGM and PWD are supported by the Biotechnology andBiological Research Council (BBM0241561 andNPRONET awards)

REFERENCES

Aeed P A Young C L Nagiec MM and Elhammer A P(2009) Inhibition of inositol phosphorylceramide synthase by thecyclic peptide aureobasidin A Antimicrobial Agents and Chemotherapy53 496ndash504Alqaisi A Q I Mbekeani A J Llorens M B Elhammer A Pand Denny PW (2017) The antifungal Aureobasidin A and an analogueare active against the protozoan parasite Toxoplasma gondii but do notinhibit sphingolipid biosynthesis Parasitology 1ndash8 doi 101017S0031182017000506Andenmatten N Egarter S Jackson A J Jullien N Herman JP and Meissner M (2013) Conditional genome engineering inToxoplasma gondii uncovers alternative invasion mechanisms NatureMethods 10 125ndash127Andrews K T Fisher G and Skinner-Adams T S (2014) Drugrepurposing and human parasitic protozoan diseases InternationalJournal for Parasitology Drugs and Drug Resistance 4 95ndash111Antczak M Dzitko K and Długon ska H (2016) Human toxoplas-mosis-searching for novel chemotherapeutics Biomedicine ampPharmacotherapy 82 677ndash684Babokhov P Sanyaolu A O Oyibo W A Fagbenro-Beyioku AF and Iriemenam N C (2013) A current analysis of chemotherapystrategies for the treatment of human African trypanosomiasis Pathogensand Global Health 107 242ndash252Baron E J (1996) Classification In Medical Microbiology 4th edn (edBaron S) University of Texas Medical Branch at Galveston GalvestonTX USA

Bartke N and Hannun Y A (2009) Bioactive sphingolipids metabol-ism and function Journal of Lipid Research 50 S91ndashS96Bermudez J Davies C Simonazzi A Pablo Real J andPalma S (2016) Current drug therapy and pharmaceutical challengesfor Chagas disease Acta Tropica 156 1ndash16Biamonte M A Wanner J and Le Roch K G (2013) Recentadvances in malaria drug discovery Bioorganic amp Medicinal ChemistryLetters 23 2829ndash2843Bisanz C Bastien O Grando D Jouhet J Marechal E andCesbron-DelauwM F (2006)Toxoplasma gondii acyl-lipid metabolismde novo synthesis from apicoplast-generated fatty acids versus scavengingof host cell precursors Biochemical Journal 394 197ndash205Bishop A (1951) Drug-resistance in malaria British Medical Bulletin 847ndash50Bjoumlrkbom A Ohvo-Rekilauml H Kankaanpaumlauml P Nyholm T KMWesterlund B and Slotte J P (2010) Characterization of membraneproperties of inositol phosphorylceramide Biochimica et Biophysica Acta(BBA) ndash Biomembranes 1798 453ndash460Black S J andMansfield JM (2016) Prospects for vaccination againstpathogenic African trypanosomes Parasite Immunology 38 735ndash743Bockelmann S Mina J Jain A Ehring K Korneev S andHolthuis J CM (2015) Molecular dissection of ceramide-inducedapoptosis using bifunctional lipid analogs Febs Journal 282 399ndash399Boggs J M (1980) Intermolecular hydrogen-bonding between lipids ndashinfluence on organization and function of lipids in membranes CanadianJournal of Biochemistry 58 755ndash770Boggs JM (1987) Lipid intermolecular hydrogen-bonding ndash influenceon structural organization and membrane-function Biochimica etBiophysica Acta 906 353ndash404Botteacute C Y Yamaryo-Botteacute Y Rupasinghe TW T Mullin K AMacRae J I Spurck T P Kalanon M Shears M J Coppel RL Crellin P K Mareacutechal E McConville M J andMcFadden G I (2013) Atypical lipid composition in the purified relictplastid (apicoplast) of malaria parasites Proceedings of the NationalAcademy of Sciences of the United States of America 110 7506ndash7511Brady R O (1978) Sphingolipidoses Annual Review of Biochemistry 47687ndash713Bray P G Barrett M P Ward S A and de Koning H P (2003)Pentamidine uptake and resistance in pathogenic protozoa past presentand future Trends in Parasitology 19 232ndash239Bromley P E Li Y N O Murphy SM Sumner CM andLynch D V (2003) Complex sphingolipid synthesis in plants character-ization of inositolphosphorylceramide synthase activity in bean micro-somes Archives of Biochemistry and Biophysics 417 219ndash226Brown D A and London E (1998) Functions of lipid rafts in biologicalmembranesAnnual Review of Cell and Developmental Biology 14 111ndash136Brown D A and London E (2000) Structure and function of sphingo-lipid- and cholesterol-rich membrane rafts Journal of Biological Chemistry275 17221ndash17224Bruzik K S (1988) Conformation of the polar headgroup of sphingo-myelin and its analogues Biochimica et Biophysica Acta (BBA) ndashBiomembranes 939 315ndash326Buccoliero R and Futerman A H (2003) The roles of ceramide andcomplex sphingolipids in neuronal cell function Pharmacological Research47 409ndash419Buckner F S Waters NC and Avery VM (2012) Recent highlightsin anti-protozoan drug development and resistance research InternationalJournal for Parasitology Drugs and Drug Resistance 2 230ndash235Carabarin-Lima A Gonzaacutelez-Vaacutezquez M C Rodriacuteguez-Morales O Bayloacuten-Pacheco L Rosales-Encina J L Reyes-Loacutepez P A and Arce-Fonseca M (2013) Chagas disease (Americantrypanosomiasis) in Mexico an update Acta Tropica 127 126ndash135CDC (2017) About Parasites Centers for Disease Control and PreventionAtlanta GA USA httpswwwcdcgovparasitesabouthtmlCenter for Food Security and Public Health C o V M Iowa StateUniversity Ames Iowa 50011 (2004) Leishmaniasis (cutaneous and vis-ceral) Center for Food Security and Public Health College of VeterinaryMedicine Iowa State University Ames Iowa 50011Chappuis F Sundar S Hailu A Ghalib H Rijal S Peeling RW Alvar J and Boelaert M (2007) Visceral leishmaniasis what arethe needs for diagnosis treatment and control Nature ReviewsMicrobiology 5 873ndash882Chellan P Sadler P J and Land KM (2017) Recent developmentsin drug discovery against the protozoal parasites Cryptosporidium andToxoplasma Bioorganic amp Medicinal Chemistry Letters 27 1491ndash1501Chen C-S Patterson M C Wheatley C L OrsquoBrien J F andPagano R E (1999) Broad screening test for sphingolipid-storage dis-eases Lancet 354 901ndash905



Chen M Han G Dietrich C R Dunn TM and Cahoon E B(2006) The essential nature of sphingolipids in plants as revealed by thefunctional identification and characterization of the Arabidopsis LCB1subunit of serine palmitoyltransferase Plant Cell 18 3576ndash3593Chen X-M Keithly J S Paya C V and LaRusso N F (2002)Cryptosporidiosis New England Journal of Medicine 346 1723ndash1731Chinappi M Via A Marcatili P and Tramontano A (2010) Onthe mechanism of chloroquine resistance in Plasmodium falciparum PlosONE 5 e14064Collins C R Das S Wong E H Andenmatten N Stallmach RHackett F Herman J P Muller S Meissner M andBlackman M J (2013) Robust inducible Cre recombinase activity inthe human malaria parasite Plasmodium falciparum enables efficient genedeletion within a single asexual erythrocytic growth cycle MolecularMicrobiology 88 687ndash701Colwell D D Dantas-Torres F andOtranto D (2011) Vector-borneparasitic zoonoses emerging scenarios and new perspectives VeterinaryParasitology 182 14ndash21Coppens I (2013) Targeting lipid biosynthesis and salvage in apicom-plexan parasites for improved chemotherapies Nature ReviewsMicrobiology 11 823ndash835Croft S L and Coombs G H (2003) Leishmaniasis ndash current chemo-therapy and recent advances in the search for novel drugs Trends inParasitology 19 502ndash508Croft S L Sundar S and Fairlamb A H (2006) Drug resistance inleishmaniasis Clinical Microbiology Reviews 19 111ndash126Cuvillier O (2002) Sphingosine in apoptosis signaling Biochimica etBiophysica Acta (BBA) ndashMolecular and Cell Biology of Lipids 1585 153ndash162Dechy-Cabaret O and Benoit-Vical F (2012) Effects of antimalarialmolecules on the gametocyte stage of Plasmodium falciparum the debateJournal of Medicinal Chemistry 55 10328ndash10344De Lederkremer RM Agusti R and Docampo R (2011)Inositolphosphoceramide metabolism in Trypanosoma cruzi as comparedto other Trypanosomatids Journal of Eukaryotic Microbiology 58 79ndash87de Oliveira C I Nascimento I P Barral A Soto M and Barral-Netto M (2009) Challenges and perspectives in vaccination against leish-maniasis Parasitology International 58 319ndash324Denny PW and Steel PG (2015) Yeast as a potential vehicle for neglectedtropical disease drug discovery Journal of Biomolecular Screening 20 56ndash63Denny PW Goulding D Ferguson M A and Smith D F (2004)Sphingolipid-free Leishmania are defective in membrane traffickingdifferentiation and infectivity Molecular Microbiology 52 313ndash327Denny PW Shams-Eldin H Price H P Smith D F andSchwarz R T (2006) The protozoan inositol phosphorylceramide syn-thase a novel drug target that defines a new class of sphingolipid synthaseJournal of Biological Chemistry 281 28200ndash28209Dondorp AM (2013) Editorial commentary single-dose primaquine asgametocytocidal treatment in patients with uncomplicated falciparummalaria Clinical Infectious Diseases 56 694ndash696Dondorp AM Fanello C I Hendriksen I C E Gomes ESeni A Chhaganlal K D Bojang K Olaosebikan RAnunobi N Maitland K Kivaya E Agbenyega T Nguah SB Evans J Gesase S Kahabuka C Mtove G Nadjm BDeen J Mwanga-Amumpaire J Nansumba M Karema CUmulisa N Uwimana A Mokuolu O A Adedoyin O TJohnson W B R Tshefu A K Onyamboko M ASakulthaew T Ngum W P Silamut K Stepniewska KWoodrow C J Bethell D Wills B Oneko M Peto T E vonSeidlein L Day N P J and White N J (2010a) Artesunate versusquinine in the treatment of severe falciparum malaria in African children(AQUAMAT) an open-label randomised trial Lancet 376 1647ndash1657Dondorp AM Yeung S White L Nguon C Day N PSocheat D and von Seidlein L (2010b) Artemisinin resistance currentstatus and scenarios for containmentNature ReviewsMicrobiology 8 272ndash280Dubey J P (1977) Toxoplasma Hammondia Besnotia Sarcocystis andother cyst-forming coccidia of man and animals In Parasitic Protozoa (edKreier J P) pp 101ndash237 Academic Press New YorkDumonteil E (2007) DNA vaccines against protozoan parasitesadvances and challenges Journal of Biomedicine and Biotechnology 2007 11Duncan SM Myburgh E Philipon C Brown E Meissner MBrewer J and Mottram J C (2016) Conditional gene deletion withDiCre demonstrates an essential role for CRK3 in Leishmania mexicanacell cycle regulation Molecular Microbiology 100 931ndash944Eyles D E and Coleman N (1953) Antibiotics in the treatment of toxo-plasmosis American Journal of Tropical Medicine and Hygiene 2 64ndash69Figueiredo JM Rodrigues D C Silva R CM C Koeller CMJiang J C Jazwinski SM Previato J O Mendonccedila-Previato LUumlrmeacutenyi T P and Heise N (2012) Molecular and functional

characterization of the ceramide synthase from Trypanosoma cruziMolecular and Biochemical Parasitology 182 62ndash74Florentino P T V Real F Bonfim-Melo A Orikaza CMFerreira E R Pessoa C C Lima B R Sasso G R S andMortara R A (2014) An historical perspective on how advances inmicroscopic imaging contributed to understanding the Leishmania sppand Trypanosoma cruzi host-parasite relationship BioMed ResearchInternational 2014 16Fridberg A Olson C L Nakayasu E S Tyler KM Almeida IC and Engman DM (2008) Sphingolipid synthesis is necessary forkinetoplast segregation and cytokinesis in Trypanosoma brucei Journal ofCell Science 121 522ndash535Futerman A H and Hannun Y A (2004) The complex life of simplesphingolipids EMBO Reports 5 777ndash782Gamo F-J Sanz LM Vidal J de Cozar C Alvarez ELavandera J-L Vanderwall D E Green D V S Kumar VHasan S Brown J R Peishoff C E Cardon L R and Garcia-Bustos J F (2010) Thousands of chemical starting points for antimalariallead identification Nature 465 305ndash310Georgopapadakou N H (2000) Antifungals targeted to sphingolipidsynthesis focus on inositol phosphorylceramide synthase Expert Opinionon Investigational Drugs 9 1787ndash1796Gerold P and Schwarz R T (2001) Biosynthesis of glycosphingoli-pids de-novo by the human malaria parasite Plasmodium falciparumMolecular and Biochemical Parasitology 112 29ndash37Ghosh S Bhattacharyya S Das S Raha S Maulik N Das DK Roy S and Majumdar S (2001) Generation of ceramide inmurine macrophages infected with Leishmania donovani alters macrophagesignaling events and aids intracellular parasitic survival Molecular andCellular Biochemistry 223 47ndash60Ghosh S Bhattacharyya S Sirkar M Sa G S Das TMajumdar D Roy S and Majumdar S (2002) Leishmania donovanisuppresses activated protein 1 and NF-kappaB activation in host macro-phages via ceramide generation involvement of extracellular signal-regu-lated kinase Infection and Immunity 70 6828ndash6838Gibaud S and Jaouen G (2010) Arsenic-based drugs from Fowlerrsquossolution to modern anticancer chemotherapy In MedicinalOrganometallic Chemistry (ed Jaouen G and Metzler-Nolte N) pp 1ndash20 Springer Berlin Heidelberg Berlin HeidelbergGreif G Harder A and Haberkorn A (2001) Chemotherapeuticapproaches to protozoa Coccidiae ndash current level of knowledge andoutlook Parasitology Research 87 973ndash975Guillas I Kirchman P A Chuard R Pfefferli M Jiang J CJazwinski SM and Conzelmann A (2001) C26-CoA-dependent cer-amide synthesis of Saccharomyces cerevisiae is operated by Lag1p andLac1p Embo Journal 20 2655ndash2665Gurr M I Harwood J L and Frayn K N (2002) LipidBiochemistry An Introduction 5th edn Blackwell Science Ltd OxfordUKHaberkant P and Holthuis J CM (2014) Fat amp fabulousBifunctional lipids in the spotlight Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids 1841 1022ndash1030Hallinan T C (1953) Drug resistance in malariaBritishMedical Journal2 135ndash136Han G Gable K Yan L Natarajan M Krishnamurthy JGupta S D Borovitskaya A Harmon JM and Dunn TM(2004) The topology of the Lcb1p subunit of yeast serine palmitoyltrans-ferase Journal of Biological Chemistry 279 53707ndash53716Hanada K (2003) Serine palmitoyltransferase a key enzyme of sphingo-lipid metabolism Biochim Biophys Acta 1632 16ndash30Hannun Y A and Obeid LM (2008) Principles of bioactive lipid sig-nalling lessons from sphingolipids Nature Reviews Molecular Cell Biology9 139ndash150Harris G H Shafiee A Cabello M A Curotto J EGenilloud O Goklen K E Kurtz M B Rosenbach MSalmon PM Thornton R A Zink D L and Mandala SM(1998) Inhibition of fungal sphingolipid biosynthesis by rustmicingalbonolide B and their new 21-hydroxy analogs Journal of Antibiotics51 837ndash844Hashidaokado T Ogawa A Endo M Takesako K and Kato I(1995) Cloning and characterization of a gene conferring resistance tothe antifungal antibiotic aureobasidin-A (R106-I) in yeast FASEBJournal 9 A1371ndashA1371Heidler S A and Radding J A (2000) Inositol phosphoryl trans-ferases from human pathogenic fungi Biochimica Et Biophysica Acta-Molecular Basis of Disease 1500 147ndash152Heung L J Luberto C and Del Poeta M (2006) Role of sphingoli-pids in microbial pathogenesis Infection and Immunity 74 28ndash39



Hojjati M R Li Z and Jiang X-C (2005) Serine palmitoyl-CoAtransferase (SPT) deficiency and sphingolipid levels in mice Biochimicaet Biophysica Acta (BBA) ndash Molecular and Cell Biology of Lipids 173744ndash51Holthuis J CM and Menon A K (2014) Lipid landscapes and pipe-lines in membrane homeostasis Nature 510 48ndash57Holthuis J CM Tafesse FG and Ternes P (2006) The multigenicsphingomyelin synthase family Journal of Biological Chemistry 281 29421ndash29425Hsu F-F Turk J Zhang K and Beverley SM (2007)Characterization of Inositol Phosphorylceramides from Leishmaniamajor by Tandem Mass Spectrometry with Electrospray IonizationJournal of the American Society for Mass Spectrometry 18 1591ndash1604Huff J (2015) The Airyscan detector from ZEISS confocal imaging withimproved signal-to-noise ratio and super-resolution Nature Methods 12 indashiiHuitema K van den Dikkenberg J Brouwers J and Holthuis JCM (2004) Identification of a family of animal sphingomyelin synthasesEmbo Journal 23 33ndash44Huwiler A Kolter T Pfeilschifter J and Sandhoff K (2000)Physiology and pathophysiology of sphingolipid metabolism and signalingBiochimica et Biophysica Acta (BBA) ndash Molecular and Cell Biology ofLipids 1485 63ndash99Ikai K Takesako K Shiomi K Moriguchi M Umeda YYamamoto J Kato I and Naganawa H (1991) Structure of aureo-basidin-A Journal of Antibiotics 44 925ndash933Ikushiro H Hayashi H and Kagamiyama H (2001) A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas pau-cimobilis EY2395T strain Purification characterization cloning and over-production Journal of Biological Chemistry 276 18249ndash18256Innes E A Bartley PM Rocchi M Benavidas-Silvan JBurrells A Hotchkiss E Chianini F Canton G and Katzer F(2011) Developing vaccines to control protozoan parasites in ruminantsdead or alive Veterinary Parasitology 180 155ndash163Jimenez-Ruiz E Wong E H Pall G S and Meissner M (2014)Advantages and disadvantages of conditional systems for characterizationof essential genes in Toxoplasma gondii Parasitology 141 1390ndash1398Kedzierski L Sakthianandeswaren A Curtis JM Andrews PC Junk P C and Kedzierska K (2009) Leishmaniasis current treat-ment and prospects for new drugs and vaccines Current MedicinalChemistry 16 599ndash614King L (2011) The Causes and Impacts of Neglected Tropical and ZoonoticDiseases Opportunities for Integrated Intervention Strategies NationalAcademies Press Washington DC USAKoeller CM and Heise N (2011) The sphingolipid biosyntheticpathway is a potential target for chemotherapy against Chagas diseaseEnzyme Research 2011 13Kol M Panatala R Nordmann M Swart L Van Suijlekom LCabukusta B Hilderink A Gabrietz T Mina J GSomerharju P Korneev S Tafesse F G and Holthuis J C(2017) Switching head group selectivity in mammalian sphingolipid bio-synthesis by active-site-engineering of sphingomyelin synthases Journalof Lipid Research 58 962ndash973Kudo N Kumagai K Matsubara R Kobayashi S Hanada KWakatsuki S and Kato R (2010) Crystal structures of the CERTSTART domain with inhibitors provide insights into the mechanism ofceramide transfer Journal of Molecular Biology 396 245ndash251Kumagai K Yasuda S Okemoto K Nishijima M Kobayashi Sand Hanada K (2005) CERT mediates intermembrane transfer ofvarious molecular species of ceramides Journal of Biological Chemistry280 6488ndash6495Kuris AM (2012) The global burden of human parasites who and whereare they How are they transmitted Journal of Parasitology 98 1056ndash1064Labaied M Dagan A Dellinger M Gegraveze M Egeacutee SThomas S L Wang C Gatt S and Grellier P (2004) Anti-Plasmodium activity of ceramide analogs Malaria Journal 3 49Lahari S and Futerman A H (2007) The metabolism and function ofsphingolipids and glycosphingolipids Cellular and Molecular Life Sciences64 2270ndash2284Layre E and Moody D B (2013) Lipidomic profiling of model organ-isms and the worldrsquos major pathogens Biochimie 95 109ndash115Levine T P Wiggins C A R and Munro S (2000) Inositol phos-phorylceramide synthase is located in the Golgi apparatus ofSaccharomyces cerevisiae Molecular Biology of the Cell 11 2267ndash2281Levy M and Futerman A H (2010) Mammalian ceramide synthasesIUBMB Life 62 347ndash356Lowther J Naismith J H Dunn TM and Campopiano D J(2012) Structural mechanistic and regulatory studies of serine palmitoyl-transferase Biochemical Society Transactions 40 547ndash554

Magee T Prinen N Alder J Pagakis S N and Parmryd I(2002) Lipid rafts cell surface platforms for T cell signaling BiologicalResearch 35 127ndash131Mandala SM Thornton R A Rosenbach M Milligan JGarcia-Calvo M Bull H G and Kurtz M B (1997) Khafrefungina novel inhibitor of sphingolipid synthesis Journal of BiologicalChemistry 272 32709ndash32714Mandala SM Thornton R A Milligan J Rosenbach MGarcia-Calvo M Bull H G Harris G Abruzzo G KFlattery AM Gill C J Bartizal K Dreikorn S and Kurtz MB (1998) Rustmicin a potent antifungal agent inhibits sphingolipid syn-thesis at inositol phosphoceramide synthase Journal of BiologicalChemistry 273 14942ndash14949Marechal E Riou M Kerboeuf D Beugnet F Chaminade Pand Loiseau PM (2011) Membrane lipidomics for the discovery ofnew antiparasitic drug targets Trends in Parasitology 27 496ndash504McAllister MM (2014) Successful vaccines for naturally occurringprotozoal diseases of animals should guide human vaccine research Areview of protozoal vaccines and their designs Parasitology 141 624ndash640Meissner M Schluumlter D and Soldati D (2002) Role of ToxoplasmagondiiMyosin A in powering parasite gliding and host cell invasion Science298 837ndash840Menard D and Dondorp A (2017) Antimalarial drug resistance athreat to malaria elimination Cold Spring Harbor Perspectives inMedicine 1ndash25 doi 101101cshperspecta025619Merrill A H and Sandhoff K (2002) Sphingolipids metabolism andcell signalling In Biochemistry of Lipids Lipoproteins and MembranesVol 36 4th edn (ed Vance D E and Vance J E) pp 373ndash407Elsevier Science AmsterdamMetzler D E (2003) Biochemistry The Chemical Reactions of LivingCells 2nd edn Elsevier Academic Press San Diego CA USAMina J G Pan S Y Wansadhipathi N K Bruce C R Shams-Eldin H Schwarz R T Steel P G and Denny PW (2009) TheTrypanosoma brucei sphingolipid synthase an essential enzyme and drugtarget Molecular and Biochemical Parasitology 168 16ndash23Mina J GMosely J A Ali H Z Shams-Eldin H Schwarz R TSteel P G and Denny PW (2010) A plate-based assay system for ana-lyses and screening of the Leishmania major inositol phosphorylceramidesynthase International Journal of Biochemistry amp Cell Biology 42 1553ndash1561Mina J G Thye J K Alqaisi A Q I Bird L E Dods R HGroftehauge M K Mosely J A Pratt S Shams-Eldin HSchwarz R T Pohl E and Denny PW (2017) Functional andphylogenetic evidence of a bacterial origin for the first enzyme in sphingo-lipid biosynthesis in a phylum of eukaryotic protozoan parasites Journal ofBiological Chemistry in press doi 101074jbcM117792374Montoya J G and Remington J S (2008) Management ofToxoplasmagondii infection during pregnancy Clinical Infectious Diseases 47 554ndash566Murcia L Carrilero B Munoz-Davila M J Thomas M CLoacutepez M C and Segovia M (2013) Risk factors and primary preven-tion of congenital Chagas disease in a nonendemic country ClinicalInfectious Diseases 56 496ndash502Nagiec MM Baltisberger J A Wells G B Lester R L andDickson R C (1994) The LCB2 gene of Saccharomyces and therelated LCB1 gene encode subunits of serine palmitoyltransferase theinitial enzyme in sphingolipid synthesis Proceedings of the NationalAcademy of Sciences of the United States of America 91 7899ndash7902Nagiec MM Nagiec E E Baltisberger J A Wells G BLester R L and Dickson R C (1997) Sphingolipid synthesis as atarget for antifungal drugs Journal of Biological Chemistry 272 9809ndash9817Newton P N Caillet C and Guerin P J (2016) A link between poorquality antimalarials and malaria drug resistance Expert Review of Anti-Infective Therapy 14 531ndash533Norcliffe J L Alvarez-Ruiz E Martin-Plaza J J Steel P G andDenny PW (2014) The utility of yeast as a tool for cell-based target-directed high-throughput screening Parasitology 141 8ndash16Ohanian J and Ohanian V (2001) Sphingolipids in mammalian cellsignalling Cellular and Molecular Life Sciences 58 2053ndash2068Ohnuki T Yano T Ono Y Kozuma S Suzuki T Ogawa Y andTakatsu T (2009) Haplofungins novel inositol phosphorylceramide syn-thase inhibitors from Lauriomyces bellulus SANK 26899 I Taxonomy fer-mentation isolation and biological activities Journal of Antibiotics 62 545ndash549Opremcak EM Scales D K and Sharpe M R (1992)Trimethoprim-sulfamethoxazole therapy for ocular toxoplasmosisOphthalmology 99 920ndash925Pal R (2015) Phase modulation nanoscopy a simple approach toenhanced optical resolution Faraday Discussions 177 507ndash515



Pankova-Kholmyansky I Dagan A Gold D Zaslavsky ZSkutelsky E Gatt S and Flescher E (2003) Ceramide mediatesgrowth inhibition of the Plasmodium falciparum parasite Cellular andMolecular Life Science 60 577ndash587Paquet C and Yudin MH (2013) Toxoplasmosis in pregnancy pre-vention screening and treatment Journal of Obstetrics and GynaecologyCanada 35 78ndash81Parija S C (2016) Drug resistance in malaria a predicament TropicalParasitology 6 1Park JW Park W J and Futerman A H (2014) Ceramidesynthases as potential targets for therapeutic intervention in human dis-eases Biochim Biophys Acta 1841 671ndash681PataM O Hannun Y A andNg C K-Y (2010) Plant sphingolipidsdecoding the enigma of the Sphinx New Phytologist 185 611ndash630Patnaik P K Field M C Menon A K Cross G A Yee M Cand Butikofer P (1993) Molecular species analysis of phospholipidsfrom Trypanosoma brucei bloodstream and procyclic forms Molecularand Biochemical Parasitology 58 97ndash105Pettus B J Chalfant C E and Hannun Y A (2002) Ceramide inapoptosis an overview and current perspectives Biochimica et BiophysicaActa (BBA) ndash Molecular and Cell Biology of Lipids 1585 114ndash125Pewzner-Jung Y Ben-Dor S and Futerman A H (2006) When doLasses (longevity assurance genes) become CerS (ceramide synthases)Insights into the regulation of ceramide synthesis Journal of BiologicalChemistry 281 25001ndash25005Pieperhoff M S Pall G S Jimenez-Ruiz E Das S Melatti CGow M Wong E H Heng J Muller S Blackman M J andMeissner M (2015) Conditional U1 gene silencing in Toxoplasmagondii Plos ONE 10 24Pierce S K (2002) Lipid rafts and B-cell activation Nature ReviewsImmunology 2 96ndash105Pratt S Wansadhipathi-Kannangara N K Bruce C R Mina JG Shams-Eldin H Casas J Hanada K Schwarz R TSonda S and Denny PW (2013) Sphingolipid synthesis and scaven-ging in the intracellular apicomplexan parasite Toxoplasma gondiiMolecular and Biochemical Parasitology 187 43ndash51Pruett S T Bushnev A Hagedorn K Adiga M Haynes C ASullards M C Liotta D C and Merrill A H (2008) Thematicreview series sphingolipids ndash biodiversity of sphingoid bases (lsquosphingo-sinesrsquo) and related amino alcohols Journal of Lipid Research 49 1621ndash1639Quintildeones W Urbina J A Dubourdieu M and LuisConcepcioacuten J (2004) The glycosome membrane of Trypanosoma cruziepimastigotes protein and lipid composition Experimental Parasitology106 135ndash149Raas-Rothschild A Pankova-Kholmyansky I Kacher Y andFuterman A H (2004) Glycosphingolipidoses beyond the enzymaticdefect Glycoconjugate Journal 21 295ndash304Ramakrishnan S Serricchio M Striepen B and Buumltikofer P(2013) Lipid synthesis in protozoan parasites a comparison between kine-toplastids and apicomplexans Progress in Lipid Research 52 488ndash512Raman M C Johnson K A Yard B A Lowther J Carter L GNaismith J H and Campopiano D J (2009) The external aldimineform of serine palmitoyltransferase structural kinetic and spectroscopicanalysis of the wild-type enzyme and HSAN1 mutant mimics Journal ofBiological Chemistry 284 17328ndash17339Ramaprasad A Mourier T Naeem R Malas T B Moussa EPanigrahi A Vermont S J Otto T D Wastling J and Pain A(2015) Comprehensive evaluation of Toxoplasma gondii VEG andNeospora caninum LIV genomes with tachyzoite stage transcriptome andproteome defines Novel transcript features Plos ONE 10 e0124473Ramstedt B and Slotte J P (2002) Membrane properties of sphingo-myelins Febs Letters 531 33ndash37Rao R P Scheffer L Srideshikan SM Parthibane VKosakowska-Cholody T Masood M A Nagashima KGudla P Lockett S Acharya U and Acharya J K (2014)Ceramide transfer protein deficiency compromises organelle function andleads to senescence in primary cells Plos ONE 9 e92142Richmond G S Gibellini F Young S A Major LDenton H Lilley A and Smith T K (2010) Lipidomic analysisof bloodstream and procyclic form Trypanosoma brucei Parasitology137 1357ndash1392Rieckmann K and Cheng Q (2002) Pyrimethamine-sulfadoxineresistance in Plasmodium falciparum must be delayed in Africa Trends inParasitology 18 293Romano J D Sonda S Bergbower E Smith M E andCoppens I (2013) Toxoplasma gondii salvages sphingolipids from thehost Golgi through the rerouting of selected Rab vesicles to the parasito-phorous vacuole Molecular Biology of the Cell 24 1974ndash1995

Rovina P Schanzer A Graf C Mechtcheriakova D Jaritz Mand Bornancin F (2009) Subcellular localization of ceramide kinaseand ceramide kinase-like protein requires interplay of their PleckstrinHomology domain-containing N-terminal regions together with C-ter-minal domains Biochimica Et Biophysica Acta-Molecular and CellBiology of Lipids 1791 1023ndash1030Ryan U andHijjawi N (2015) New developments in Cryptosporidiumresearch International Journal for Parasitology 45 367ndash373Sandmeier E Hale T I and Christen P (1994) Multiple evolution-ary origin of pyridoxal-5prime-phosphate-dependent amino acid decarboxy-lases European Journal of Biochemistry 221 997ndash1002Sandosham A A Eyles D E and Montgomery R (1964) Drug-resistance in falciparum malaria in South-East Asia Medicinal Journal ofMalayasia 18 172ndash183Schmidt T J Khalid S A Romanha A J Alves TMBiavatti MW Brun R Da Costa F B de Castro S LFerreira V F de Lacerda M V G Lago J H G Leon L LLopes N P Amorim R C D Niehues M Ogungbe I VPohlit AM Scotti M T Setzer WN Soeiro M D CSteindel M and Tempone A G (2012) The potential of secondarymetabolites from plants as drugs or leads against protozoan neglected dis-eases ndash part I Current Medicinal Chemistry 19 2128ndash2175Seacutegui B Andrieu-Abadie N Jaffreacutezou J-P Benoist H andLevade T (2006) Sphingolipids as modulators of cancer cell deathpotential therapeutic targets Biochimica et Biophysica Acta (BBA) ndashBiomembranes 1758 2104ndash2120Serpeloni M Jimenez-Ruiz E Vidal NM Kroeber CAndenmatten N Lemgruber L Morking P Pall G SMeissner M and Avila A R (2016) UAP56 is a conserved crucialcomponent of a divergent mRNA export pathway in Toxoplasma gondiiMolecular Microbiology 102 672ndash689Sevova E S Goren M A Schwartz K J Hsu F F Turk JFox B G and Bangs J D (2010) Cell-free synthesis and functionalcharacterization of sphingolipid synthases from parasitic trypanosomatidprotozoa Journal of Biological Chemistry 285 20580ndash20587Sheridan C (2011) Industry continues dabbling with open innovationmodels Nature Biotechnology 29 1063ndash1065Sidik SM Huet D Ganesan SM Huynh M-H Wang TNasamu A S Thiru P Saeij J P J Carruthers V B Niles J Cand Lourido S (2016) A genome-wide CRISPR screen in toxoplasmaidentifies essential apicomplexan genes Cell 166 1423ndash1435e1412Sims P F G (2009) Drug Resistance in Toxoplasma gondii InAntimicrobial Drug Resistance Clinical and Epidemiological Aspects (edMayers D L) pp 1121ndash1126 Humana Press Totowa NJSmith T K and Buumltikofer P (2010) Lipid metabolism in Trypanosomabrucei Molecular and Biochemical Parasitology 172 66ndash79Sonda S Sala G Ghidoni R Hemphill A and Pieters J (2005)Inhibitory effect of Aureobasidin A on Toxoplasma gondii AntimicrobialAgents and Chemotherapy 49 1794ndash1801Soto J and Berman J (2006) Treatment of NewWorld cutaneous leish-maniasis with miltefosine Transactions of the Royal Society of TropicalMedicine and Hygiene 100 S34ndashS40Sow S O Muhsen K Nasrin D Blackwelder W C Wu YFarag T H Panchalingam S Sur D Zaidi A KMFaruque A S G Saha D Adegbola R Alonso P LBreiman R F Bassat Q Tamboura B Sanogo DOnwuchekwa U Manna B Ramamurthy T Kanungo SAhmed S Qureshi S Quadri F Hossain A Das S KAntonio M Hossain M J Mandomando I Nhampossa TAcaacutecio S Omore R Oundo J O Ochieng J B Mintz E DOrsquoReilly C E Berkeley L Y Livio S Tennant SMSommerfelt H Nataro J P Ziv-Baran T Robins-Browne RM Mishcherkin V Zhang J Liu J Houpt E R Kotloff K Land Levine MM (2016) The burden of cryptosporidium diarrhealdisease among children lt24 months of age in ModerateHigh MortalityRegions of Sub-Saharan Africa and South Asia utilizing data from theGlobal Enteric Multicenter Study (GEMS) PLOS Neglected TropicalDiseases 10 e0004729Spassieva S Seo J G Jiang J C Bielawski J Alvarez-Vasquez F Jazwinski SM Hannun Y A and Obeid LM(2006) Necessary role for the Lag1p motif in (dihydro)ceramide synthaseactivity Journal of Biological Chemistry 281 33931ndash33938Sugi T Kato K and Weiss LM (2016) An improved method forintroducing site-directed point mutation into the Toxoplasma gondiigenome using CRISPRCas9 Parasitology International 65 558ndash562Sugimoto Y Sakoh H and Yamada K (2004) IPC synthase as auseful target for antifungal drugs Current Drug Targets InfectiousDisorders 4 311ndash322



Sunder S Jha T K Thakur C P Engel J Sindermann HFischer C Jungle K Bryceson A and Berman J (2002) Oral mil-tefosine for Indian visceral leishmaniasisNew England Journal of Medicine347 1739ndash1746Sutterwala S S Creswell C H Sanyal S Menon A K andBangs J D (2007) De novo sphingolipid synthesis is essential for viabil-ity but not for transport of glycosylphosphatidylinositol-anchored pro-teins in African trypanosomes Eukaryot Cell 6 454ndash464Sutterwala S S Hsu F F Sevova E S Schwartz K J Zhang KKey P Turk J Beverley SM and Bangs J D (2008)Developmentally regulated sphingolipid synthesis in African trypano-somes Molecular Microbiology 70 281ndash296Tafesse F G Vacaru AM Bosma E F Hermansson MJain A Hilderink A Somerharju P and Holthuis J CM(2014) Sphingomyelin synthase-related protein SMSr is a suppressor ofceramide-induced mitochondrial apoptosis Journal of Cell Science 127445ndash454Takesako K Kuroda H Inoue T Haruna F Yoshikawa YKato I Uchida K Hiratani T and Yamaguchi H (1993)Biological properties of aureobasidin-A a cyclic depsipeptide antifungalantibiotic Journal of Antibiotics 46 1414ndash1420Talbott CM Vorobyov I Borchman D Taylor K G DuPreacute DB and Yappert M C (2000) Conformational studies of sphingolipids byNMR spectroscopy II Sphingomyelin Biochimica et Biophysica Acta(BBA) ndash Biomembranes 1467 326ndash337Tanaka A K Valero V B Takahashi H K and Straus A H(2007) Inhibition of Leishmania (Leishmania) amazonensis growth andinfectivity by aureobasidin A Journal of Antimicrobial Chemotherapy 59487ndash492Tatematsu K Tanaka Y Sugiyama M Sudoh M andMizokami M (2011) Host sphingolipid biosynthesis is a promisingtherapeutic target for the inhibition of hepatitis B virus replicationJournal of Medical Virology 83 587ndash593Thudichum J LW (1884)ATreatise on the Chemical Constitution of theBrain Archon Books Hamden ConnTorgerson P R and Macpherson CN L (2011) The socioeconomicburden of parasitic zoonoses global trendsVeterinary Parasitology 182 79ndash95Vacaru AM Tafesse F G Ternes P Kondylis VHermansson M Brouwers J Somerharju P Rabouille C andHolthuis J CM (2009) Sphingomyelin synthase-related proteinSMSr controls ceramide homeostasis in the ER Journal of Cell Biology185 1013ndash1027Vance D E and Vance J E (2002) Biochemistry of Lipids Lipoproteinsand Membranes 4th ed Elsevier ScienceVerma N K and Dey C S (2004) Possible mechanism of miltefosine-mediated death of Leishmania donovani Antimicrobial Agents andChemotherapy 48 3010ndash3015Wang W Yang X Tangchaiburana S Ndeh R Markham J ETsegaye Y Dunn TM Wang G L Bellizzi M Parsons J F

Morrissey D Bravo J E Lynch D V and Xiao S (2008) An inosi-tolphosphorylceramide synthase is involved in regulation of plant pro-grammed cell death associated with defense in Arabidopsis Plant Cell20 3163ndash3179Ward E N and Pal R (2017) Image scanning microscopy an overviewJournal of Microscopy 266 221ndash228Watson D G (2010) The potential of mass spectrometry for the globalprofiling of parasite metabolomes Parasitology 137 1409ndash1423Webster J P Kaushik M Bristow G C and McConkey G A(2013) Toxoplasma gondii infection from predation to schizophrenia cananimal behaviour help us understand human behaviour Journal ofExperimental Biology 216 99ndash112Weiss B and Stoffel W (1997) Human and murine serine-palmitoyl-CoA transferase European Journal of Biochemistry 249 239ndash247Welti R Mui E Sparks A Wernimont S Isaac G Kirisits MRoth M Roberts CW Botte C Marechal E and McLeod R(2007) Lipidomic analysis of Toxoplasma gondii reveals unusual polarlipids Biochemistry 46 13882ndash13890WHO (2004) The World Health Report 2004 Changing History WHOGenevaWHO (2016)Malaria ndash Fact Sheet (Dec 2016) WHO Media Centre vol2017Wuts P GM Simons L J Metzger B P Sterling R CSlightom J L and Elhammer A P (2015) Generation of Broad-Spectrum Antifungal Drug Candidates from the Natural ProductCompound Aureobasidin A ACSMedicinal Chemistry Letters 6 645ndash649Yatsu FM (1971) SPHINGOLIPIDOSES California Medicine 1141ndashampYoung S A Mina J G Denny PW and Smith T K (2012)Sphingolipid and ceramide homeostasis potential therapeutic targetsBiochemistry Research International 2012 12Zhang K Showalter M Revollo J Hsu F F Turk J andBeverley SM (2003) Sphingolipids are essential for differentiationbut not growth in Leishmania Embo Journal 22 6016ndash6026Zhang K Hsu F-F Scott D A Docampo R Turk J andBeverley SM (2005) Leishmania salvage and remodelling of hostsphingolipids in amastigote survival and acidocalcisome biogenesisMolecular Microbiology 55 1566ndash1578Zhang K Bangs J D and Beverley SM (2010) Sphingolipids inparasitic protozoa Advances in Experimental Medicine and Biology 688238ndash248Zhou L J Xia J Wei H X Liu X J and Peng H J (2017) Risk ofdrug resistance in Plasmodium falciparum malaria therapy-a systematicreview and meta-analysis Parasitology Research 116 781ndash788Zofou D Nyasa R B Nsagha D S Ntie-Kang F Meriki H DAssob J C N and Kuete V (2014) Control of malaria and othervector-borne protozoan diseases in the tropics enduring challengesdespite considerable progress and achievements Infectious Diseases ofPoverty 3 1



Everybody needs sphingolipids right Mining for new drugtargets in protozoan sphingolipid biosynthesis

JOHN G M MINA and P W DENNY

Department of Biosciences Lower Mountjoy Stockton Road Durham DH1 3LE UK

(Received 10 April 2017 revised 15 May 2017 accepted 18 May 2017)

SUMMARY

Sphingolipids (SLs) are an integral part of all eukaryotic cellular membranes In addition they have indispensable functions assignallingmolecules controlling a myriad of cellular events Disruption of either the de novo synthesis or the degradation path-ways has been shown to have detrimental effects The earlier identification of selective inhibitors of fungal SL biosynthesispromised potent broad-spectrum anti-fungal agents which later encouraged testing some of those agents against protozoanparasites In this review we focus on the key enzymes of the SL de novo biosynthetic pathway in protozoan parasites of theApicomplexa and Kinetoplastidae outlining the divergence and interconnection between host and pathogen metabolismThe druggability of the SL biosynthesis is considered alongside recent technology advances that will enable the dissectionand analyses of this pathway in the parasitic protozoa The future impact of these advances for the development of newtherapeutics for both globally threatening and neglected infectious diseases is potentially profound

Key words sphingolipids ceramide drug targets protozoan parasites apicomplexa kinetoplastidae

INTRODUCTION

Protozoan parasites and the global burden of theirdiseases

Protozoa (kingdom Protista) are single-cell organismsthat can be free-living or parasitic in nature (Baron1996) Out of more than 50 000 protozoan speciesthat have been described to-date relatively few havebeen identified as major contributors to the globalburden of human diseases (Kuris 2012) and animalagriculture (Dubey 1977) The protozoa represent19 of all human parasites (83 out of 437 species to-date) and are associated with 30 of parasite-inducedhuman morbidity-mortality (Kuris 2012)Of the four groups of infectious protozoa (CDC

2017) the Mastigophora (flagellates) and Sporozoacontain theKinetoplastidae andApicomplexa respect-ively It is to these two phyla that belong many ofthe causative agents of disease Mastigophora ndash theinsect vector-borne kinetoplastids Trypanosomabrucei (Human African Trypanosomiasis HAT)Leishmania spp (leishmaniasis cutaneous andvisceral)and Trypanosoma cruzi (American trypanosomiasisChagasrsquo disease) Sporozoa ndash the apicomplexanToxoplasma gondii (toxoplasmosis) Cryptosporidiumspp (cryptosporidiosis) andEimeria spp (coccidiosisinpoultry andcattle)Theileria spp (EastCoastFeverin cattle) andPlasmodium spp includingPlasmodiumfalciparum the causative agent of severe malaria andone of the lsquoBig Threersquo global infectious diseases

alongside HIV and tuberculosis (Torgerson ampMacpherson 2011)Historically the diseases caused by some of these

parasites have been classified as Neglected TropicalDiseases (NTDs) or Neglected Zoonotic Diseases(King 2011) and were associated with the classicalmodel of the lsquopoverty traprsquo covering tropical andsub-tropical regions in Africa Latin America andthe Indian subcontinent (Kuris 2012) Howeverwith global changes in climate and human demo-graphics and associated practices the classicalmodels do not promise safe boundaries that mightcontain andor stop the further global spread ofmany of these parasitic diseases (Colwell et al 2011)The problems associated with these pathogens arefurther aggravated by the lack of effective vaccines(Dumonteil 2007 Innes et al 2011 McAllister2014 BlackampMansfield 2016) and the paucity of reli-able drugs (Zofou et al 2014) in addition to thedifficulties of vector or reservoir control (Colwellet al 2011) Therefore there is a recognized need tofind new therapeutic targets in these causative agentsin order to develop effective treatment regimens toavoid potentially catastrophic outbreaks both interms of human health and economic impactThis review presents sphingolipid (SL) biosyn-

thesis and ceramide (CER) homoeostasis as a poten-tial gold mine of tractable drug targets for theseprotozoan parasites

State-of-the-art treatment of apicomplexan andkinetoplastid diseases

In general available treatments for the diseasescaused by the Kinetoplastidae and Apicomplexa

Corresponding author Department of BiosciencesLower Mountjoy Stockton Road Durham DH1 3LEUK E-mail jgmminadurhamacuk

1SPECIAL ISSUE REVIEW

Parasitology Page 1 of 14 copy Cambridge University Press 2017 This is an Open Access article distributed under the terms of theCreative Commons Attribution licence (httpcreativecommonsorglicensesby40) which permits unrestricted re-use distributionand reproduction in any medium provided the original work is properly citeddoi101017S0031182017001081





































SL METABOLISM

















Ceramide synthase





























PERSPECTIVE









ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES
























































SL METABOLISM

















Ceramide synthase





























PERSPECTIVE









ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES































Ceramide synthase





























PERSPECTIVE









ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES






































PERSPECTIVE









ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES
























ACKNOWLEDGMENTS


FINANCIAL SUPPORT


REFERENCES





















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