Living in a phagolysosome;metabolism of LeishmaniaamastigotesMalcolm J. McConville1,2, David de Souza1,2, Eleanor Saunders1,2,Vladimir A. Likic2 and Thomas Naderer1,2

1 Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia2 Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia

Review TRENDS in Parasitology Vol.23 No.8

Leishmania amastigotes primarily proliferate withinmacrophages in the mammalian host. Genome-basedmetabolic reconstructions, combined with biochemical,reverse genetic and mRNA or protein profiling studiesare providing new insights into the metabolism of thisintracellular stage. We propose that the complex nutri-tional requirements of amastigotes have contributed tothe tropism of these parasites for the amino acid-richphagolysosome of macrophages. Amastigote metab-olism in this compartment is robust because manymetabolic mutants are capable of either growing nor-mally or persisting long term in susceptible animals.New approaches for measuring amastigote metabolismin vivo are discussed.

The enemy withinSeveral important human microbial pathogens proliferatewithin macrophages (Mø). Although most of these patho-gens evade the microbicidal responses of the Mø host bysubverting or escaping from the phagocytic pathway, pro-tozoan parasites belonging to the genus Leishmania man-age both to survive and to proliferate within the maturephagolysosome compartment of Mø. The infection of Mø byLeishmania is initiated by flagellated, metacyclic (non-dividing) promastigotes, which are injected into the skinof themammalian host by the phlebotomine sandfly vector.Metacyclic promastigotes are phagocytosed by Mø eitherdirectly or after cycling through a wave of neutrophils(which are rapidly recruited to the site of the sandfly bitebut then undergo apoptosis) [1]. Promastigotes interna-lized byMø are delivered to the phagolysosome, where theydifferentiate into small, aflagellated amastigotes. Amasti-gotes are a replicative stage that periodically escape fromthe host cell by a poorly defined mechanism and rapidlyreinfect other phagocytes (i.e. Mø or dendritic cells) andsome non-phagocytic cell types (i.e. fibroblasts) [2,3].Amastigotes are the cause of acute disease (ranging fromself-healing cutaneous infections to severe disfiguringmucocutaneous and lethal visceral disease), as well aschronic or latent infections that can persist for the lifetime of the host. Despite the importance of amastigotes in

Corresponding author: McConville, M.J. (malcolmm@unimelb.edu.au).Available online 2 July 2007.

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perpetuating disease and as the target of antileishmanialdrugs, comparatively little is known about either themetabolism of this stage in vivo or the biochemical com-position of the phagolysosome. Although in vitro cultivatedamastigotes (derived from in vitro differentiated promas-tigotes or lesion amastigotes) have been used to investigatesome aspects of amastigote metabolism, these stages aretypically grown in rich medium that is unlikely to mimicthe biochemical milieu of the phagolysosome. Analysis ofamastigote metabolism in vivo is further complicated bythe fact that different species of Leishmania reside withindifferent populations ofMø and can inducemorphologicallydistinct phagolysosomes within the same population of Mø[3]. For example, members of the Leishmania mexicanacomplex induce spacious communal vacuoles rather thanthe tight fitting individual phagolysosomes occupied bymany other species (e.g. Leishmania major, Leishmaniadonovani). Moreover, the L. mexicana-occupied phagolyso-somes become filled with electron-dense material over thecourse of several days, thus indicating temporal changesin the biochemical composition of the phagolysosomelumen [4]. In this review, we highlight recent insights intothe in vivo metabolism of intracellular amastigotes thathave emerged from genome-based metabolic reconstruc-tions, the generation of metabolic mutants and profilingapproaches.

Nutrient availability and uptake in thephagolysosomeIntracellular stages of Leishmania must scavenge all oftheir carbon source and micronutrient requirements fromthe lumen or limiting membrane of the Mø phagosome(Figure 1). Based on the known nutrient requirements ofcultured stages [5] and genome-based reconstructions of theLeishmaniametabolome [6,7], it is predicted that Leishma-nia amastigotes must scavenge all their purine require-ments, many vitamins and at least ten essential aminoacids from the Mø phagolysosome (Figure 2). The uptakeof these essential metabolites is mediated by families ofpolytopic membrane transporters, which are often protonsymporters, enabling high affinity uptake of substrates inthe acidic phagolysosome [5,8–10] (Table 1). In some cases,distinct transporters enable uptake of different forms ofthe same metabolite class, providing a high degree of

d. doi:10.1016/j.pt.2007.06.009

Figure 1. Life in the phagolysosome. Leishmania amastigotes proliferate within

tight-fitting, individual or large communal phagolysosomes. Essential nutrients,

cations and carbon sources are delivered to the phagolysosome via the endocytic

pathway or directly from the macrophage cytosol. Amastigotes might internalize low

molecular weight nutrients (such as hexose, amino acids, polyamines, purines and

vitamins) and cations (Fe2+, Ms2+) via plasma membrane transporters, often in

competition with phagolysosome membrane transporters. The also internalize large

macromolecules (such as proteins, carbohydrates, DNA and RNA by endocytosis.

Heme can be obtained by endocytosis of host proteins or through uptake of free

heme by FP receptors. A tight junction (shown on the right) might form between the

posterior membrane of the amastigote and the phagolysosome membrane, and be

involved in scavenging host lipids. Abbreviations: Lyso, parasite lysosome; FP,

flagellar pocket; PV, parasitophorous vacuole/phagolysosome.

Review TRENDS in Parasitology Vol.23 No.8 369

redundancy in nutrient salvage pathways [11–14]. Forexample, distinct transporters are involved in internalizingnucleobases, nucleosides and possibly nucleotides, all ofwhich are potential precursors for purine salvage. As aresult, single or double deletions of these transporters havelittle effect on amastigote growth [15]. Redundancy is alsoapparent in the uptake of hexoses, polyamines and folates(Table 1, Figure 2). In addition to small metabolites, Leish-mania amastigotes can scavenge complex lipids and(glyco)proteins from the phagolysosome. All Leishmaniaamastigotes seem to incorporate host glycosphingolipidsinto their plasma membrane [16]. These or other hostsphingolipids might be internalized by intracellular amas-tigotes and used to synthesize endogenous sphingolipids,

Table 1. Cloned transporters expressed in Leishmania amastigote

Nutrient Gene(s)a Comm

Monosaccharides GT1, GT2 and GT3 GT1 an

GT2 de

myo-inositol D1 Expres

Amino acids AAP3 Arginin

PAT1 Amast

Polyamines POT1 Relate

Nucleosides NT1 Adeno

NT2 Inosine

Nucleobases NT3 (Hypo)

NT4 Undefi

Biopterin BT1 Deletio

Folic acid FT1 Transp

Iron Lit1 RequiraAbbreviations: AAP3, amino acid permease 3; BT, biopterin transporter; D1, inositol tra

PAT1, putative amino acid transporter 1; POT1, polyamine transporter.

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such as inositolphosphoceramide [17]. Essential aminoacids and hememight also be obtained from the proteolysisof host phagolysosome proteins [18] and exogenous proteinsdelivered to the phagolysosome via endocytosis and autop-hagy [19]. Digestion of glycoproteins in the highly activeamastigote lysosome would provide a source of essentialamino acids and sugars [4,20]. Endocytic uptake is import-ant because disruption of either endocytosis or lysosomalfunction markedly reduces amastigote virulence [21–23](Table 2). Although these analyses suggest that both lipidsand amino acids are important carbon sources for amasti-gotes, the L. major genome lacks two key enzymes in theglyoxylate pathway, which is required for net conversion ofacetyl-CoA, the end-product of fatty acid b-oxidation, intosugars [6]. The absence of this pathway in Leishmania,together with recent metabolic labeling experiments [24],indicates thatamastigotesareunable toutilize fattyacidsastheir major carbon source.

The nutritional requirements of Leishmaniaamastigotes, briefly summarized here, are considerablymore complex than those of most prokaryotes and fungalpathogens, and might have prevented Leishmania amas-tigotes from occupying many intracellular niches in themammalian host. In particular, they are likely to haveprecludedLeishmania from colonizing the early endosomalor non-hydrolytic vacuoles of Mø, which are rich in lipidsbut contain low levels of sugars and amino acids [25,26]. Bycontrast, the phagolysosomes of Mø are predicted to con-tain relatively high levels of amino acids, as shown bytranscript profiling studies of phagocytosed fungal patho-gens [25,27]. Mø could be unusual in this respect, asphagocytosis of Candida albicans by neutrophils inducesa strong amino acid starvation response in the fungus [27].Host-specific differences in the nutrient composition of thehost cell phagosomes could be part of the reason whyLeishmania promastigotes cannot proliferate or differen-tiate to amastigotes in neutrophils [1] but can in Mø.Similar considerations might dictate the tissue tropismof other microbial pathogens. For example, the gram-nega-tive bacterium, Coxiella burnetti, is one of the few othermicrobial pathogens to inhabit the phagolysosome of Mø.Like Leishmania, C. burnetti is an auxotroph for manyamino acids (i.e. it needs these amino acids externally,having no de novo pathway for their synthesis) andlacks the glyoxylate pathway [28]. As with Leishmania,

stages

ents Refs

d 3 expressed in all stages [72]

creased in amastigotes

sion induced by low inositol levels [9]

e uptake [12]

igote-specific amino acid permease [10]

d transporters probably expressed in amastigotes [8]

sine, pyrimidine [11]

, guanosine [11]

xanthine, adenine, guanine [11]

ned

n results in increased virulence [14]

orts folate and methotrexate [14]

ed for intracellular growth [41]

nsporter; FT, folate transporter; GT, glucose transporters; NT, purine transporters;

Figure 2. Metabolism of Leishmania amastigotes. Metabolic pathways known to be operative in Leishmania amastigotes based on biochemical analysis and infectivity

phenotypes of mutant strains. Metabolic steps are represented by arrows. Unbroken arrows indicate direct steps in a pathway, broken arrows indicate multiple steps in a

pathway not shown. The subcellular localization of specific pathways is based on the subcellular localization of one or more enzymes in the pathway and/or the presence of

targeting presequences in those enzymes. Note that many glycosomal enzymes have a dual localization in the cytosol. Reactions that can be disrupted without loss of

infectivity in the mammalian host are indicated in blue, whereas those that are required for lesion-development are indicated in red. Amino acids are indicated in the three-

letter code. Abbreviations: AcCoA, acetyl-CoA; AMP, adenosine monophosphate; CpB, cysteine proteinase B family; DHAP, dihydroxyacetone phosphate; DPM, dolichol

phosphate mannose; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; F6P, fructose-6-phosphate; FA-CoA, fatty acyl-CoA; Fru, fructose;

GAP, glyceraldehyde-3-phosphate; GDP-Man, GDP–mannose; Glc, glucose; GMP, guanosine monophosphate; GroP, glycerol-3-phosphate; G6P, glucose-6-phosphate; GPI,

glycosylphosphatidylinositol; HMG-CoA, 3-hydroxy-3-methylglutaryl CoA; IMP, inosine monophosphate; Ino, myo-inositol; Ino3P, inositol-3-phosphate; IPC,

inositolphosphoceramide; aKG, a-ketoglutarate; LPG, lipophosphoglycan; M1P, mannose-1-phosphate; M6P, mannose-6-phosphate; Mann/Mann+1, b1–2mannan

oligomers; OAA, oxaloacetate; OAc, acetate; Orn, ornithine; PEP, phosphoenolpyruvate; PRPP, phosphoribose pyrophosphate; PPG, proteophosphoglycan; PPi,

pyrophosphate; PtdIno, phosphatidylinositol; Put, putrescine; Pyr, pyruvate; Rib, ribose; Rib5P, ribose-5-phosphate; AldoMet, S-adenosylmethionine; SL, sphingolipid;

Sper, spermidine; Succ, succinate; TAG, triacylglycerol; T[SH]2 and TS2, trypanothione (reduced and non-reduced); UDP-Gal, UDP-galactopyranose. Major metabolic end-

products are highlighted in black.

370 Review TRENDS in Parasitology Vol.23 No.8

C. burnetti might have to trade increased exposure to hostmicrobicidal processes for access to nutrients.

Use of stable mutants to identify metabolic pathwaysrequired for virulenceThe genetic disruption of specificmetabolic pathways is themost rigorous approach for demonstrating a requirementfor intracellular growth (but see Box 1 for pros and cons).The Leishmaniamutants listed in Table 2 can be classifiedinto three categories. PAV (promastigote avirulent)mutants are poorly infective as promastigotes, but fullyinfective if viable amastigotes can be generated. Thesemutants characteristically generate lesions after a delayof several weeks. AAV (amastigote avirulent) mutants are

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unable to survive in susceptible animals (or macrophages)as promastigotes and/or amastigotes. APER (amastigotepersistent) mutants differentiate to amastigotes and cansurvive long term in infected macrophages or susceptiblemice, but are unable to induce lesions.

A major group of L. major PAV mutants are unable tosynthesize components of the complex cell surface glycoca-lyx of promastigotes, such as the lipophosphoglycan (LPG),glycosylphosphatidylinositol (GPI)-anchored proteins andfree GPI glycolipids (GIPLs) (Table 2). Because amastigotesare coated by a much simpler glycocalyx (lacking LPG andGPI-anchored proteins) [29], it is not surprising that loss ofenzymes involved in their synthesis has little affect onamastigote virulence (Table 2). Although intracellular

Table 2. Metabolic pathways required for Leishmania infectivity

Pathwaya Gene(s) Speciesb Virulence Refs

Proc Amad

PAV (promastigote avirulent) mutants

LPG1 (Galf-transferase) LPG biosynthesis Lmj � +/+ [73]

Lmx + +/+

UDP-Gal mutase LPG/GIPL biosynthesis Lmj � +/+ [74]

Alkyl-DHAP synthase Ether lipid Lmj � +/+ [37]

Ser:palmitoyltransferase Sphingolipid Lmj � +/+ [17]

LmxPK4 (MAP kinase kinase) Protein kinase Lmx � +/+ [49]

AAV (amastigote avirulent) mutants

Trypanothione reductase Trypanothione regeneration

(partial k/o or dominant negative)

Ldn � �/� [75]

hgprt xprt double mutant Purine scavenging Ldn � [76]

Hexose transporter-1,-2 & -3 Glucose uptake Lmx � �/� [13]

Phosphomannomutase Mannose metabolism Lmx � �/� [67]

GDP–mannose pyrophosphorylase Mannose metabolism Lmx � �/� [33]

DHAP acyltransferase Acylglycerolipid biosynthesis Lmj � �/� [77]

Inositol-3-phosphate synthase Myo-inositol biosynthesis Lmx � �/� [78]

AP1 s / m subunits Endocytosis Lmx � �/� [22]

LMPK (MAP kinase) Protein kinase Lmx � �/� [50]

APER (amastigote persistent) mutants

Fructose-1,6-bisphosphatase Gluconeogenesis Lmj + +/� [24]

dhfr-ts Thymidine synthesis Lmj + +/� [38]

LitA Iron uptake Lam + +/� [42]

cpB Lysosome protease Lmx – +/� [21]

cpA Lysosome protease Linf – +/� [79]

LPG2 (GDP–mannose transporter)e Phosphoglycan biosynthesis Lmj � +/� [31]

Lmx + +/+ [30]aOnly metabolic pathways required for virulence are included in this list (metabolic enzymes required for growth in rich medium are not included). Abbreviations; AP1,

adaptor protein 1; CpB, cysteine proteinase B family; DHAP, dihydroxyacetone phosphate; hgprt, hypoxanthine-guanine phosphoribosyltransferase; xprt, xanthine

phosphoribosyltransferase.bLmj, L. major; Lmx, L. mexicana; Ldn, L. donovani; Lam, L. amazonensis; Linf, L. infantum.cPromastigote (Pro) infectivity. + Promastigotes infect Mø and differentiate to amastigotes similar to wild type strains.� Promastigotes poorly infective in Mø and/or unable to

differentiate to amastigotes.dAmastigote (Ama) infectivity; +/+ amastigotes infect Mø and induce lesions in mice with similar kinetics to wild-type parasites. +/� amastigotes survive in macrophages and

susceptible mice, but do not induce lesions. �/� amastigotes unable to survive in Mø or susceptible mice.eDeletions of the lpg1 and lpg2 genes in L. mexicana have no effect on virulence, whereas comparable deletions in L. major induce a PAV and an APER phenotype, respectively.

Review TRENDS in Parasitology Vol.23 No.8 371

amastigotes retain a surface glycocalyx of GIPLs [29], theymight be able to compensate for the loss of these glycolipidsby scavenging glycosphingolipids from the host cell (seebelow). Promastigote and amastigote stages also expresson their surfaces and secrete several distinct classes ofproteophosphoglycans (PPGs). Although loss of both LPGand PPG glycosylation in L. major (owing to ablation of thelpg2 gene encoding a Golgi GDP–mannose transporter)resulted in a marked reduction in both promastigoteand amastigote virulence, disruption of the lpg2 gene inL. mexicana has no measurable effect on either promasti-gote or amastigote virulence [30,31]. These studies initiallysuggested that PPG is a virulence factor inL.major, but notinL.mexicana. However, analysis of anotherL.majorLPG/PPG mutant (defective in a Golgi UDP-galactopyranosetransporter) has revealed that PPG biosynthesis per se isnot required for amastigote virulence [32]. Thus, none of themajor surface glycoconjugates of Leishmania amastigotesseems to be essential for amastigote survival and growthin vivo.

In contrast to the surface coat mutants, L. mexicanamutants with global defects in sugar nucleotide biosyn-thesis and carbohydrate metabolism constitute an import-ant group of AAVmutants (Table 2). In particular, deletionof enzymes involved in mannose and inositol metabolismresult in effective loss of promastigote and amastigoteinfectivity (Table 2). In the case of the mannose mutants,

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loss of infectivity was attributed to the cumulative loss ofall major surface glycoconjugates [33]. However, morerecent studies have highlighted the potential importanceof a unique, intracellular mannan reserve material in L.mexicana that might be important for intracellular survi-val [34]. Defects in mannan synthesis [35] and possiblyother glycosylation pathways, such as protein N-glycosyl-ation, could therefore contribute to the severe loss ofinfectivity of the mannose mutants. The importance ofcarbohydrate metabolism more generally is supported bythe finding that a L. mexicana mutant lacking three glu-cose transporters (GTs) is avirulent in Mø [13]. The sever-ity of the L. mexicana Dgt mutant virulence phenotype issurprising given that Leishmania amastigotes are capableof de novo glucose biosynthesis via the gluconeogenic path-way [24]. It is possible that gluconeogenesis is insufficientto provide all of the glucose requirements of intracellularamastigotes or that a minimum level of glucose uptake isrequired for nutrient sensing. Other AAV mutants incl-ude those with defects in trypanothione metabolism, pur-ine salvage, glycerolipid biosynthesis and endocytosis(Table 2). Trypanothione is involved in many metabolicprocesses and is a key component in the oxidative defensesof Leishmania, whereas purine salvage is essential forgrowth in the absence of de novo purine biosynthetic path-ways. The reason why disruption of dihydroxyacetonephosphate acyltransferase (an early enzyme in glycerolipid

Box 1. The pros and cons of Leishmania metabolic mutants

A variety of approaches have been used to genetically disrupt

Leishmania metabolism. In most cases, the gene of interest is

deleted by targeted replacement of the chromosomal alleles with

drug resistance cassettes [60]. Antisense approaches, chemical and

transposon mutagenesis, and the expression of proteins with

dominant-negative function [60,61] have also been used. Although

L. major lacks the machinery for RNA interference, the enzymes

needed for this pathway are present in members of the Leishmania

braziliensis complex [62], suggesting that it could be possible to use

RNA interference to knock down gene expression in the future. The

analysis of Leishmania metabolic mutants has been informative, but

interpreting the phenotypes can be complicated.

First, because most gene targeting manipulations are performed

on the promastigote stage, disruption of metabolic processes that

selectively perturb promastigote infectivity in macrophages and/or

the capacity of promastigotes to differentiate into amastigotes

might be classified as being essential when, in fact, they are not

required for amastigote survival. Several L. major mutants lacking

the capacity to synthesize surface lipophosphoglycan have this

phenotype [63]. A small number of these mutants survive as

promastigotes and transform into amastigotes, which then grow

at the same rate as wild-type amastigotes in macrophages [63].

Although difficult to detect in in vitro infection experiments, these

mutants often display a characteristic delayed lesion phenotype in

animal models [37,63].

Second, the disruption of a single metabolic enzyme or pathway

can often be compensated for by other metabolic reactions resulting

in a silent growth or virulence phenotype [14,15,64]. However, the

disrupted metabolic steps might still be important at stages of

infection not measured by the investigator. Alternatively, disruption

of a particular pathway might result in more drastic compensatory

changes in metabolic networks and the selection of bypass mutants.

Compensatory mutants of L. major Dlpg2 and L. mexicana Dpk4 lack

the pathway or the protein that was initially deleted, but have fully

restored virulence [49,65].

Third, gene deletion studies are time consuming and are often

performed in laboratory-adapted strains that might not be repre-

sentative of clinical isolates. Examples exist where the deletion of a

particular gene was readily accomplished in one strain of Leishma-

nia but not another [66,67]. The consequences of deleting a gene in

one species might also be different in another species. For example,

markedly different virulence phenotypes were observed when

genes for lipophosphoglycan (lpg1) and phosphoglycan (lpg2)

biosynthesis were disrupted in L. major and L. mexicana (Table 2).

As noted in the text, the differences in the virulence phenotype of

the L. major and L. mexicana Dlpg2 mutants might reflect

perturbation of additional, but undefined, pathways in L. major.

These studies show that it is not always possible to extrapolate the

finding of gene deletion studies from one species to another.

372 Review TRENDS in Parasitology Vol.23 No.8

biosynthesis) results in loss of virulence is unclear, giventhat amastigotes can scavenge fatty acids from the hostand genetic disruption of other genes in glycerolipid bio-synthesis has no affect on virulence [36,37]. The require-ment for endocytosis might also be complex. Endocytosedhost proteins are likely to be an important source ofnutrients, whereas endocytic membrane transport andlysosome function are required for autophagy and turnoverof parasite proteins and organelles during differentiationand normal growth [23].

APER mutants are able to invade Mø and differentiateinto amastigotes, but fail to proliferate in the Mø in vitro(Table 2). These mutants can persist for long periods oftime on susceptible hosts, but generally fail to inducelesions despite the presence of reasonable parasite num-bers. Several of these mutants are auxotrophic for nutri-ents normally synthesized by wild-type parasites,

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providing insights into the nutrient environment of theMø phagolysosome. For example, the APER phenotype ofL. major mutants deficient in gluconeogenesis [24] anddeoxythymidylate synthesis [38] indicates that the Møphagolysosome contains low levels of hexoses and thymi-dine, respectively (both mutants grow normally in mediumsupplemented with these nutrients). By contrast, Leish-mania mutants that are auxotrophic for polyamines [39],methionine [40] or biopterin [41] proliferate normally inMø and susceptible mice, indicating that amastigotes canscavenge all of these nutrients from theMø phagolysosomein vivo. Other APER mutants include Leishmania amazo-nensis and L. mexicana mutants deficient in the irontransporter (Lit) [42] and in the cysteine proteinase B(CpB) family of lysosomal proteases [21], thus highlightingthe importance of cation uptake and lysosomal degradationin amastigote nutrition. These mutants are of interest asattenuated live vaccines because of their potential to elicita long-term protective immune response [38]. Furtheranalysis of these mutants will also be useful for definingthe relationship between parasite growth rate and lesionformation.

Gene and protein expression profiling of amastigotestagesTranscript and protein profiling approaches have beenused to probe the physiological state of many intracellularpathogens, often revealing the concerted up- and down-regulation of entiremetabolic pathways, such as glyoxylateshunt and gluconeogenesis [25]. However, Leishmania andother trypanosomatids lack a conventional network oftranscription factors and most genes are constitutivelytranscribed [43]. Although there are mechanisms for reg-ulating mRNA levels in a stage- or growth-dependentmanner [20], transcript profiling studies of differentgrowth stages of L. major, L. mexicana and L. donovaniindicate remarkable stability in the transcriptome [44–46].In most of these studies, fewer than 5% of mRNA tran-scripts vary by more than twofold when log- and station-ary-phase promastigotes were compared with lesionamastigotes. By comparison, >25% of genes in the yeastSaccharomyces cerevisiae change by more than twofoldduring the log–stationary phase transition [47]. Interest-ingly, most of the stage-regulated transcripts in Leishma-nia encode surface or highly abundant proteins, withenzymes involved in central carbon metabolism beingunder-represented. Comparative proteomic analyses ofpromastigote and intracellular amastigote stages also sup-port the idea thatmost proteins involved inmetabolism areexpressed at similar levels in all developmental stages[46]. However, these analyses only cover a minor pro-portion of the proteome and probably miss many lowabundance enzymes. Indeed, ammonium sulfate prefrac-tionation of extracts from cultured Leishmania infantumpromastigotes and amastigotes revealed a greater numberof stage-specific changes in protein expression levels andalso stage-specific changes in the post-translational modi-fication of amastigote proteins [48]. Although our under-standing of signaling pathways and post-translationalregulatory mechanisms in Leishmania (and other trypa-nosomatids) remains rudimentary, recent studies have

Review TRENDS in Parasitology Vol.23 No.8 373

shown that the mitogen-activated protein (MAP) kinasefamily are important for promastigote–amastigote differ-entiation [49] and intracellular survival of amastigotes [50](Table 2). The constitutive expression of core anabolic andcatabolic pathways in all life-cycle stages seems wastefulat first glance, but might enable Leishmania to respondrapidly to changes in nutrient availability in both theinsect vector and Mø phagolysosome. In support of thenotion that nutrient levels fluctuate in the Mø phagolyso-some, exposure of infected Mø to T-helper cell type-2 (Th2)cytokines stimulates the synthesis of ornithine and poly-amines in the host cell, and promotes amastigote growth[51]. Conversely, activation of infected Mø with T-helpercell type-1 (Th1) cytokines stimulates the synthesis ofnitric oxide at the expense of polyamine synthesis. Thecombined effect of increased nitric oxide production anddepletion of polyamines in the phagolysosome has a nega-tive effect on amastigote growth [51].

New approaches for defining the physiological stateof intracellular pathogensChanges in the physiological state of amastigotes can alsobe assessed bymeasuring the steady-state levels of cellularmetabolites. Metabolite profiling, or metabolomics, refersto the quantitative analysis of low molecular weightmetabolites (Box 2), and is increasingly being used tocomplement transcript and protein-profiling approaches,

Box 2. Tools for metabolomics

Metabolite profiling or metabolomics is a rapidly emerging area of

systems biology that refers to the quantitative analysis of low-

molecular-weight metabolites in a biological system [52]. A variety

of analytical platforms are employed in metabolite profiling, ranging

from high performance liquid chromatography (HPLC) to nuclear

magnetic resonance (NMR) spectroscopy, to hyphenated [gas

chromatography (GC)-, liquid chromatography (LC)- and capillary

electrophoresis (CE)-] mass spectrometric (MS) techniques. Each of

these techniques differs in the number and type of metabolite

analyzed, as well as reproducibility and quantitation. For example,

traditional HPLC analyses of specific classes of metabolites typically

measure a relatively small number of metabolites (typically <10)

and can be highly quantitative (targeted metabolite profiling). At the

other extreme, hyphenated MS methods can measure 102–104

metabolites in a single run [68], but at the cost of accurate

quantitation (metabolite profiling and fingerprinting approaches).

Of the techniques listed above, the most commonly used are NMR

and GC-MS. NMR can, in principal, be used to detect all metabolites,

without the need for derivatization and with high precision [69]. In

practice, it is limited by low sensitivity (fewer than �50 analytes

detected in typical analyses) but is useful for measuring polar or

charged metabolites and metabolic fluxes when the biological

system has been labeled with stable isotopes [69].

GC-MS is capable of the simultaneous detection of up to 1000

metabolites [70]. The instrumentation is robust, amenable to

automation and extensive mass spectral libraries are available for

metabolite identification. Although some volatile metabolites can be

detected directly by GC-MS, most cellular metabolite extracts are

derivatized to increase their volatility before analysis.

The total number of metabolites in a cell is not known.

Approximately 20 000 metabolites have been described in prokar-

yotic and eukaryotic microbes [71], although the number of

physiologically relevant metabolites within any one cell type is

likely to be much smaller. Leishmania is predicted to contain �1000

enzymes, suggesting that the metabolome is considerably smaller

than the proteome and that it is possible to observe a reasonable

proportion of the parasite metabolome.

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as well as being an important technique in its own right[52]. Metabolite profiling approaches might be particularlyuseful for assessing the physiological state of trypanoso-matid parasites given the stable expression of most mRNAtranscripts and proteins in these pathogens, and the find-ing that there is often a poor positive correlation betweenin vitro enzyme activities and metabolic fluxes in vivo [53].Metabolite profiling also has the potential to detect novelmetabolites, as well as those derived from the host cell.

Several targeted metabolite-profiling studies onLeishmania support the idea that broader metabolitestudies will be useful for identifying new metabolic path-ways and the metabolic state of different developmentalstages. Analysis of metabolite levels in different develop-mental stages of L. donovani using 13C- or 1H-NMR led tothe identification of unanticipated metabolites (betaine,b-hydroxybutyrate), as well as marked differences insteady-state metabolite levels of cultured promastigote/amastigote stages and lesion amastigotes [54,55]. Highlevels of short chain polyphosphates, pyrophosphate phos-phocholine, glycerophosphocholine and phosphoargininewere detected in perchloric acid extracts of L. major pro-mastigotes using 31P-NMR [56]. The presence of phos-phoarginine, a potentially important phosphagen [57],had not been predicted from the initial annotation of theL. major genome-based analyses. Targeted analyses ofthiols [58] and oligosaccharides [34] have also revealedspecies- and stage-specific changes in these metabolites.Lesion amastigotes were found to accumulate high levels(�10 mM) of b1–2-mannan oligomers, the major carbo-hydrate reserve material of these parasites, suggestingthat the intracellular stages are growth limited [34]. Bycontrast, high performance thin layer chromatographyanalysis of the apolar (lipid) extracts of lesion amastigotesdemonstrated that intracellular amastigotes scavengelipids from the host cell [16,59]. Host-derived glycosphin-golipids are likely to contribute to the surface glycocalyx ofintracellular amastigotes and could compensate for theloss of amastigote GIPLs and sphingolipids in Leishmanialipid mutants (Table 2) [37,17].

Conclusions and perspectivesIt is oftenassumed that themetabolic repertoire ofmicrobialpathogens is the result of adaptations to nutrient conditionsencountered in their respective hosts. An alternative (andnotmutually exclusive) view is that themetabolic repertoireof microbial pathogens might define the range of niches inthe host that can be successfully colonized. We suggest thatthe complex nutritional requirements of Leishmania andtheir inability to use fatty acids as their primary carbonsource has severely restricted the range of niches in themammalian host that can sustain parasite growth, account-ing for the remarkable tropism of these parasites forthe phagolysosome of Mø. The fact that many Leishmaniaauxotrophs either display no loss of virulence or can persistlong-term in susceptible animals indicates that the phago-lysosome is a nutritionally permissive environment. As aresult, the metabolism of Leishmania amastigotes seems tobe both robust and resistant to perturbation, with signifi-cant implications for drug development. The finding thatsome metabolic mutants fail to induce lesions, but can

374 Review TRENDS in Parasitology Vol.23 No.8

achievehighparasitemiaandpersist for longperiods of time(APER mutants), is of interest with regard to the develop-ment of live attenuated vaccines and to understandingfactors that underlie lesion development. Finally, mostgenes encoding metabolic enzymes are constitutivelyexpressed in the different developmental stages of Leish-mania spp., suggesting that post-translational processeshave a key role in regulating metabolism. This strategymight enable more rapid responses to nutrient fluctuationsin both the sandfly and mammalian hosts. In future, theextension of these profiling strategies to includemetaboliteswill be needed to identify more precisely the metabolicpathways that are active in Leishmania amastigotes, howthese pathways are regulated in the absence of tight tran-scriptional control, and to what extent amastigote metab-olism varies in acute and latent stages of infection.

AcknowledgementsWe were unable to reference all relevant work owing to space constraints.Our laboratory is supported by the Australian National Health andMedical Research Council.

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