Living in a phagolysosome; metabolism of Leishmania amastigotes

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Vol.23 No.8

Living in a phagolysosome; metabolism of Leishmania amastigotes Malcolm J. McConville1,2, David de Souza1,2, Eleanor Saunders1,2, Vladimir A. Likic2 and Thomas Naderer1,2 1 2

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

Leishmania amastigotes primarily proliferate within macrophages in the mammalian host. Genome-based metabolic reconstructions, combined with biochemical, reverse genetic and mRNA or protein profiling studies are providing new insights into the metabolism of this intracellular stage. We propose that the complex nutritional requirements of amastigotes have contributed to the tropism of these parasites for the amino acid-rich phagolysosome of macrophages. Amastigote metabolism in this compartment is robust because many metabolic mutants are capable of either growing normally or persisting long term in susceptible animals. New approaches for measuring amastigote metabolism in vivo are discussed. The enemy within Several important human microbial pathogens proliferate within macrophages (Mø). Although most of these pathogens evade the microbicidal responses of the Mø host by subverting or escaping from the phagocytic pathway, protozoan parasites belonging to the genus Leishmania manage both to survive and to proliferate within the mature phagolysosome compartment of Mø. The infection of Mø by Leishmania is initiated by flagellated, metacyclic (nondividing) promastigotes, which are injected into the skin of the mammalian host by the phlebotomine sandfly vector. Metacyclic promastigotes are phagocytosed by Mø either directly or after cycling through a wave of neutrophils (which are rapidly recruited to the site of the sandfly bite but then undergo apoptosis) [1]. Promastigotes internalized by Mø are delivered to the phagolysosome, where they differentiate into small, aflagellated amastigotes. Amastigotes are a replicative stage that periodically escape from the host cell by a poorly defined mechanism and rapidly reinfect other phagocytes (i.e. Mø or dendritic cells) and some non-phagocytic cell types (i.e. fibroblasts) [2,3]. Amastigotes are the cause of acute disease (ranging from self-healing cutaneous infections to severe disfiguring mucocutaneous and lethal visceral disease), as well as chronic or latent infections that can persist for the life time of the host. Despite the importance of amastigotes in Corresponding author: McConville, M.J. ([email protected]). Available online 2 July 2007. www.sciencedirect.com

perpetuating disease and as the target of antileishmanial drugs, comparatively little is known about either the metabolism of this stage in vivo or the biochemical composition of the phagolysosome. Although in vitro cultivated amastigotes (derived from in vitro differentiated promastigotes or lesion amastigotes) have been used to investigate some aspects of amastigote metabolism, these stages are typically grown in rich medium that is unlikely to mimic the biochemical milieu of the phagolysosome. Analysis of amastigote metabolism in vivo is further complicated by the fact that different species of Leishmania reside within different populations of Mø and can induce morphologically distinct phagolysosomes within the same population of Mø [3]. For example, members of the Leishmania mexicana complex induce spacious communal vacuoles rather than the tight fitting individual phagolysosomes occupied by many other species (e.g. Leishmania major, Leishmania donovani). Moreover, the L. mexicana-occupied phagolysosomes become filled with electron-dense material over the course of several days, thus indicating temporal changes in the biochemical composition of the phagolysosome lumen [4]. In this review, we highlight recent insights into the in vivo metabolism of intracellular amastigotes that have emerged from genome-based metabolic reconstructions, the generation of metabolic mutants and profiling approaches. Nutrient availability and uptake in the phagolysosome Intracellular stages of Leishmania must scavenge all of their carbon source and micronutrient requirements from the lumen or limiting membrane of the Mø phagosome (Figure 1). Based on the known nutrient requirements of cultured stages [5] and genome-based reconstructions of the Leishmania metabolome [6,7], it is predicted that Leishmania amastigotes must scavenge all their purine requirements, many vitamins and at least ten essential amino acids from the Mø phagolysosome (Figure 2). The uptake of these essential metabolites is mediated by families of polytopic membrane transporters, which are often proton symporters, enabling high affinity uptake of substrates in the acidic phagolysosome [5,8–10] (Table 1). In some cases, distinct transporters enable uptake of different forms of the same metabolite class, providing a high degree of

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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.

redundancy in nutrient salvage pathways [11–14]. For example, distinct transporters are involved in internalizing nucleobases, nucleosides and possibly nucleotides, all of which are potential precursors for purine salvage. As a result, single or double deletions of these transporters have little effect on amastigote growth [15]. Redundancy is also apparent in the uptake of hexoses, polyamines and folates (Table 1, Figure 2). In addition to small metabolites, Leishmania amastigotes can scavenge complex lipids and (glyco)proteins from the phagolysosome. All Leishmania amastigotes seem to incorporate host glycosphingolipids into their plasma membrane [16]. These or other host sphingolipids might be internalized by intracellular amastigotes and used to synthesize endogenous sphingolipids,

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such as inositolphosphoceramide [17]. Essential amino acids and heme might also be obtained from the proteolysis of host phagolysosome proteins [18] and exogenous proteins delivered to the phagolysosome via endocytosis and autophagy [19]. Digestion of glycoproteins in the highly active amastigote lysosome would provide a source of essential amino acids and sugars [4,20]. Endocytic uptake is important because disruption of either endocytosis or lysosomal function markedly reduces amastigote virulence [21–23] (Table 2). Although these analyses suggest that both lipids and amino acids are important carbon sources for amastigotes, the L. major genome lacks two key enzymes in the glyoxylate pathway, which is required for net conversion of acetyl-CoA, the end-product of fatty acid b-oxidation, into sugars [6]. The absence of this pathway in Leishmania, together with recent metabolic labeling experiments [24], indicates that amastigotes are unable to utilize fatty acids as their major carbon source. The nutritional requirements of Leishmania amastigotes, briefly summarized here, are considerably more complex than those of most prokaryotes and fungal pathogens, and might have prevented Leishmania amastigotes from occupying many intracellular niches in the mammalian host. In particular, they are likely to have precluded Leishmania from colonizing the early endosomal or non-hydrolytic vacuoles of Mø, which are rich in lipids but contain low levels of sugars and amino acids [25,26]. By contrast, the phagolysosomes of Mø are predicted to contain relatively high levels of amino acids, as shown by transcript profiling studies of phagocytosed fungal pathogens [25,27]. Mø could be unusual in this respect, as phagocytosis of Candida albicans by neutrophils induces a strong amino acid starvation response in the fungus [27]. Host-specific differences in the nutrient composition of the host cell phagosomes could be part of the reason why Leishmania promastigotes cannot proliferate or differentiate to amastigotes in neutrophils [1] but can in Mø. Similar considerations might dictate the tissue tropism of other microbial pathogens. For example, the gram-negative bacterium, Coxiella burnetti, is one of the few other microbial pathogens to inhabit the phagolysosome of Mø. Like Leishmania, C. burnetti is an auxotroph for many amino acids (i.e. it needs these amino acids externally, having no de novo pathway for their synthesis) and lacks the glyoxylate pathway [28]. As with Leishmania,

Table 1. Cloned transporters expressed in Leishmania amastigote stages Nutrient Monosaccharides

Gene(s) a GT1, GT2 and GT3

myo-inositol Amino acids

D1 AAP3 PAT1 POT1 NT1 NT2 NT3 NT4 BT1 FT1 Lit1

Polyamines Nucleosides Nucleobases Biopterin Folic acid Iron

Comments GT1 and 3 expressed in all stages GT2 decreased in amastigotes Expression induced by low inositol levels Arginine uptake Amastigote-specific amino acid permease Related transporters probably expressed in amastigotes Adenosine, pyrimidine Inosine, guanosine (Hypo)xanthine, adenine, guanine Undefined Deletion results in increased virulence Transports folate and methotrexate Required for intracellular growth

Refs [72] [9] [12] [10] [8] [11] [11] [11] [14] [14] [41]

a Abbreviations: AAP3, amino acid permease 3; BT, biopterin transporter; D1, inositol transporter; FT, folate transporter; GT, glucose transporters; NT, purine transporters; PAT1, putative amino acid transporter 1; POT1, polyamine transporter.

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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 threeletter 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 endproducts are highlighted in black.

C. burnetti might have to trade increased exposure to host microbicidal processes for access to nutrients. Use of stable mutants to identify metabolic pathways required for virulence The genetic disruption of specific metabolic pathways is the most rigorous approach for demonstrating a requirement for intracellular growth (but see Box 1 for pros and cons). The Leishmania mutants listed in Table 2 can be classified into three categories. PAV (promastigote avirulent) mutants are poorly infective as promastigotes, but fully infective if viable amastigotes can be generated. These mutants characteristically generate lesions after a delay of several weeks. AAV (amastigote avirulent) mutants are www.sciencedirect.com

unable to survive in susceptible animals (or macrophages) as promastigotes and/or amastigotes. APER (amastigote persistent) mutants differentiate to amastigotes and can survive long term in infected macrophages or susceptible mice, but are unable to induce lesions. A major group of L. major PAV mutants are unable to synthesize components of the complex cell surface glycocalyx of promastigotes, such as the lipophosphoglycan (LPG), glycosylphosphatidylinositol (GPI)-anchored proteins and free GPI glycolipids (GIPLs) (Table 2). Because amastigotes are coated by a much simpler glycocalyx (lacking LPG and GPI-anchored proteins) [29], it is not surprising that loss of enzymes involved in their synthesis has little affect on amastigote virulence (Table 2). Although intracellular

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Table 2. Metabolic pathways required for Leishmania infectivity Pathway a PAV (promastigote avirulent) mutants LPG1 (Galf-transferase)

Species b

Gene(s)

LPG biosynthesis

Lmj Lmx

Virulence

Refs

Pro c

Ama d +/+ +/+

[73]

+

LPG/GIPL biosynthesis Ether lipid Sphingolipid Protein kinase

Lmj Lmj Lmj Lmx

+/+ +/+ +/+ +/+

[74] [37] [17] [49]

Ldn

/

[75]

hgprt xprt double mutant Hexose transporter-1,-2 & -3 Phosphomannomutase GDP–mannose pyrophosphorylase DHAP acyltransferase Inositol-3-phosphate synthase AP1 s / m subunits LMPK (MAP kinase)

Trypanothione regeneration (partial k/o or dominant negative) Purine scavenging Glucose uptake Mannose metabolism Mannose metabolism Acylglycerolipid biosynthesis Myo-inositol biosynthesis Endocytosis Protein kinase

Ldn Lmx Lmx Lmx Lmj Lmx Lmx Lmx

/ / / / / / /

[76] [13] [67] [33] [77] [78] [22] [50]

APER (amastigote persistent) mutants Fructose-1,6-bisphosphatase dhfr-ts LitA cpB cpA LPG2 (GDP–mannose transporter) e

Gluconeogenesis Thymidine synthesis Iron uptake Lysosome protease Lysosome protease Phosphoglycan biosynthesis

+/ +/ +/ +/ +/ +/ +/+

[24] [38] [42] [21] [79] [31] [30]

UDP-Gal mutase Alkyl-DHAP synthase Ser:palmitoyltransferase LmxPK4 (MAP kinase kinase) AAV (amastigote avirulent) mutants Trypanothione reductase

Lmj Lmj Lam Lmx Linf Lmj Lmx

+ + + – – +

a

Only 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. b Lmj, L. major; Lmx, L. mexicana; Ldn, L. donovani; Lam, L. amazonensis; Linf, L. infantum. c Promastigote (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. d Amastigote (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. e Deletions 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.

amastigotes retain a surface glycocalyx of GIPLs [29], they might be able to compensate for the loss of these glycolipids by scavenging glycosphingolipids from the host cell (see below). Promastigote and amastigote stages also express on their surfaces and secrete several distinct classes of proteophosphoglycans (PPGs). Although loss of both LPG and PPG glycosylation in L. major (owing to ablation of the lpg2 gene encoding a Golgi GDP–mannose transporter) resulted in a marked reduction in both promastigote and amastigote virulence, disruption of the lpg2 gene in L. mexicana has no measurable effect on either promastigote or amastigote virulence [30,31]. These studies initially suggested that PPG is a virulence factor in L. major, but not in L. mexicana. However, analysis of another L. major LPG/ PPG mutant (defective in a Golgi UDP-galactopyranose transporter) has revealed that PPG biosynthesis per se is not required for amastigote virulence [32]. Thus, none of the major surface glycoconjugates of Leishmania amastigotes seems to be essential for amastigote survival and growth in vivo. In contrast to the surface coat mutants, L. mexicana mutants with global defects in sugar nucleotide biosynthesis and carbohydrate metabolism constitute an important group of AAV mutants (Table 2). In particular, deletion of enzymes involved in mannose and inositol metabolism result in effective loss of promastigote and amastigote infectivity (Table 2). In the case of the mannose mutants, www.sciencedirect.com

loss of infectivity was attributed to the cumulative loss of all major surface glycoconjugates [33]. However, more recent studies have highlighted the potential importance of a unique, intracellular mannan reserve material in L. mexicana that might be important for intracellular survival [34]. Defects in mannan synthesis [35] and possibly other glycosylation pathways, such as protein N-glycosylation, could therefore contribute to the severe loss of infectivity of the mannose mutants. The importance of carbohydrate metabolism more generally is supported by the finding that a L. mexicana mutant lacking three glucose transporters (GTs) is avirulent in Mø [13]. The severity of the L. mexicana Dgt mutant virulence phenotype is surprising given that Leishmania amastigotes are capable of de novo glucose biosynthesis via the gluconeogenic pathway [24]. It is possible that gluconeogenesis is insufficient to provide all of the glucose requirements of intracellular amastigotes or that a minimum level of glucose uptake is required for nutrient sensing. Other AAV mutants include those with defects in trypanothione metabolism, purine salvage, glycerolipid biosynthesis and endocytosis (Table 2). Trypanothione is involved in many metabolic processes and is a key component in the oxidative defenses of Leishmania, whereas purine salvage is essential for growth in the absence of de novo purine biosynthetic pathways. The reason why disruption of dihydroxyacetone phosphate acyltransferase (an early enzyme in glycerolipid

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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 representative of clinical isolates. Examples exist where the deletion of a particular gene was readily accomplished in one strain of Leishmania 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.

biosynthesis) results in loss of virulence is unclear, given that amastigotes can scavenge fatty acids from the host and genetic disruption of other genes in glycerolipid biosynthesis has no affect on virulence [36,37]. The requirement for endocytosis might also be complex. Endocytosed host proteins are likely to be an important source of nutrients, whereas endocytic membrane transport and lysosome function are required for autophagy and turnover of parasite proteins and organelles during differentiation and normal growth [23]. APER mutants are able to invade Mø and differentiate into amastigotes, but fail to proliferate in the Mø in vitro (Table 2). These mutants can persist for long periods of time on susceptible hosts, but generally fail to induce lesions despite the presence of reasonable parasite numbers. Several of these mutants are auxotrophic for nutrients normally synthesized by wild-type parasites, www.sciencedirect.com

providing insights into the nutrient environment of the Mø phagolysosome. For example, the APER phenotype of L. major mutants deficient in gluconeogenesis [24] and deoxythymidylate synthesis [38] indicates that the Mø phagolysosome contains low levels of hexoses and thymidine, respectively (both mutants grow normally in medium supplemented with these nutrients). By contrast, Leishmania mutants that are auxotrophic for polyamines [39], methionine [40] or biopterin [41] proliferate normally in Mø and susceptible mice, indicating that amastigotes can scavenge all of these nutrients from the Mø phagolysosome in vivo. Other APER mutants include Leishmania amazonensis and L. mexicana mutants deficient in the iron transporter (Lit) [42] and in the cysteine proteinase B (CpB) family of lysosomal proteases [21], thus highlighting the importance of cation uptake and lysosomal degradation in amastigote nutrition. These mutants are of interest as attenuated live vaccines because of their potential to elicit a long-term protective immune response [38]. Further analysis of these mutants will also be useful for defining the relationship between parasite growth rate and lesion formation. Gene and protein expression profiling of amastigote stages Transcript and protein profiling approaches have been used to probe the physiological state of many intracellular pathogens, often revealing the concerted up- and downregulation of entire metabolic pathways, such as glyoxylate shunt and gluconeogenesis [25]. However, Leishmania and other trypanosomatids lack a conventional network of transcription factors and most genes are constitutively transcribed [43]. Although there are mechanisms for regulating mRNA levels in a stage- or growth-dependent manner [20], transcript profiling studies of different growth stages of L. major, L. mexicana and L. donovani indicate remarkable stability in the transcriptome [44–46]. In most of these studies, fewer than 5% of mRNA transcripts vary by more than twofold when log- and stationary-phase promastigotes were compared with lesion amastigotes. By comparison, >25% of genes in the yeast Saccharomyces cerevisiae change by more than twofold during the log–stationary phase transition [47]. Interestingly, most of the stage-regulated transcripts in Leishmania encode surface or highly abundant proteins, with enzymes involved in central carbon metabolism being under-represented. Comparative proteomic analyses of promastigote and intracellular amastigote stages also support the idea that most proteins involved in metabolism are expressed at similar levels in all developmental stages [46]. However, these analyses only cover a minor proportion of the proteome and probably miss many low abundance enzymes. Indeed, ammonium sulfate prefractionation of extracts from cultured Leishmania infantum promastigotes and amastigotes revealed a greater number of stage-specific changes in protein expression levels and also stage-specific changes in the post-translational modification of amastigote proteins [48]. Although our understanding of signaling pathways and post-translational regulatory mechanisms in Leishmania (and other trypanosomatids) remains rudimentary, recent studies have

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shown that the mitogen-activated protein (MAP) kinase family are important for promastigote–amastigote differentiation [49] and intracellular survival of amastigotes [50] (Table 2). The constitutive expression of core anabolic and catabolic pathways in all life-cycle stages seems wasteful at first glance, but might enable Leishmania to respond rapidly to changes in nutrient availability in both the insect vector and Mø phagolysosome. In support of the notion that nutrient levels fluctuate in the Mø phagolysosome, exposure of infected Mø to T-helper cell type-2 (Th2) cytokines stimulates the synthesis of ornithine and polyamines in the host cell, and promotes amastigote growth [51]. Conversely, activation of infected Mø with T-helper cell type-1 (Th1) cytokines stimulates the synthesis of nitric oxide at the expense of polyamine synthesis. The combined effect of increased nitric oxide production and depletion of polyamines in the phagolysosome has a negative effect on amastigote growth [51]. New approaches for defining the physiological state of intracellular pathogens Changes in the physiological state of amastigotes can also be assessed by measuring the steady-state levels of cellular metabolites. Metabolite profiling, or metabolomics, refers to the quantitative analysis of low molecular weight metabolites (Box 2), and is increasingly being used to complement 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 lowmolecular-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 prokaryotic 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. www.sciencedirect.com

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as well as being an important technique in its own right [52]. Metabolite profiling approaches might be particularly useful for assessing the physiological state of trypanosomatid parasites given the stable expression of most mRNA transcripts and proteins in these pathogens, and the finding that there is often a poor positive correlation between in vitro enzyme activities and metabolic fluxes in vivo [53]. Metabolite profiling also has the potential to detect novel metabolites, as well as those derived from the host cell. Several targeted metabolite-profiling studies on Leishmania support the idea that broader metabolite studies will be useful for identifying new metabolic pathways and the metabolic state of different developmental stages. Analysis of metabolite levels in different developmental stages of L. donovani using 13C- or 1H-NMR led to the identification of unanticipated metabolites (betaine, b-hydroxybutyrate), as well as marked differences in steady-state metabolite levels of cultured promastigote/ amastigote stages and lesion amastigotes [54,55]. High levels of short chain polyphosphates, pyrophosphate phosphocholine, glycerophosphocholine and phosphoarginine were detected in perchloric acid extracts of L. major promastigotes using 31P-NMR [56]. The presence of phosphoarginine, a potentially important phosphagen [57], had not been predicted from the initial annotation of the L. major genome-based analyses. Targeted analyses of thiols [58] and oligosaccharides [34] have also revealed species- and stage-specific changes in these metabolites. Lesion amastigotes were found to accumulate high levels (10 mM) of b1–2-mannan oligomers, the major carbohydrate reserve material of these parasites, suggesting that the intracellular stages are growth limited [34]. By contrast, high performance thin layer chromatography analysis of the apolar (lipid) extracts of lesion amastigotes demonstrated that intracellular amastigotes scavenge lipids from the host cell [16,59]. Host-derived glycosphingolipids are likely to contribute to the surface glycocalyx of intracellular amastigotes and could compensate for the loss of amastigote GIPLs and sphingolipids in Leishmania lipid mutants (Table 2) [37,17]. Conclusions and perspectives It is often assumed that the metabolic repertoire of microbial pathogens is the result of adaptations to nutrient conditions encountered in their respective hosts. An alternative (and not mutually exclusive) view is that the metabolic repertoire of microbial pathogens might define the range of niches in the host that can be successfully colonized. We suggest that the complex nutritional requirements of Leishmania and their inability to use fatty acids as their primary carbon source has severely restricted the range of niches in the mammalian host that can sustain parasite growth, accounting for the remarkable tropism of these parasites for the phagolysosome of Mø. The fact that many Leishmania auxotrophs either display no loss of virulence or can persist long-term in susceptible animals indicates that the phagolysosome is a nutritionally permissive environment. As a result, the metabolism of Leishmania amastigotes seems to be both robust and resistant to perturbation, with significant implications for drug development. The finding that some metabolic mutants fail to induce lesions, but can

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achieve high parasitemia and persist for long periods of time (APER mutants), is of interest with regard to the development of live attenuated vaccines and to understanding factors that underlie lesion development. Finally, most genes encoding metabolic enzymes are constitutively expressed in the different developmental stages of Leishmania spp., suggesting that post-translational processes have a key role in regulating metabolism. This strategy might enable more rapid responses to nutrient fluctuations in both the sandfly and mammalian hosts. In future, the extension of these profiling strategies to include metabolites will be needed to identify more precisely the metabolic pathways that are active in Leishmania amastigotes, how these pathways are regulated in the absence of tight transcriptional control, and to what extent amastigote metabolism varies in acute and latent stages of infection. Acknowledgements We were unable to reference all relevant work owing to space constraints. Our laboratory is supported by the Australian National Health and Medical Research Council.

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