Parasitic protozoa belonging to the genus Leishmania are the cause of a spectrum of diseases in humans, as well as chronic long-term infections. These parasites exhibit a remarkable capacity to survive and proliferate within the phagolysosome compartment of host macrophages. Studies with defined Leishmania mutants in mouse models of infection have highlighted processes that are required for parasite survival in macrophages. Parasite mutants have been identified that (i) are poorly virulent when the insect (promastigote) stage is used to initiate infection, but retain wild-type virulence following transformation to the obligate intracellular amastigote stage, (ii) are highly attenuated when either promastigotes or amastigotes are used, and (iii) are unable to induce characteristic lesion granulomas, but can persist within macrophages in other tissues. From these analyses it can be concluded that promastigote stages of some species require the surface expression of lipophosphoglycan, but not other surface components. Survival and subsequent proliferation of Leishmania in macrophages requires the activation of heat-shock responses (involving the up-regulation and/or phosphorylation of heat-shock proteins), the presence of oxidative and nitrosative defence mechanisms, and uptake and catabolism of carbon sources (glycoproteins, hexoses and amino acids) and essential nutrients (purines, amino acids and vitamins). Parasite mutants with defects in specific kinase/phosphatase-dependent signalling pathways are also severely attenuated in amastigote virulence, highlighting the potential importance of post-translational regulatory mechanisms in parasite adaptation to this host niche.
Leishmania spp. comprise an important group of trypanosomatid parasites that are transmitted by sandfly vectors and cause a range of diseases in humans . Depending on the infecting species and a number of other factors (host immunity etc.), these parasites may induce localized, self-curing, cutaneous lesions (cutaneous leishmaniasis) or disseminate to facial mucosal tissues or the liver and spleen to cause serious and life-threatening mucocutaneous and visceral forms of leishmaniasis. Parasites can persist long term in the host, even after apparent resolution of the disease, a factor that may be important in the development of long-term immunity to subsequent infection, but also the cause of re-occuring infections in immunocompromised individuals. Transmission occurs when an infected sandfly takes a bloodmeal on a human or another animal host, resulting in the injection of pathogenic metacyclic promastigotes into the subdermal layers of the skin. Promastigotes can be phagocytosed by a number of host cell types (neutrophils, dendritic cells and fibroblasts), but disease is propagated primarily within macrophages. Internalized promastigotes are delivered to the mature phagolysosome compartment of host macrophages where they differentiate to the non-motile amastigote stage. Depending on the Leishmania species, amastigotes can reside within individual tight-fitting phagolysosomes or within large communal phagolysosomes (Figure 1). Propagation of intracellular amastigotes to other macrophages occurs following cell division or lysis of the host cell. Leishmania are stealthy pathogens that either avoid or actively suppress macrophage microbicidal processes. Although much is known about the Leishmania–macrophage interactions, we are only just beginning to understand how amastigotes adapt to life in this hostile host niche [2,3]. In recent years, an increasing number of Leishmania mutants have been generated that can be cultured as promastigotes in vitro, but show reduced levels of growth and survival in macrophages and mice. These mutants have revealed processes that are required for Leishmania pathogenesis, as well as insights into the nature of the intracellular niches occupied by these parasites (Table 1). Although it is often difficult to define whether loss of virulence is due to impaired promastigote differentiation or amastigote survival in macrophages, the generation of novel molecular tools promises to distinguish between the different processes in the future . In the present chapter, we summarize some of the key processes that are required for parasite survival in mammalian macrophages, tissue tropism and pathogenesis in susceptible hosts.
Genes required for parasite survival in macrophages
Promastigote stages are coated with a densely packed surface glycocalyx that protects these stages from host hydrolases and serum complement components in the insect bloodmeal and digestive tract. Leishmania mutants lacking specific surface components were among the first class of mutants to be generated and have helped to define the functions of different surface components in the insect vector and the mammalian host. The major component of the promastigote surface glycocalyx is a complex lipoglycoconjugate termed LPG (lipophosphoglycan). LPG comprises a long phosphoglycan chain (made of oligosaccharide–phosphate repeat units) that is anchored to the plasma membrane via a novel GPI (glycosylphosphatidylinositol) anchor . Leishmania mutants lacking LPG are unable to colonize their sandfly vector, primarily because they cannot bind to the mid-gut wall of the digestive tract and are rapidly cleared with the remains of the bloodmeal. Leishmania major and Leishmania donovani mutants lacking LPG are also poorly virulent in mammalian macrophages and exhibit a characteristic delayed lesion phenotype in susceptible BALB/c mice. Studies by Spath et al.  have suggested that the attenuated virulence of these mutants primarily reflects their increased susceptibility to ROS (reactive oxygen species) transiently generated during macrophage invasion. Although other functions for LPG have been proposed (as a ligand for macrophage receptors, inhibition of phagolysosome maturation and inhibition of host cell signalling pathways), these functions appear to be less important or redundant . Remarkably, the surface glycocalyx of LPG is completely lost following promastigote differentiation to amastigotes, suggesting that LPG is not required for amastigote virulence . Indeed, promastigote stages of Leishmania LPG− mutants that manage to differentiate to amastigotes are as virulent as wild-type parasites, explaining why these mutants characteristically show a delay of several weeks before inducing normal lesions in susceptible mice (Table 1). Interestingly, LPG does not appear to be required for virulence in members of the Leishmania mexicana complex . These parasites may be intrinsically more resistant to oxidative stress. Alternatively, these species induce quite a different vacuolar structure in the host macrophage (Figure 1) and may thus be exposed to lower levels of ROS during invasion.
Promastigotes express a number of other surface molecules that contribute to the surface glycocalyx, but do not appear to be essential for survival in macrophages. These include a number of GPI-anchored proteins and a highly abundant family of glycolipids, termed GIPLs (glycosylphosphatidylinositol lipids), that are structurally related to the protein and LPG GPI anchors . GIPLs are abundant plasma membrane components in both promastigote and amastigote stages, and the absence of a detectable loss-of-virulence phenotype in an L. major GIPL mutant was surprising. It is likely that these parasite glycolipids are functionally replaced by host glycosphingolipids that accumulate in the plasma membrane of intracellular amastigotes . How amastigotes acquire these host lipids is unclear. One possibility is that phagolysosome membrane lipids are flipped across tight junctions that form between the posterior end of the amastigote and the vacuolar membrane (Figure 2). The acquisition of host glycolipids may protect the parasite membrane from lysosomal hydrolases. There is also evidence that scavenged host lipids can be used to synthesize parasite lipids. Specifically, the L. major Δspt mutant with a defect in de novo sphingolipid biosynthesis is able to infect and induce lesions in mice, despite displaying severe growth defects when cultivated in sphingolipid-free medium in vitro . Amastigote stages of this mutant synthesize complex parasite-specific sphingolipids, such as inositol-phosphoceramide, suggesting that scavenged sphingosine bases are internalized to the amastigote Golgi apparatus and are re-used by lipid biosynthetic enzymes in this organelle . Collectively, these studies suggest that amastigote lipid uptake is important for pathogenesis, conferring ‘host-like' characteristics to the parasite plasma membrane, as well as providing a source of precursors for complex lipid biosynthesis.
The heat-shock response pathway
Leishmania thermotolerance is essential for survival at the elevated temperatures (33–37°C) encountered in mammalian hosts. Elevated temperature is also one of the key environmental cues, together with acidification, that induces promastigote-to-amastigote differentiation in vitro [9,10]. Recent studies have suggested that HSPs (heat-shock proteins) play a key role in co-ordinating responses to heat shock and differentiation. Interestingly, in contrast with the situation in other organisms, the major HSP families of Leishmania, including HSP70, HSP90 and STI1 (stress-inducible protein 1)/HOP (HSP organizer protein), are constitutively expressed in both life cycle stages and only marginally up-regulated during heat shock . However, these abundant cytoplasmic proteins are extensively phosphorylated and assemble into large protein complexes following promastigote-to-amastigote differentiation . From detailed proteomic and genetic studies, it has been suggested that phosphorylation may control the interaction of HSP proteins with client proteins involved in regulating a variety of cellular processes . Recruitment to HSP complexes may directly regulate the activities of these client proteins and/or their susceptibility to proteosomal degradation. Consistent with this proposal, pharmacological inhibition of L. donovani HSP90 with geldanamycin or radicicol results in spontaneous amastigote differentiation under promastigote culture conditions and increased expression of other protein chaperones . The Leishmania heat-shock response may thus be regulated by heat-induced kinases instead of heat-shock transcriptional factors, as in other eukaryotes. The identity of these kinases and the precise signals that lead to their activation have yet to be identified. Finally, it is worth noting that the expression of some stress proteins, including HSP100, a mitochondrial chaperone, and the ER (endoplasmic reticulum) A2 stress protein, are up-regulated during promastigote-to-amastigote differentiation . Most of these proteins (i.e. HSP100 and A2) are not essential for promastigote growth, but are required for survival in the mammalian host .
Protection against oxidative stress
Intracellular stages are exposed to oxidative and nitrosative stresses as a result of the production of ROS and NOS (nitrosative species) by the macrophage. Metabolic reactions within the parasite, such as mitochondrial respiration, may also lead to oxidative stress. As expected, partial or complete deletion of any of the oxidative defence mechanisms of Leishmania leads to a dramatic loss of virulence. For example, the major thiol of Leishmania (and other trypanosomes) is trypanothione, a conjugate of two glutathione molecules linked by the polyamine spermidine [14–16]. Trypanothione is linked to many processes performed by glutathione in other organisms, such as the metabolism of peroxides, biosynthesis of deoxyribonucleotides, detoxification of methylglyoxal and detoxification of zenobiotics. Although enzymes involved in the synthesis and recycling of oxidized trypanothione are essential, Leishmania mutants with single allele deletions in trypanothione reductase are avirulent in macrophages [14,15]. Significantly, this enzyme is the major target of front-line anti-leishmanial drugs, such as trivalent and pentavalent antimony . Similarly, deletion of a single allele of a glycosomal Leishmania chagasi iron SOD (superoxide dismutase) results in an attenuated virulence phenotype . Collectively, these observations suggest that the oxidative defence mechanisms of Leishmania, while comprehensive, can be overwhelmed if part of the network is inhibited.
Amastigote metabolism and nutrient scavenging
Although the metabolism of in vitro cultivated promastigotes has been extensively studied, comparatively little is known about the metabolism of the intracellular amastigote stages. Transcriptomic analyses of promastigote and intracellular amastigotes stages indicate that most genes are constitutively expressed in all life cycle stages . Of the few genes that are differentially regulated, most are down-regulated in amastigote stages, suggesting that this stage may enter a growth-restricted state . In view of this transcriptional hardwiring, it is likely that stage-specific changes in metabolism are regulated by changes in protein expression and post-translational modifications. Indeed, proteomic analyses have revealed co-ordinated stage-specific changes in the level of expression of enzymes involved in a number of key pathways of carbon-source utilization, as well as alterations in the abundance and distribution of different post-translational modifications [20–22]. Analyses of the virulene phenotype of Leishmania metabolic mutants have allowed further dissection of metabolic pathways that are important for intracellular survival (Table 1). For example, disruption of glucose uptake in L. mexicana results in loss of virulence in macrophages and susceptible BALB/c mice, suggesting that glucose uptake is essential for virulence . However, levels of free sugars in the macrophage endo-lysosomal system are thought to be low, suggesting that glucose will not be a major carbon source for amastigotes. Indeed, a L. major mutant with a defect in gluconeogenesis is highly attenuated in macrophages and mice, confirming that sugar levels in vivo are insufficient to sustain amastigote growth . Metabolomic analyses of L. mexicana amastigotes suggest that this stage is capable of gluconeogenesis, but that the flux through this pathway is insufficient for growth in the absence of exogenous glucose (E. Saunders, T. Naderer and M. McConville, unpublished work). Amastigotes may therefore need to salvage sugars for specific pathways of carbohydrate metabolism, such as the pentose phosphate pathway (supplying essential NADPH-reducing equivalents), protein N-glycosylation (required for thermotolerance) and/or synthesis of the major intracellular reserve carbohydrate mannogen [25,26].
The phagolysosome is a major site of protein degradation, raising the possibility that amino acids are an important carbon source for intracellular amastigotes (Figure 2). Leishmania are also auxotrophic for many amino acids, supporting the notion that this compartment contains a rich supply of amino acids. Amino acids may be taken up directly by amino acid permeases in the amastigote plasma membrane. Alternatively, amastigotes may internalize host proteins and degrade them in enlarged lysosomal compartments, termed megasomes. Amastigote cysteine proteases are required for virulence, suggesting that parasite lysosomal degradation is important for amastigote growth . The possibility that amastigotes depend on amino acid catabolism as a major source of energy is further supported by the finding that mitochondrial metabolism is essential for intracellular survival. In particular, mitochondrial respiratory chain complexes, such as the COX (cytochrome c oxidase complex) have been shown to contain novel protein subunits that are essential for amastigote virulence [28,29]. Intriguingly, amino acid uptake by intracellular amastigotes may affect host cell responses. For example, arginine uptake and catabolism by intracellular amastigotes may lead to depletion of arginine levels in the host cell and inhibition of key microbicidal processes such as NO production (Figure 2) [30,31].
Leishmania amastigotes express all of the enzymes involved in fatty acid β-oxidation, suggesting that lipids derived from internalized lipoproteins or the phagolysosome membrane could be used for energy or as carbon sources . However, Leishmania lacks the metabolic capability to utilize fatty acids as a major carbon source, and direct evidence that fatty acids are used for energy generation in vivo has yet to be obtained . Other nutrients scavenged by amastigotes include purines, vitamins and haem. Some of these metabolite salvage systems have been well characterized and shown to be essential for intracellular growth. Not surprisingly, many of these pathways exhibit a high degree of redundancy. For example, purine salvage is initiated by a number of surface and secreted nucleotidases and a redundant network of nucleotide synthases. Interestingly, genetic disruption of the purine salvage network in L. donovani resulted in the rapid selection of suppressor strains during animal infections . The latter finding highlights the genomic flexibility of these parasites and their capacity to adapt to the loss of a particular metabolic pathway as a result of gene amplification.
Host sensing and signal transduction pathways
All microbial pathogens must sense and respond to changes in their environment. Leishmania express a large number of protein kinases and phosphatases, some of which appear to be involved in parasite adaptations to life in the mammalian host . Many of these proteins share homology with well characterized signalling kinases/phosphatases involved in regulating cellular responses to stress and nutritional responses in other eukaryotes. However, Leishmania and other trypanosomatids lack most of the cell surface receptors that initiate these signalling cascades (i.e. G-protein-coupled receptors), as well as the major downstream effectors (i.e. transcription factors) , suggesting that signalling pathways in these parasites will differ substantially from those in other eukaryotes.
The most intensively studied signalling kinases in Leishmania to date are the MAPKs (mitogen-activated protein kinases). Leishmania is predicted to contain 23 MAPKKKs (MAPK kinase kinases), seven MAPKKs (MAPK kinases) and 15 MAPKs, considerably more than in other eukaryotes . Wiese  has shown that a number of these kinases may have important roles in regulating differentiation, as well as stress responses important for survival in the mammalian host. MPK1 (mitogen-activated protein kinase 1) was discovered serendipitously and shown to be essential for survival in BALB/c mice, but not for promastigote growth in culture [35a]. On the other hand, MPK4 is essential for both developmental stages, whereas MPK7 and MPKK4 are selectively expressed in promastigote stages and reduce amastigote viability when overexpressed in this stage . The deleterious effect of MPK7 expression on amastigote growth was dependent on MAPK activity and associated with reduced protein translation and growth arrest. It is possible that MPK7 promotes parasite growth but causes parasite death when conditions are not optimal for rapid proliferation.
The TOR (target of rapamycin) serine/threonine-specific kinase is a master regulator of many processes in other eukaryotic cells, including cell growth, energy balance and metabolism. Leishmania express at least three putative TOR kinases (compared with one in mammals and two in yeast). L. major TOR1 and TOR2 appear to be essential for promastigote growth . In contrast, loss of TOR3 has little effect on promastigote growth, but is essential for infectivity in macrophages and mice . Parasite mutants lacking TOR3 are more sensitive to glucose starvation than wild-type parasites, providing a possible explanation for the loss of virulence in mice (see amastigote metabolism). Loss of TOR3 also leads to defects in the biogenesis of acidocalcisomes, an organelle involved in the storage of polyphosphates, divalent cations (Ca2+) and amino acids in many protists. Other Leishmania mutants with defects in acidocalcisome biogenesis  are attenuated in virulence, providing an additional explanation for why TOR3 is required for intracellular survival.
Leishmania also express a large number of as yet uncharacterized serine/threonine-specific protein phosphatases . On the other hand, a non-receptor PTPase (protein tyrosine phosphatase), PTP1, has been shown to be essential for virulence of L. donovani amastigotes in BALB/c mice, but not for growth of promastigotes or their differentiation to amastigotes in vitro . Intriguingly, the Trypanosoma brucei PTP1 is required for sensing extracellular nutrients and stage differentiation . The T. brucei PTP1 appears to act on a serine/threonine-specific phosphatase located in modified peroxisomes (glycosomes), providing a direct link between the plasma membrane sensor and potential intracellular targets. A related pathway is predicted to occur in Leishmania  and may play a role in regulating amastigote responses to altered nutrient levels in the phagolysosome. Collectively, these studies highlight both the importance of phosphorylation during parasite colonization of the mammalian host, but also the potential opportunities to exploit these pathways in developing new anti-parasite therapeutics.
Genes required for lesion formation
Leishmania infections in susceptible mouse strains and in many human infections are commonly associated with the development of large lesions or granulomas at the site of infection or in the liver. The formation of a lesion granuloma is driven by the host immune response and the massive recruitment of macrophages and lymphocytes to the site of infection. A number of Leishmania mutants have been generated that are able to persist long term in circulating macrophages (in lymph nodes, etc.), but do not induce the formation of granulomatous lesions. These persistent mutants include Leishmania strains with defects in gluconeogenesis (L. major ΔFBP), iron uptake (Leishmania amazonensis ΔLit1), serine biosynthesis (L. major ΔGCS) and lysosome cysteine proteases (L. mexicana ΔCPB). All of these mutants have a defect in their capacity to scavenge various nutrients and may grow more slowly than wild-type parasites in vivo. As a result they may fail to reach a threshold parasite load needed to induce a robust immune response and granuloma formation. Alternatively, nutrient levels in granuloma macrophages may be more restrictive than in other macrophage populations, preventing the establishment of a core of infected macrophages.
Genes involved in induction of visceral disease
Although a number of factors are thought to determine the outcome of Leishmania infections, the identity of the infecting species is a key factor in determining whether infections remain localized in the skin or disseminate to other tissues to cause mucocutaneous (infections of the mucosa) or visceral (infections of the liver, spleen and bone marrow) leishmaniasis. Comparative genome analysis of species associated with cutaneous (L. major), mucocutaneous (Leishmania braziliensis) and visceral disease (Leishmania infantum) have revealed a limited number of genes that are selectively expressed in L. infantum and L. donovani, but not in the other species . These include the A2 protein, a well characterized marker of amastigote stages in visceralizing species. A2 has recently been localized to the ER and shown to interact with the major ER protein chaperone BiP (immunoglobulin heavy-chain-binding protein) . A2 expression is induced by heat shock and chemical inducers of protein misfolding, suggesting that it may bolster the amastigote heat-shock response and allow these species to tolerate the elevated temperatures in the liver, as well as the fever induced by visceral infections. Supporting this notion, overexpression of the A2 protein in L. major increases the capacity of this species to metastasize and survive in the liver and spleen of mice. A number of other L. infantum/L. donovani proteins have been shown to increase the metastatic potential of L. major when ectopically expressed in this species [41,43]. One of these proteins (a putative regulator of membrane transport) is normally expressed in L. major, but at much lower levels than in L. donovani. Species-specific differences in disease pathology may therefore not reflect the unique expression of a limited number of specific proteins, but rather differences in protein expression of a large number of shared proteins .
Leishmania parasites proliferate within the mature phagolysosome compartment of macrophages, a hostile host niche occupied by a very few other pathogens. Analysis of the virulence phenotype of Leishmania mutants has provided insights into the properties of the phagolysosome and parasites factors required for virulence. Not surprisingly, proteins required for thermotolerance, protection against oxidative stress and salvage of essential carbon sources and nutrients have been shown to be important for pathogenesis. Remarkably, many of these processes may be regulated post-translationally, providing opportunities for development of new therapeutics. Major questions that remain to be answered are (i) the physiological state of amastigotes at different stages of infection, (ii) the identity of the signalling pathways that allow these parasites to adapt to changes in their intracellular niches during acute and chronic stages of infection, and (iii) processes that allow some species to metastasize to non-cutaneous tissues and cause life-threatening disease.
• Infections of the mammalian host are initiated by sandfly-transmitted promastigote stages. The LPG surface coat of this stage is required during the initial stages of infection of some Leishmania species.
• Genes involved in thermotolerance (particularly HSPs), protection against oxidative stress (trypanothione synthesis and metabolism, SODs) and metabolism (nutrient salvage, hexose and amino acid catabolism) are required for survival in macrophages and susceptible animal models.
• Many of these processes are regulated post-translationally. In particular, HSPs are phosphorylated in a stage-specific manner.
• Leishmania express a large number of kinases and phosphatases that are likely to interact with novel upstream and downstream effectors, and to be key virulence determinants.
We thank other members of the McConville group for discussions and apologize to colleagues whose references we have not cited due to space constraints. Work from M.J.M.'s laboratory was supported by the Australian National Health and Medical Research Council.
- © The Authors Journal compilation © 2011 Biochemical Society