Surface determinants of Leishmania parasites and their role in infectivity in the mammalian host

Leishmania are intracellular protozoan parasites that reside primarily in host mononuclear phagocytes. Infection of host macrophages is initiated by infective promastigote stages and perpetuated by an obligate intracellular amastigote stage. Studies undertaken over the last decade have shown that the composition of the complex surface glycocalyx of these stages (comprising lipophosphoglycan, GPI-anchored glycoproteins, proteophosphoglycans and free GPI glycolipids) changes dramatically as promastigotes differentiate into amastigotes. Marked stage-specific changes also occur in the expression of other plasma membrane components, including type-1, polytopic and peripheral membrane proteins, reflecting the distinct microbicidal responses and nutritional environments encountered by these stages. More recently, a number of Leishmania mutants lacking single or multiple surface components have been generated. While some of these mutants are less virulent than wild type parasites, many of these mutants exhibit only mild or no loss of virulence. These studies suggest that, 1) the major surface glycocalyx components of the promastigote stage (i.e. LPG, GPI-anchored proteins) only have a transient or minor role in macrophage invasion, 2) that there is considerable functional redundancy in the surface glycocalyx and/or loss of some components can be compensated for by the acquisition of equivalent host glycolipids, 3) the expression of specific nutrient transporters is essential for life in the macrophage and 4) the role(s) of some surface components differ markedly in different Leishmania species. These mutants will be useful for identifying other surface or intracellular components that are required for virulence in macrophages.     

Stage-specific pathways of Leishmania infantum chagasi entry and phagosome maturation in macrophages

The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival.     

Role of hexosamine biosynthesis in Leishmania growth and virulence

Leishmania parasites incorporate N-acetylglucosamine (GlcNAc) into surface-expressed glycosylphosphatidylinositol (GPI) glycolipids and N-linked glycans. To investigate whether these glycoconjugates are required for infectivity of promastigote and intracellular amastigote stages, we generated a Leishmania major mutant lacking the gene encoding glutamine : fructose-6-phosphate amidotransferase (GFAT). The L. major Δ gfat mutant is unable to synthesize GlcN-6-phosphate de novo and is auxotrophic for GlcN or GlcNAc. GlcN starvation leads to the rapid depletion of dolichol-linked oligosaccharides and GPI precursors, hypersensitivity to elevated temperatures encountered in the mammalian host and eventual parasite death. Short-term tunicamycin treatment induces a similar hypersensitivity to temperature, indicating that N-linked glycans are required for thermotolerance and viability. L. major Δ gfat promastigotes are unable to proliferate in ex vivo infected macrophages, demonstrating that GlcN(Ac) levels in the phagolysosome are low. In contrast, Δ gfat amastigotes grow as well as wild-type amastigotes in macrophages and induce lesions in susceptible mice. These stages still require GlcN(Ac) for viability but can apparently scavenge all of their glucosamine requirements from the macrophage phagolysosome. These results highlight significant differences in the nutrient requirements of promastigote and amastigote stages and suggest that enzymes involved in UDP-GlcNAc biosynthesis are essential for pathogenesis in the mammalian host.     

Developmentally regulated sphingolipid degradation in Leishmania major

Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs) through de novo synthesis (to produce inositol phosphorylceramide) and salvage (to obtain sphingomyelin from the host). A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like) enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity) and sphingomyelin (the SMase activity). Recent studies of a L. major ISCL-null mutant (iscl(-)) indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl(-) mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl(-) promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl(-). These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.     

Leishmania mexicana pifanoi: analysis of the antigenic relationships between promastigotes and amastigotes by gel diffusion, immunoelectrophoresis, and immunoprecipitation

The antigenic relationships between Leishmania mexicana pifanoi promastigotes, axenically grown amastigotes, and amastigotes isolated from the footpads of infected hamsters or from a J774 macrophage cell line were studied by three serologic methods. Amastigote and promastigote antigens were disrupted by freeze-thawing of intact cells in a lysis buffer. Antisera were prepared in rabbits by repeated subcutaneous inoculations of the parasite antigens in complete Freund's adjuvant and were tested against the homologous and heterologous antigens in a series of gel diffusion experiments. Negative results were obtained in all control experiments. In each instance, the homologous antigen-antiserum reactions yielded the largest numbers of precipitin lines. A pattern of cross-reactivity was also observed in the heterologous systems. Results indicated that the amastigote and promastigote forms had unique and common antigens. The two parasite antigen-antiserum systems were also examined by immunoelectrophoresis. Qualitative and quantitative differences between the promastigote and amastigote antigens were readily demonstrable by this technique. Results indicated that each parasite form had specific and many common antigens. In the homologous system, major proteins, with molecular weights (MW) of 23, 52, and 68 kd, were demonstrated in the promastigotes by immunoprecipitation of lactoperoxidase-catalyzed radioiodinated cells. In a similar (homologous) system, axenically grown amastigotes were found to contain three proteins with MW of 38, 70, and 74 kd and, therefore, different from those demonstrated for the promastigotes. All the results suggested that the three amastigote stages of different origins are antigenically similar to one another, but differ from the promastigote forms.     

Leishmania mexicana pifanoi: Analysis of the Antigenic Relationships Between Promastigotes and Amastigotes by Gel Diffusion,Immunoelectrophoresis, and Immunoprecipitation1

ABSTRACT. The antigenic relationships between Leishmania mexicana pifanoi promastigotes, axenically grown amastigotes, and amastigotes isolated from the footpads of infected hamsters or from a J774 macrophage cell line were studied by three serologic methods. Amastigote and promastigote antigens were disrupted by freeze-thawing of intact cells in a lysis buffer. Antisera were prepared in rabbits by repeated subcutaneous inoculations of the parasite antigens in complete Freund's adjuvant and were tested against the homologous and heterologous antigens in a series of gel diffusion experiments. Negative results were obtained in all control experiments. In each instance, the homologous antigen-antiserum reactions yielded the largest numbers of precipitin lines. A pattern of cross-reactivity was also observed in the heterologous systems. Results indicated that the amastigote and promastigote forms had unique and common antigens. The two parasite antigen-antiserum systems were also examined by immunoelectrophoresis. Qualitative and quantitative differences between the promastigote and amastigote antigens were readily demonstrable by this technique. Results indicated that each parasite form had specific and many common antigens. In the homologous system, major proteins, with molecular weights (MW) of 23, 52, and 68 kd, were demonstrated in the promastigotes by immunoprecipitation of lactoperoxidase-catalyzed radioiodinated cells. In a similar (homologous) system, axenically grown amastigotes were found to contain three proteins with MW of 38, 70, and 74 kd and, therefore, different from those demonstrated for the promastigotes. All the results suggested that the three amastigote stages of different origins are antigenically similar to one another, but differ from the promastigote forms.     

The surface membrane of Leishmania mexicana mexicana: comparison of amastigote and promastigote using freeze-fracture cytochemistry

The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages of Leishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to beta-hydroxysterols content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of beta-hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.     

Effects of long-term in vitro cultivation on Leishmania donovani promastigotes

Promastigotes of Leishmania donovani that had been subcultured in modified Tobie's medium for 2 to 3 years showed decreased infectivity and lack of virulence for hamsters and mice compared to newly transformed promastigotes. Amastigotes derived from these long-term promastigote cultures decreased in number rapidly in hamsters, but only slightly in mice, over a 48-day period. In cultured mouse and hamster macrophages infected in vitro, amastigotes derived from long-term cultures rapidly decreased to low numbers, which persisted. The same pattern was seen in macrophages treated with catalase, an inhibitor of the oxygen-dependent killing mechanism of the macrophage. Promastigotes from long-term cultures also differed from virulent first-passage promastigotes in size, growth patterns in Tobie's medium, and in the quantities of some of their antigens.     

Influence of parasite encoded inhibitors of serine peptidases in early infection of macrophages with Leishmania major

Ecotin is a potent inhibitor of family S1A serine peptidases, enzymes lacking in the protozoan parasite Leishmania major . Nevertheless, L. major has three ecotin-like genes, termed inhibitor of serine peptidase (ISP). ISP1 is expressed in vector-borne procyclic and metacyclic promastigotes, whereas ISP2 is also expressed in the mammalian amastigote stage. Recombinant ISP2 inhibited neutrophil elastase, trypsin and chymotrypsin with K _is between 7.7 and 83 nM. L. major ISP2–ISP3 double null mutants (Δ isp 2/3) were created. These grew normally as promastigotes, but were internalized by macrophages more efficiently than wild-type parasites due to the upregulation of phagocytosis by a mechanism dependent on serine peptidase activity. Δ isp 2/3 promastigotes transformed to amastigotes, but failed to divide for 48 h. Intracellular multiplication of Δ isp 2/3 was similar to wild-type parasites when serine peptidase inhibitors were present, suggesting that defective intracellular growth results from the lack of serine peptidase inhibition during promastigote uptake. Δ isp 2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became equivalent as the infection progressed. These data support the hypothesis that ISPs of L. major target host serine peptidases and influence the early stages of infection of the mammalian host.     

Lysosomal degradation of Leishmania hexose and inositol transporters is regulated in a stage-, nutrient- and ubiquitin-dependent manner

Leishmania parasites experience variable nutrient levels as they cycle between the extracellular promastigote stage in the sandfly vector and the obligate intracellular amastigote stage in the mammalian host. Here we show that the surface expression of three Leishmania mexicana hexose and myo-inositol transporters is regulated in both a stage-specific and nutrient-dependent manner. GFP-chimeras of functionally active hexose transporters, LmGT2 and LmGT3, and the myo-inositol transporter, MIT, were primarily expressed in the cell body plasma membrane in rapidly dividing promastigote stages. However MIT-GFP was mostly rerouted to the multivesicular tubule (MVT)-lysosome when promastigotes reached stationary phase growth and all three nutrient transporters were targeted to the amastigote lysosome following transformation to in vitro differentiated or in vivo imaged amastigote stages. This stage-specific decrease in surface expression of GFP-tagged transporters correlated with decreased hexose or myo-inositol uptake in stationary phase promastigotes and amastigotes. The MVT-lysosme targeting of the MIT-GFP protein was reversed when promastigotes were deprived of myo-inositol, indicating that nutrient signals can override stage-specific changes in transporter distribution. The surface expression of the hexose and myo-inositol transporters was not regulated by interactions with the subpellicular cytoskeleton, as both classes of transporters associated with detergent-resistant membranes. LmGT3-GFP and MIT-GFP proteins C-terminally modified with mono-ubiquitin were constitutively transported to the MVT-lysosome, suggesting that ubiquitination may play a key role in regulating the subcellular distribution of these transporters and parasite adaptation to different nutrient conditions.     

Evidence that intracellular beta1-2 mannan is a virulence factor in Leishmania parasites

The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular beta1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold increase in mannan levels. Developmental stages with low mannan levels or L. mexicana mutants lacking mannan do not survive heat shock and are unable to differentiate to amastigotes or infect macrophages in vitro. In contrast, a L. mexicana mutant deficient only in components of the mannose-rich surface glycocalyx differentiates normally and infects macrophages in vitro. Collectively, these data provide strong evidence that mannan accumulation is important for parasite differentiation and survival in macrophages.     

Differential survival of Leishmania donovani amastigotes in human monocytes

Leishmania donovani is an important intracellular protozoal pathogen of man; it is found solely within macrophages in its amastigote stage in humans, and exists in its extracellular, flagellated promastigote stage in the sandfly, its arthropod vector. To determine if either stage of L. donovani was capable of surviving within monocytes--the oxidatively active precursors of tissue macrophages--interactions of the parasite with human monocytes were studied in vitro. Amastigotes and promastigotes were ingested to a comparable degree by monocytes; whereas 79% of promastigotes were killed within 48 hr, however, amastigotes survived and multiplied threefold over 5 days. Promastigotes, which have been shown to be sensitive to hydrogen peroxide-peroxidase-halide microbicidal mechanisms, elicited a phagocytic oxidative burst that was 49% of the response to serum-opsonized zymosan, as assessed by luminol-enhanced chemiluminescence. NBT was reduced to formazan in 71% of monocytes exposed to promastigotes. The death of promastigotes within monocytes could be attributed at least in part to oxidative microbicidal mechanisms because there was no significant decrease in the number of cell-associated parasites in monocytes from donors with chronic granulomatous disease of childhood. In contrast to promastigotes, amastigotes survived within monocytes, despite eliciting an oxidative response that was 27% of the response produced by serum-opsonized zymosan; this response was not significantly different from that produced by promastigotes. In a phagocyte-free system, amastigotes were found to be sevenfold more resistant than were promastigotes to the lethal effects of hydrogen peroxide. The survival of L. donovani in human monocytes is thus dependent on the parasite stage; promastigotes are ingested, they elicit an oxidative burst, and the majority are killed by oxidative microbicidal mechanisms, whereas amastigotes are ingested and survive to parasitize human monocytes successfully, despite eliciting a phagocytic oxidative burst.     

Life in vacuoles--nutrient acquisition by Leishmania amastigotes

Leishmania have a digenetic life cycle, involving a motile, extracellular stage (promastigote) which parasitises the alimentary tract of a sandfly vector. Bloodfeeding activity by an infected sandfly can result in transmission of infective (metacyclic) promastigotes to mammalian hosts, including humans. Leishmania promastigotes are rapidly phagocytosed but may survive and transform into non-motile amastigote forms which can persist as intracellular parasites. Leishmania amastigotes multiply in an acidic intracellular compartment, the parasitophorous vacuole. pH plays a central role in the developmental switch between promastigote and amastigote stages, and amastigotes are metabolically most active when their environment is acidic, although the cytoplasm of the amastigote is regulated at near-neutral pH by an active process of proton extrusion. A steep proton gradient is thus maintained across the amastigote surface and all membrane processes must be adapted to function under these conditions. Amastigote uptake systems for glucose, amino acids, nucleosides and polyamines are optimally active at acidic pH. Promastigote uptake systems are kinetically distinct and function optimally at more neutral environmental pH, indicating that membrane transport activity is developmentally regulated. The nutrient environment encountered by amastigotes is not well understood but the parasitophorous vacuole can fuse with endosomes, phagosomes and autophagosomes, suggesting that a diverse range of macromolecules will be present. The parasitophorous vacuole is a hydrolytic compartment in which such material will be rapidly degraded to low molecular weight components which are typical substrates for membrane transporters. Amastigote surface transporters must compete for these substrates with equivalent host transporters in the membrane of the parasitophorous vacuole. The elaboration of accumulative transporters with high affinity will be beneficial to amastigotes in this environment. The influence of environmental pH on membrane transporter function is discussed, with emphasis on the potential role of a transmembrane proton gradient in active, high affinity transport.     

Quantitative proteomic profiling of the promastigotes and the intracellular amastigotes of Leishmania donovani isolates identifies novel proteins having a role in Leishmania differentiation and intracellular survival

Protozoan parasites of the genus Leishmania are important human pathogens that cycle between an extracellular promastigote stage residing in the sandflies and an intracellular amastigote stage colonizing the phagolysosomal compartment of the mammalian macrophages. Here, we used the isobaric tagging method to quantify the global proteomic differences between the promastigotes and the intracellular amastigotes of three different Leishmania donovani clones derived from the THP-1 human macrophage cell line. We identified a substantial number of differentially modulated proteins involved in nutrient acquisition and energy metabolism, cell motility and cytoskeleton, transport, cell signaling and stress response. Proteins involved in vesicular trafficking and endocytosis like the rab7 GTP binding protein, GTP-binding proteins of the Ras superfamily and developmentally regulated GTP-binding protein 1 revealed enhanced expression and also a putative dynein heavy chain protein was found to be up-regulated in the amastigotes and it probably has a role in cargo transport inside the vesicles. Significantly, in the amastigotes the expression of a protein involved in glucose transport was increased eight to fifteen-fold, whereas concentrations of several proteins associated with cell motility and cytoskeleton were reduced. Thus, the quantitative proteomic analysis of L. donovani isolates sheds light on some novel proteins that may have a role in Leishmania differentiation and intracellular survival.     

Antiproliferative Activity and Ultrastructural Changes in Promastigote and Amastigote forms of Leishmania amazonensis Caused by Limonene-Acylthiosemicarbazide Hybrids

Leishmaniasis is a tropical zoonotic disease. It is found in 98 countries, with an estimated 1.3 million people being affected annually. During the life cycle, the Leishmania parasite alternates between promastigote and amastigote forms. The first line treatment for leishmaniasis are the pentavalent antimonials, such as N-methylglucamine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®). These drugs are commonly related to be associated with dangerous side effects such as cardiotoxicity, nephrotoxicity, hepatotoxicity, and pancreatitis. Considering these aspects, this work aimed to obtain a new series of limonene-acylthiosemicarbazides hybrids as an alternative for the treatment of leishmaniasis. For this, promastigotes, axenic amastigotes, and intracellular amastigotes of Leishmania amazonensis were used in the antiproliferative assay; J774-A1 macrophages for the cytotoxicity assay; and electron microscopy techniques were performed to analyze the morphology and ultrastructure of parasites. ATZ−S-04 compound showed the best result in both tests. Its IC₅₀, in promastigotes, axenic amastigotes and intracellular amastigotes was 0.35±0.08 μM, 0.49±0.06 μM, and 15.90±2.88 μM, respectively. Cytotoxicity assay determined a CC₅₀ of 16.10±1.76 μM for the same compound. By electron microscopy, it was observed that ATZ−S-04 affected mainly the Golgi complex, in addition to morphological changes in promastigote forms of L. amazonensis.     

Centrin gene disruption impairs stage-specific basal body duplication and cell cycle progression in Leishmania

Centrin is a calcium-binding cytoskeletal protein involved in the duplication of centrosomes in higher eukaryotes. To explore the role of centrin in the protozoan parasite Leishmania, we created Leishmania deficient in the centrin gene (LdCEN). Remarkably, centrin null mutants (LdCEN(-/-)) showed selective growth arrest as axenic amastigotes but not as promastigotes. Flow cytometry analysis confirmed that the mutant axenic amastigotes have a cell cycle arrest at the G(2)/M stage. The axenic amastigotes also showed failure of basal body duplication and failure of cytokinesis resulting in multinucleated "large" cells. Increased terminal deoxy uridine triphosphate nick end labeling positivity was observed in centrin mutant axenic amastigotes compared with wild type cells, suggesting the activation of a programmed cell death pathway. Growth of LdCEN(-/-) amastigotes in infected macrophages in vitro was inhibited and also resulted in large multinucleated parasites. Normal basal body duplication and cell division in the LdCEN knockout promastigote is unique and surprising. Further, this is the first report where disruption of a centrin gene displays stage-specific/cell type-specific failure in cell division in a eukaryote. The centrin null mutant defective in amastigote growth could be useful as a vaccine candidate against leishmaniasis.     

Leishmania tropica: differences in the antigenicity of promastigotes and amastigotes

In vitro bioassays were used to compare T-cell responses induced by the intramacrophage amastigote stage and the sandfly-borne promastigote stage of Leishmania tropica. Lymph node cells from mice immunized with sonicated promastigotes or amastigotes incorporated in Freund's complete adjuvant were assayed for their ability to proliferate and to release interleukin 2 following in vitro challenge with promastigote or amastigote antigen. The levels of the proliferative and interleukin 2 synthetic responses of cells from promastigote and amastigote immunized mice were quite distinct. Cells from mice immunized with promastigotes demonstrated a vigorous in vitro response to the homologous antigen, but a reduced response to the potentially cross-reactive amastigotes. In contrast, cells from mice immunized with amastigotes mounted a weak response to the homologous antigen, but a consistently greater response to promastigote antigen. This unusual response was similar when 8 M urea extracts were used as immunogens and test antigens. In general, interleukin 2 production by immune mice paralleled the results from the T-cell proliferation assays. These results are discussed in relation to evasion of host immunologic detection by the intramacrophage amastigote stage.     

Leishmania mexicana: Energy metabolism of amastigotes and promastigotes

The utilisation of substrates by Leishmania mexicana amastigotes and promastigotes differed significantly. The rates of uptake and catabolism of nonesterified fatty acids were up to 10-fold higher with amastigotes. Almost all the available exogenous fatty acids were consumed during amastigote transformation and by stationary phase of promastigote growth. The results suggest that fatty acids are important energy substrates for amastigotes, whereas promastigote utilisation may reflect the requirement for these substrates in anabolism. Glucose was utilised by amastigotes and promastigotes but the rate of catabolism was up to 10-fold higher in promastigotes. Uptake of glucose occurred throughout amastigote transformation and growth in vitro of promastigotes. High-subpassage promastigotes exhibited markedly lower glucose but higher amino acid utilisation than low-subpassage promastigotes. Asparagine, glutamine, glutamate, leucine, lysine, methionine, and threonine were consumed in large quantities by amastigotes and promastigotes, whereas alanine and glycine were excreted. Proline was catabolised to CO₂ by amastigotes and promastigotes but only at a low rate, and it was excreted in large amounts throughout promastigote growth. The major end products of energy metabolism were found to be CO₂ and succinate with both forms of the parasite and there was a secretion of up to 12 and 16% of the total protein synthesised by transforming amastigotes and growing promastigotes, respectively. Catabolism in amastigotes and promastigotes was found to be sensitive to cyanide and amytal, whereas 2-mercaptoacetate and 4-pentenoate primarily affected β-oxidation in the amastigote.