Leishmania survival in the macrophage: where the ends justify the means

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ScienceDirect Leishmania survival in the macrophage: where the ends justify the means Guillermo Arango Duque1,2 and Albert Descoteaux1,2 Macrophages are cells of the immune system that mediate processes ranging from phagocytosis to tissue homeostasis. Leishmania has evolved ingenious ways to adapt to life in the macrophage. The GP63 metalloprotease, which disables key microbicidal pathways, has recently been found to disrupt processes ranging from antigen cross-presentation to nuclear pore dynamics. New studies have also revealed that Leishmania sabotages key metabolic and signaling pathways to fuel parasite growth. Leishmania has also been found to induce DNA methylation to turn off genes controlling microbicidal pathways. These novel findings highlight the multipronged attack employed by Leishmania to subvert macrophage function. Addresses 1 INRS-Institut Armand-Frappier, Laval, QC H7 V 1B7, Canada 2 Centre for Host-Parasite Interactions, Laval, QC H7 V 1B7, Canada Corresponding authors: Arango Duque, Guillermo ([email protected], [email protected]) and Descoteaux, Albert ([email protected])

Current Opinion in Microbiology 2015, 26:32–40 This review comes from a themed issue on Host-microbe interactions: parasites Edited by Ira Blader

http://dx.doi.org/10.1016/j.mib.2015.04.007 1369-5274/# 2015 Published by Elsevier Ltd.

Introduction Macrophages: sentinels of the immune system

Macrophages are crucial to the immune response and their absence would allow infections to progress uncontrollably to the detriment and eventual demise of the host [1]. The importance of these phagocytic cells in antimicrobial defence and development was recognized and documented by Ilya Metchnikoff [2], an achievement that merited him the 1908 Nobel Prize in Physiology or Medicine. Macrophages are found in every tissue, and most develop from bone marrow myeloid precursor cells. Phagocytosis is at the helm of macrophage biology [3]. This process is essential for nutrient recycling, organismal homeostasis and defense from pathogens [4]. For example, macrophages phagocytose circulating erythrocytes in order to recycle iron back into circulation [5]. Resting Current Opinion in Microbiology 2015, 26:32–40

macrophages can be chemoattracted to the site of infection, and can be activated by cytokines such as interferon gamma (IFN-g) and by microbial compounds [6,7]. In their first-of-the-line role in infections, macrophages are some of the first cells to come in contact with, recognize and kill microbes. Macrophages can also present antigens to lymphocytes [8] via major histocompatibility complex (MHC) molecules at the macrophage’s plasmalemma. Leishmania parasites have evolved to conquer macrophages

Pathogens have evolved to circumvent and conquer many of the antimicrobial strategies mounted by macrophages [9,10]. Living inside of the macrophage is an ideal way to escape the immune system, obtain nutrients, and proliferate. Leishmania parasites, which cause the leishmaniases, constitute one such example of a microorganism that has successfully adapted to life in the macrophage. The leishmaniases constitute a spectrum of diseases caused by flagellate protozoa. These intracellular parasites cause cutaneous, mucocutaneous and visceral pathologies in humans and other animals [11]. The leishamaniases are prevalent in 98 countries spread over 5 continents [12]. The parasite has a digenetic life cycle and is transmitted to humans by infected hematophagous female sand flies. Promastigotes, which are the extracellular forms of the parasite, are injected into the host via the proboscis of the sand fly [13]. Parasites in the skin are ingested primarily by macrophages, and also by neutrophils and dendritic cells. Although many promastigotes are destroyed by macrophages, some evade the microbicidal power of the phagolysosome, thereby transforming this powerful organelle into a parasitophorous vacuole (PV) that fosters parasite growth [14–16]. Within PVs, promastigotes transform into amastigotes that multiply via binary fission. When the host cell becomes overwhelmed by parasites, it may either rupture and release amastigotes, or become apoptotic and pass the amastigote cargo to surrounding macrophages [17]. Either way, amastigotes metastasize and cause pathology. The life cycle is completed when a previously uninfected sand fly takes a blood meal containing amastigotes or infected phagocytes. Leishmania-macrophage interactions are multifaceted and involve a number of pathogenicity factors that allow parasites to use the phagolysosome to obtain nutrients, hijack antimicrobial pathways, and replicate [18]. The zinc metalloprotease GP63 is the most abundant molecule on the promastigote surface. It is a pathogenicity factor that cleaves host proteins, enabling Leishmania to www.sciencedirect.com

Leishmania sabotages macrophage biology for survival Arango Duque and Descoteaux 33

subvert processes such as transcription and translation [16]. In the process of conquering the macrophage, Leishmania has also been known to hijack signaling pathways [19] that would otherwise kill the parasite in the phagolysosome and impede dissemination [14]. This review discusses recent studies (2013–2015) that cast light on how Leishmania sabotages macrophage functions for survival. It will focus on new discoveries concerning how the GP63 protease alters the macrophage’s membrane fusion machinery to tamper with processes that are essential to macrophage physiology and function. Furthermore, it will discuss how Leishmania disrupts various host metabolic and signaling pathways, as well as DNA accessibility, in order to promote intracellular survival.

The onslaught of the GP63 protease: from membrane trafficking to nuclear pore dynamics Manipulation of membrane trafficking to subvert antigen presentation and cytokine secretion

Phagocytosis is governed by sequential interactions with cell organelles. These interactions are spatiotemporally regulated by a complex molecular machinery involving proteins that mediate vesicle fusion [20]. Proteins regulating neurotransmitter release, notably those of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) family [21,22], are of salient importance in coordinating membrane trafficking in cells of the immune system. During vesicle fusion, SNAREs in vesicles and target membranes interact specifically to pull two membranes into close proximity [21,22]. SNAREs are selectively distributed in different organelles. VAMP8, which forms complexes with syntaxin-7, syntaxin-8 and Vti1b, participates in homotypic fusion of late endosomes and is recruited to phagosomes [23,24]. It also mediates the recruitment of gp91phox — a component of the NOX2 oxidase — to the phagosome [25]. This in turn implies that VAMP8 plays an important role in modulating intraphagosomal function and host defence. Antigen crosspresentation is an important function of phagosomes [26,27]. Cross-presentation of microbial peptides on major histocompatibility (MHC) I molecules is essential to activate CD8+ T cells. Using cells from Vamp8–/– mice, Matheoud et al. discovered that VAMP8 plays an essential role in antigen cross-presentation in both bone marrowderived macrophages (BMM) and dendritic cells (BMDC) [25]. Interestingly, infection of Vamp8–/– mice with L. major parasites results in larger footpad lesions [25]. Bacteria have evolved numerous ways to hamper SNARE function. For instance, clostridial toxins, which block synaptic transmission in peripheral cholinergic synapses, cleave SNAREs [28]. Using gp63-KO L. major parasites, Matheoud and colleagues found that VAMP8 and VAMP3 were directly cleaved by GP63 [25]. Given the importance of SNAREs in mediating trafficking to and from www.sciencedirect.com

the phagosome, the authors sought to identify the consequences of SNARE cleavage on cross-presentation. Feeding L. major-infected macrophages with ovalbumin (OVA)-coated beads, the authors found that GP63 strongly inhibits OVA cross-presentation in both BMMs and BMDCs. Detailed analysis of these phagosomes revealed that cross-presentation is hindered due to decreased gp91phox recruitment, which leads to decreased intraphagosomal oxidation, increased proteolytic activity and altered pH (Figure 1a). In sum, by targeting SNAREs, Leishmania impairs crucial processes involved in phagolysosome biogenesis that are required for antigen crosspresentation. Synaptotagmins (Syts) are membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis [29,30] and phagocytosis [30–32]. They regulate SNARE activity by mediating membrane fusion in a Ca2+-dependent manner [33]. All Syts possess two conserved tandem Ca2+-binding C2 domains. However, Syt XI contains a conserved serine in its C2A domain that precludes this Syt from mediating vesicle fusion [34]. In macrophages, Syt XI is a recycling endosome-associated and lysosome-associated protein that negatively regulates the secretion of tumour necrosis factor (TNF) and IL-6 [1,22,30]. The roles of Syts in vesicle trafficking make these proteins great targets for attack by intracellular pathogens. For instance, the parasite Trypanosoma cruzi uses Syt VII, a Ca2+-dependent regulator of lysosome exocytosis, to invade target cells [35]. It had previously been observed that Leishmania promastigotes of certain species are able to trigger TNF and IL-6 secretion postinfection [36–40]. Although the parasite signal that induces TNF and IL-6 secretion is not known, synthesis and secretion of these cytokines may be initially triggered by engagement of TLR2 and TLR3 with parasite molecules [41]. Given the role of Syt XI in cytokine secretion, it was hypothesized that Leishmania could affect Syt XI function to deregulate cytokine release. Using L. major strains that express or lack GP63, Arango Duque et al. found that the GP63 protease degrades Syt XI and positively regulates the post-infection release of TNF and IL-6 [42]. Using RNA interference, it was shown that cytokine release was induced by GP63-mediated degradation of Syt XI. At the forefront of these findings is the observation that, early during infection, GP63 induces the release of TNF and IL-6 in vivo. Importantly, injection of GP63-expressing parasites induces the influx of neutrophils and inflammatory monocytes to the inoculation site. This is likely to be a consequence of increased TNF and IL-6 release. Infection of recruited inflammatory monocytes [43] can trigger IL-10 secretion [44,45], and induce their differentiation into immunosuppressive, arginase-expressing alternatively activated macrophages [46]. The role of Syt XI in the recruitment of these phagocyte populations is to be elucidated. This new role for GP63 implicates this protease in the manipulation of Current Opinion in Microbiology 2015, 26:32–40

34 Host-microbe interactions: parasites

Figure 1

(a) Decreased loading on MHC class I

Alkaline pH

H2O2 Less proteolytic activity

Hampered cross-presentation

Increased proteolytic activity

H+ Loading on MHC class I

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2O– Low oxidation

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GP63 action leads to increased phagocyte recruitment (possibly due to increased cytokine release)

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Leishmania disrupts antigen cross-presentation and cytokine secretion. The GP63 protease degrades key proteins that mediate vesicle trafficking. (a) Cleavage of VAMP8 hinders antigen cross-presentation. Phagosomes (left panel) recruit the gp91phox via VAMP8. The action of gp91phox results in increased intraphagosomal oxidation, which helps keep a more alkaline pH in the phagosome. The consequent decrease in proteolytic activity allows processed antigens to be loaded onto MHC class I molecules. Cross-presentation plays an important role in the stimulation of T cells and

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Leishmania sabotages macrophage biology for survival Arango Duque and Descoteaux 35

cytokine trafficking, which may play a pivotal role in the phagocyte accrual observed at the early stages of infection [42,43] (Figure 1b). Dok proteins are important for dampening the signaling program induced by microbial molecules and cytokines on macrophages. This is achieved by recruiting inhibitory molecules following receptor activation [47]. These important adaptor proteins are recruited to latex bead and zymosan phagosomes, and may be important in phagolysosomal function. Similar to VAMP8 and Syt XI, the levels of Dok proteins are diminished in macrophages infected with GP63-expressing promastigotes [48]. Interestingly, infected IFN-g-primed Dok-1/Dok-2 / macrophages release less NO and TNF in comparison to WT macrophages; this effect is independent of GP63. Although the functions of Dok proteins in the context of phagosome maturation and cytokine secretion are not yet known, the findings by A´lvarez et al. suggest that Dok-1 and Dok-2 regulate the secretion of immune effectors triggered by Leishmania infection. The role of these proteins in an in vivo infection model is yet to be investigated. Manipulating membrane trafficking thus emerges as an efficient strategy to curb the microbicidal power of the macrophage. Another way to manipulate this process is during parasite egress and transmission to uninfected cells. Using live cell imaging, Real et al. discovered that after long-term infections, zeiotic macrophages extrude amastigote-containing PVs that can be ingested by other macrophages [17]. These extruded PVs were found to be strongly associated with LAMP1. Extruded PVs were found to induce IL-10 secretion from the ingesting macrophages, and this was found to be dependent on the presence of LAMP1. In the in vivo setting, this mode of transmission — and LAMP1-induced IL-10 secretion — is likely to play a key role in the immunosuppression of the host and in the maintenance of chronic infection. Impact of GP63 on nuclear integrity and physiology

Recent studies have revealed that GP63 is found in the vicinity of the nucleus [49], raising the possibility that GP63 could be interfering with nuclear dynamics. This is supported by the fact that cleavage of the AP-1 transcription factor occurs at the nucleus [50], suggesting that GP63 enters and disrupts this compartment. Isnard and colleagues provided evidence that L. major GP63 localizes to the nuclear envelope of infected macrophages and is active in this compartment and in the nucleoplasm [51]. This nuclear confinement is nuclear localization signal

(NLS)-independent, and occurs instead via interactions with Nup members of the nuclear pore complex. Indeed, proteins such as Nup62 and Nup368 were found to be cleaved by GP63, and mass spectrometry analyses revealed that the levels of several nuclear pore complex-associated proteins are altered. Importantly, GP63 was also found to alter the nucleoplasm proteome, with proteins involved in translational regulation and DNA accessibility severely diminished in the presence of GP63. It was also found that L. mexicana, a species that also causes cutaneous disease, alters nuclear physiology more severely than L. major. Further work will be needed to elucidate the consequences of GP63-induced changes in proteins associated with the nucleus. To further support the argument that Leishmania infection manipulates the expression of proteins found in the nucleus, Singh et al. also used proteomics to gain a global understanding of how L. donovani infection reprograms cells [52]. It was observed that histone proteins such as H2A, H2B and H4 were upregulated in infected cells, indicating that infection impacts chromatin remodelling and host gene regulation. Moreover, the expression of several proteins involved in DNA repair and replication, translation and RNA splicing were also found to be deregulated post-L. donovani infection.

Leishmania hijacks metabolic pathways in the macrophage to promote survival Retention of intracellular iron fuels amastigote survival

Iron metabolism encompasses processes including intestinal iron absorption, iron transport and storage into cells, and macrophage-mediated iron recycling from erythrocytes [53]. A dynamic balance for iron in the circulation among the different compartments is maintained, with most iron released by the decomposition of hemoglobin from senescent erythrocytes. At the end of their lives, senescent erythrocytes show a series of biochemical changes that trigger their phagocytosis by macrophages, mainly in the spleen [53,54]. During this process, hemoglobin is degraded and Fe2+ is released. Recycled Fe2+ is then transported back to circulation via ferroportin, oxidized by ceruloplasmin to Fe3+, bound to transferrin, and reused for hemoglobin synthesis. The main regulator for iron metabolism is the hormone hepcidin, which exerts its function by triggering the endocytosis and subsequent intralysosomal degradation of ferroportin. The bioactive form is produced mainly by the liver, but also by macrophages where it acts in an autocrine fashion. Hepcidin action causes iron trapping in macrophages, enterocytes, and hepatocytes [53,54]. Intracellular pathogens have evolved approaches to manipulate iron recycling to fit

(Figure 1 Legend Continued) is essential in the immune response. When macrophages and dendritic cells are infected with GP63-expressing L. major (right panel), VAMP8 is degraded and recruitment of gp91phox to the phagosome is hindered. This results in a lower pH that fosters increased proteolytic activity, but leads to decreased cross-presentation of antigens. (b) GP63 degrades Syt XI, a negative regulator of TNF and IL-6 secretion. The ensuing post-infection release of TNF and IL-6 can help recruit inflammatory monocytes and neutrophils to the infection site. Infection of these inflammatory phagocytes promotes the establishment of chronic infections. www.sciencedirect.com

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36 Host-microbe interactions: parasites

their needs [54]. Leishmania is an example of a pathogen that requires iron for survival and replication [55]. A number of studies have started to elucidate how iron is transported from the phagolysosome into amastigotes. Three channels known as LFR1, LIT1 and LABCG5 have been implicated in this process [55]. However, whether Leishmania modulates iron dynamics in macrophages was not known. A study by Ben-Othman et al. demonstrated that L. amazonensis is capable of inhibiting iron efflux [56] (Figure 2). The authors observed that amastigote infection lowered ferroportin at the transcriptional and protein level. Knowing that hepcidin plays a crucial role in ferroportin turnover, the authors found that hepcidin levels were higher in infected macrophages and in the livers and footpads of infected mice. The effects of infection on ferroportin and hepcidin levels were found to be TLR4-dependent. The increase in hepcidin was correlated with higher intracellular iron

levels. Importantly, overexpression of a dominant negative ferroportin mutant that fails to go to the macrophage plasmalemma resulted in increased parasite survival. The consequent increase in intracytoplasmic iron also allowed the proliferation of parasites that uptake iron poorly. Conversely, the authors showed that overexpression of hepcidin-insensitive ferroportin resulted in decreased parasite survival. This study is the first to show how an intracellular protozoan manipulates the recycling of iron of the host cell. The mechanism by which Leishmania augments hepcidin levels is to be elucidated. Increased expression of hepcidin is observed under inflammatory conditions and can be triggered by IL-6 [57]. This raises the possibility that the GP63-mediated increase in IL-6 secretion [30] could increase hepcidin expression in infected macrophages. How iron availability and recycling influences the various leishmanial pathologies also remains to be explored [56].

Figure 2

Fe2+ Fe2+

Fe2+

Hepcidin induces ferroportin degradation

Ferroportin

Decreased ferroportin leads to Fe2+ accumulation and favours parasite replication

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Leishmania upregulates hepcidin levels

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Leishmania prevents iron efflux via hepcidin upregulation. Infection with L. amazonensis induces hepcidin levels. Hepcidin, a peptide hormone that binds to ferroportin outside of the cell, induces ferroportin degradation. The consequent increase in intracellular iron promotes parasite growth. Current Opinion in Microbiology 2015, 26:32–40

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Leishmania sabotages macrophage biology for survival Arango Duque and Descoteaux 37

Disruption of cholesterol dynamics favours Leishmania growth

Cholesterol is an essential lipid for membrane biogenesis and cellular physiology. Leishmania promastigotes use LPG to disrupt cholesterol-rich lipid rafts to gain access to the cell, disrupt signaling pathways, and weaken the phagolysosome [58–60]. It had been observed that patients afflicted with visceral leishmaniasis have lower levels of circulating cholesterol and other lipids [61]. The importance of cholesterol during Leishmania infection was highlighted by a study that showed that GP63 cleaves DICER1, thence lowering miR-122 levels in hepatocytes of infected mice [62]. Diminished levels of miR-122, an miRNA and master regulator of lipid synthesis in the liver, ensues in lower cholesterol levels and parasite survival. Restitution of miR-122 rescues cholesterol levels and inhibits parasite growth. Whether miR-122 controls cholesterol metabolism in macrophages remains to be investigated [62,63]. Infected macrophages are defective in stimulating T cells and in assembling IFN receptor subunits at the surface [64]. T cell stimulation and proper IFN-g signaling are restored when infected macrophages are supplied with liposomal cholesterol. This treatment also activates infected macrophages and promotes parasite killing [65]. These findings raised the possibility that receptor movement at the cell surface is disrupted by L. donovani infection. To address this question, Ghosh et al. transfected macrophages with a PLCd1-GFP fusion protein and monitored the protein’s distribution and lateral movement using quantitative live cell imaging [66]. PLCd1 is found in lipid rafts. Albeit infection does not affect the expression or plasmalemmal localization of PLCd1-GFP, its motion in the plasma membrane was augmented. Recovery after photobleaching was also faster in infected cells. Normal PLCd1-GFP motion is restored after macrophages are given liposomal cholesterol. This finding could be explained by the observation that L. donovani infection disrupts actin filaments and lowers F-actin protein levels; this phenomenon is also reversible by liposomal cholesterol [66]. Increased receptor movement and altered actin dynamics may all lead to a disruption in signal transduction from the cell surface and the cytoskeleton. Improved understanding on the interplay between host actin and cholesterol will shed further light on how Leishmania survives in the macrophage. These studies may also foreshadow the use of liposomal cholesterol for the treatment of the leishmaniases. Manipulation of the host’s energy resources promotes parasite survival

Nutrient acquisition and conversion into energy is essential to the life every cell, and organisms have evolved diverse mechanisms to utilize what is available in their environment. Hence, it comes as no surprise that eukaryotic parasites compete with their hosts for some of the same nutrients required for the survival of both entities. Moreira and colleagues elegantly demonstrated that www.sciencedirect.com

Leishmania hijacks mitochondrial pathways required for glucose metabolism [67]. Early during infection, L. infantum was found to augment aerobic glycolysis, which was followed by a switch to mitochondrial metabolism and oxidative phosphorylation later during infection; infection progression was accompanied by increased mitochondria biogenesis. This claim is supported by proteomic mapping of L. donovani-infected cells, which revealed that several enzymes involved in beta-oxidation, the tricarboxylic acid cycle and mitochondrial respiration are upregulated [52]. To explain the drastic shift from glycolysis to mitochondrial metabolism, the Moreira et al. focused on the AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis in the cell [68]. AMPK is activated with rising AMP levels. Indeed, infection augmented the AMP/ATP ratio and induced the phosphorylation and subsequent activation of AMPK. This activation leads to increased levels of glucose transporters (GLUTs) and increased glucose uptake. LKB1 mediates phosphorylation of AMPK, and is required for Leishmania-induced activation of AMPK. The SIRT1 protein, a sensor of NAD+/NADH levels in cell, was found to regulate AMPK activation by L. infantum. At the helm of this research is the finding that genetic ablation of AMPK, LKB1 or SIRT1 promotes parasite clearance in in vivo and in vitro models of infection, hence highlighting the importance of these key metabolic regulators in the progression of visceral leishmaniasis [67]. DNA methylation is a strategy to shut down genes involved in host defence

Epigenetic regulation is central for the proper spatiotemporal regulation of gene expression in most vertebrates. DNA methylation is the addition of a methyl group to cytosine molecules, and is associated with gene silencing. DNA methylation can prevent promoter recognition by polymerases, and is associated with decreased nuclear clustering of chromatin [69]. Base methylation results in the silencing of a particular gene or set of genes. Microorganisms can alter the epigenome of host cells [70]. For instance, hepatitis B infection ensues in the hypermethylation of the urokinase-type plasminogen activator promoter, which is essential for hepatocyte growth and tissue repair [71]. A study by Marr and colleagues [72] found that L. donovani induces changes in DNA methylation to modulate genes involved in antimicrobial pathways (Figure 3). Using macrophages infected with either live or heat-killed L. donovani, it was found that live parasites induced significant DNA methylation changes at 443 CpG sites. Affected sites were located at proximalpromoter and at non-promoter regions. The affected genes were found to participate in processes such as calcium, chemokine and Notch signaling, and actin cytoskeleton assembly. Genes such as IRAK2, which encodes the interleukin-1 receptor-associated kinase 2 and promotes upregulation of NF-kB, was found to be methylated and its transcription was decreased. This in turn Current Opinion in Microbiology 2015, 26:32–40

38 Host-microbe interactions: parasites

Figure 3

Pathways affected by Leishmania-induced methylation changes: JAK/STAT, MAPK, NF-κB, mTOR, calcium and Notch signaling, chemokine signaling, endocytosis and cell adhesion...

Vesicles could contain parasitederived methyltransferases or methylation inhibitors Parasite exosomes??

L. donovani PV

Nucleus Leishmania-induced methylation changes affect the expression of genes controlling antimicrobial pathways

Infection provokes changes in DNA methylation ↓ ↓ Macrophage activation and ability to eliminate infection

Vesicles emanating from PV??

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Leishmania induces changes in the host macrophage methylome for survival. Infection with L. donovani alters the DNA methylation status of many host genes. Several of these genes, which partake in important antimicrobial functions, are shut down as a result.

contributes to decreased production of microbicidal molecules, and complements other Leishmania-mediated mechanisms that result in hindered NF-kB signaling [16,19]. How the parasite affects host methylation remains to be investigated. Given the evidence presented by Isnard et al. [51], it is possible for nuclear GP63 to indirectly affect the availability of DNA to methylating enzymes, or the enzymes themselves. It is also probable that parasites export DNA methyltransferases or methylation inhibitors from the PV into the cytoplasm, or via parasite-derived exosomes [72].

Conclusion Elucidating the mechanisms by which Leishmania alters the function of its host cell is essential for the design of novel and effective approaches to prevent or treat leishmaniases. In particular, future studies will be required to understand how Leishmania effectors exit the PV and traffic within the host cell. Further understanding on how Leishmania reprograms key metabolic pathways is required in order to identify new targets for the treatment of the leishmaniases.

Acknowledgements This work was funded by Canadian Institutes of Health Research (CIHR) grant MOP-125990. G.A.D. was supported by a CIHR Frederick Banting and Charles Best Doctoral Award. We kindly acknowledge the work of Current Opinion in Microbiology 2015, 26:32–40

many colleagues whose research was not cited due to space limitations. We thank Dr. N. Moradin for stimulating discussions.

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