A divergent protein kinase A regulatory subunit essential for morphogenesis of the human pathogen Leishmania.

Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the midgut of female sand flies, which they colonize as elongated extracellular promastigotes. Previous studies indicated that protein kinase A (PKA) plays an important role in the initial steps of promastigote differentiation into amastigotes. Here, we describe a novel regulatory subunit of PKA (which we have named PKAR3) that is unique to Leishmania and most (but not all) other Kinetoplastidae. PKAR3 is localized to subpellicular microtubules (SPMT) in the cell cortex, where it recruits a specific catalytic subunit (PKAC3). Promastigotes of pkar3 or pkac3 null mutants lose their elongated shape and become rounded but remain flagellated. Truncation of an N-terminal formin homology (FH)-like domain of PKAR3 results in its detachment from the SPMT, also leading to rounded promastigotes. Thus, the tethering of PKAC3 via PKAR3 at the cell cortex is essential for maintenance of the elongated shape of promastigotes. This role of PKAR3 is reminiscent of PKARIß and PKARIIß binding to microtubules of mammalian neurons, which is essential for the elongation of dendrites and axons, respectively. Interestingly, PKAR3 binds nucleoside analogs, but not cAMP, with a high affinity similar to the PKAR1 isoform of Trypanosoma. We propose that these early-diverged protists have re-purposed PKA for a novel signaling pathway that spatiotemporally controls microtubule remodeling and cell shape.

Macrophage metallothioneins participate in the antileishmanial activity of antimonials.

Host cell functions that participate in the pharmacokinetics and pharmacodynamics (PK/PD) of drugs against intracellular pathogen infections are critical for drug efficacy. In this study, we investigated whether macrophage mechanisms of xenobiotic detoxification contribute to the elimination of intracellular Leishmania upon exposure to pentavalent antimonials (SbV). Primary macrophages from patients with cutaneous leishmaniasis (CL) (n=6) were exposed ex vivo to L. V. panamensis infection and SbV, and transcriptomes were generated. Seven metallothionein (MT) genes, potent scavengers of heavy metals and central elements of the mammalian cell machinery for xenobiotic detoxification, were within the top 20 up-regulated genes. To functionally validate the participation of MTs in drug-mediated killing of intracellular Leishmania, tandem knockdown (KD) of MT2-A and MT1-E, MT1-F, and MT1-X was performed using a pan-MT shRNA approach in THP-1 cells. Parasite survival was unaffected in tandem-KD cells, as a consequence of strong transcriptional upregulation of MTs by infection and SbV, overcoming the KD effect. Gene silencing of the metal transcription factor-1 (MTF-1) abrogated expression of MT1 and MT2-A genes, but not ZnT-1. Upon exposure to SbV, intracellular survival of Leishmania in MTF-1KD cells was significantly enhanced. Results from this study highlight the participation of macrophage MTs in Sb-dependent parasite killing.

Morphogenesis Dynamics in Leishmania Differentiation.

Leishmania, the causative agent of leishmaniasis, is an obligatory intracellular parasite that cycles between phagolysosome of mammalian macrophages, where it resides as round intracellular amastigotes, and the midgut of female sandflies, where it resides as extracellular elongated promastigotes. This protozoan parasite cytoskeleton is composed of stable and abundant subpellicular microtubules (SPMT). This study aims to determine the kinetics of developmental morphogenesis and assess whether microtubules remodelling is involved in this process. Using image-streaming technology, we observed that rounding of promastigotes during differentiation into amastigotes was initiated promptly after exposure to the differentiation signal. Stabilizing microtubules with taxol sped rounding, but later killed differentiating parasites if taxol was not removed. Microtubule destabilizers such as vinblastine had no effect on the rate of rounding, nor on the viability of differentiating parasites. In the reverse process, elongation is initiated after a delay of 7.5 and completed 72 h after exposure to the amastigote to the promastigote differentiation signal. During the delay, parasites became highly sensitive to treatment with microtubule destabilizers. The addition of vinblastine during the first 7.5 h halted differentiation and killed parasites. Between 8 and 24 h, parasites gradually became resistant to vinblastine and, in parallel, started to elongate. In contrast, taxol had no effect on parasite elongation, nor on the viability of these cells. In a parallel study, we showed that the Leishmania-specific protein kinase A (PKA) holoenzyme containing the LdPKAR3-C3 complex is essential for promastigote elongation. Mutant promastigotes lacking either of these proteins are round, but maintain their flagella. Here, we observed that during differentiation into amastigotes, these mutants round at the same rate as the wild type, but never exceed the WT density of round amastigotes. In the reverse process, these mutants undergo the same initial delay and then elongate at the same rate as the WT. They stop elongating when they reach 20% of elongated cells in mature promastigotes. Our analysis indicates that while promastigote rounding into amastigotes did not require microtubule remodelling, morphogenesis of round amastigotes into elongated promastigotes required microtubule rearrangement before elongation was initiated. This is the first study that investigates the dynamics of microtubules during parasite development.

Arginine sensing in intracellular parasitism of Leishmania.

Protozoa of the genus Leishmania are intracellular parasites that cause human leishmaniasis, a disease spread mostly in the tropics and subtropics. Leishmania cycle between the midgut of female sand flies and phagolysosome of mammalian macrophages. During their life cycle they constantly encounter changing nutritional environments. To monitor the external concentration of essential nutrients, the invading parasites employ sensors that report on the availability of these nutrients; but to-date only a few sensing pathways have been identified in Leishmania. This review focuses on the Arginine Deprivation Response, which both extracellular and intracellular Leishmania utilize to monitor environmental arginine and adjust their arginine transporter (AAP3) levels accordingly.

Lysosome Sensing Is a Key Mechanism in Leishmania Intracellular Development.

Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, this organism encounters dramatic environmental changes. These include heat shock (from 26°C in the vector to 33°C or 37°C in the host, for cutaneous and visceral species, respectively) and acidic pH typical to the lysosome and nutrient availability. Leishmania cells developed ways to sense the lysosome-specific environment (acidic pH and body temperature) as means of recognition and, subsequently, initiation of differentiation into the intracellular form. Recent studies have indicated that protein kinase A plays a role as the gatekeeper that enables differentiation initiation. This review provides an update on the lysosome signaling pathway-mediated Leishmania intracellular development.

Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.

In Vitro Culture for Differentiation Simulation of Leishmania spp.

In the 1990s my laboratory discovered that Leishmania promastigotes can combine two environmental cues, typical to lysosomes, acidic pH (~5.5) and body temperature (37 °C) into a single signal that induced differentiation. Based on this concept, we modified EARLS-based medium 199 to become an amastigote-specific medium. Shifting promastigotes to this medium followed by incubation in a CO2 incubator induced differentiation. Axenic amastigotes reach maturation within 5 days, resembling the time it takes in vivo. This chapter provides a complete protocol we developed for L. donovani promastigote-to-amastigote differentiation. This protocol should be useful for both old-world and new-world species of Leishmania.

Host-Free Systems for Differentiation of Axenic Leishmania.

This chapter describes, in detail, the method our laboratory developed to differentiate L. donovani promastigotes into amastigotes in a host-free culture. This method is based on previous observations that Leishmania promastigotes can combine two environmental signals, typical to lysosomes, acidic pH (~5.5) and body temperature (37 °C), into a signal that induces differentiation. Based on this concept, we have modified medium 199 to make it into an amastigote-specific medium. Shifting promastigotes to this medium, followed by incubation in a CO2 incubator, induced differentiation. Axenic amastigotes reach maturation within 5 days, resembling the time it takes in vivo. This chapter provides a complete protocol that should be useful for both Old and New World species of Leishmania.

The arginine sensing and transport binding sites are distinct in the human pathogen Leishmania.

The intracellular protozoan parasite Leishmania donovani causes human visceral leishmaniasis. Intracellular L. donovani that proliferate inside macrophage phagolysosomes compete with the host for arginine, creating a situation that endangers parasite survival. Parasites have a sensor that upon arginine deficiency activates an Arginine Deprivation Response (ADR). L. donovani transport arginine via a high-affinity transporter (LdAAP3) that is rapidly up-regulated by ADR in intracellular amastigotes. To date, the sensor and its ligand have not been identified. Here, we show that the conserved amidino group at the distal cap of the arginine side chain is the ligand that activates ADR, in both promastigotes and intracellular amastigotes, and that arginine sensing and transport binding sites are distinct in L. donovani. Finally, upon addition of arginine and analogues to deprived cells, the amidino ligand activates rapid degradation of LdAAP3. This study provides the first identification of an intra-molecular ligand of a sensor that acts during infection.

Sensing What's Out There - Kinetoplastid Parasites.

Kinetoplastid parasites such as trypanosomes and Leishmania must adapt to their environments to survive within their hosts, yet they do not express many of the well established families of signal transduction receptors. Evidence suggests that other membrane proteins, including transporters and channels, play central roles in environmental sensing in these parasites.

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 µM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-ß-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 µM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion.

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

Protein kinase A signaling during bidirectional axenic differentiation in Leishmania.

Parasitic protozoa of the genus Leishmania are obligatory intracellular parasites that cycle between the phagolysosome of mammalian macrophages, where they proliferate as intracellular amastigotes, and the midgut of female sand flies, where they proliferate as extracellular promastigotes. Shifting between the two environments induces signaling pathway-mediated developmental processes that enable adaptation to both host and vector. Developmentally regulated expression and phosphorylation of protein kinase A subunits in Leishmania and in Trypanosoma brucei point to an involvement of protein kinase A in parasite development. To assess this hypothesis in Leishmania donovani, we determined proteome-wide changes in phosphorylation of the conserved protein kinase A phosphorylation motifs RXXS and RXXT, using a phospho-specific antibody. Rapid dephosphorylation of these motifs was observed upon initiation of promastigote to amastigote differentiation in culture. No phosphorylated sites were detected in axenic amastigotes. To analyse the kinetics of (re)phosphorylation during axenic reverse differentiation from L. donovani amastigotes to promastigotes, we first established a map of this process with morphological and molecular markers. Upon initiation, the parasites rested for 6-12 h before proliferation of an asynchronous population resumed. After early changes in cell shape, the major changes in molecular marker expression and flagella biogenesis occurred between 24 and 33 h after initiation. RXXS/T re-phosphorylation and expression of the regulatory subunit PKAR1 correlated with promastigote maturation, indicating a promastigote-specific function of protein kinase A signaling. This is supported by the localization of PKAR1 to the flagellum, an organelle reduced to a remnant in amastigote forms. We conclude that a significant increase in protein kinase A-mediated phosphorylation is part of the ordered changes that characterise the amastigote to promastigote differentiation.

Size does matter: 18 amino acids at the N-terminal tip of an amino acid transporter in Leishmania determine substrate specificity.

Long N-terminal tails of amino acid transporters are known to act as sensors of the internal pool of amino acids and as positive regulators of substrate flux rate. In this study we establish that N-termini of amino acid transporters can also determine substrate specificity. We show that due to alternative trans splicing, the human pathogen Leishmania naturally expresses two variants of the proline/alanine transporter, one 18 amino acid shorter than the other. We demonstrate that the longer variant (LdAAP24) translocates both proline and alanine, whereas the shorter variant (∆18LdAAP24) translocates just proline. Remarkably, co-expressing the hydrophilic N-terminal peptide of the long variant with ∆18LdAAP24 was found to recover alanine transport. This restoration of alanine transport could be mediated by a truncated N-terminal tail, though truncations exceeding half of the tail length were no longer functional. Taken together, the data indicate that the first 18 amino acids of the negatively charged N-terminal LdAAP24 tail are required for alanine transport and may facilitate the electrostatic interactions of the entire negatively charged N-terminal tail with the positively charged internal loops in the transmembrane domain, as this mechanism has been shown to underlie regulation of substrate flux rate for other transporters.

Proteomic analysis of posttranslational modifications using iTRAQ in Leishmania.

iTRAQ is a high coverage quantitative proteomics technique identifies and quantitates abundance changes of multiple (up to eight) distinct protein samples. To date, one iTRAQ-MS/MS assay can identify up to quarter of cells proteome. Each of the eight tags covalently binds to the N-terminus as well as arginine and lysine side chains of peptides, enabling labeling of the entire peptide population in each sample. Following tagging, the various protein samples are mixed and subjected to LC-MS/MS analysis. In the first round identical peptides from the different protein populations focus in a single pick. Subsequently, sequence of each peptide is determined. The tags whose m/z is similar to that of natural amino acids are used to determine relative abundance. To date, iTRAQ enabled identification of almost 2,000 Leishmania proteins. Here, we provide protocols for protein abundance changes and for phosphoproteomics analysis in Leishmania parasites.

Quinone-amino acid conjugates targeting Leishmania amino acid transporters.

The aim of the present study was to investigate the feasibility of targeting Leishmania transporters via appropriately designed chemical probes. Leishmania donovani, the parasite that causes visceral leishmaniasis, is auxotrophic for arginine and lysine and has specific transporters (LdAAP3 and LdAAP7) to import these nutrients. Probes 1-15 were originated by conjugating cytotoxic quinone fragments (II and III) with amino acids (i.e. arginine and lysine) by means of an amide linkage. The toxicity of the synthesized conjugates against Leishmania extracellular (promastigotes) and intracellular (amastigotes) forms was investigated, as well their inhibition of the relevant amino acid transporters. We observed that some conjugates indeed displayed toxicity against the parasites; in particular, 7 was identified as the most potent derivative (at concentrations of 1 µg/mL and 2.5 µg/mL residual cell viability was reduced to 15% and 48% in promastigotes and amastigotes, respectively). Notably, 6, while retaining the cytotoxic activity of quinone II, displayed no toxicity against mammalian THP1 cells. Transport assays indicated that the novel conjugates inhibited transport activity of lysine, arginine and proline transporters. Furthermore, our analyses suggested that the toxic conjugates might be translocated by the transporters into the cells. The non-toxic probes that inhibited transport competed with the natural substrates for binding to the transporters without being translocated. Thus, it is likely that 6, by exploiting amino acid transporters, can selectively deliver its toxic effects to Leishmania cells. This work provides the first evidence that amino acid transporters of the human pathogen Leishmania might be modulated by small molecules, and warrants their further investigation from drug discovery and chemical biology perspectives.

Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids.

Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the up-take of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter.

Regulation dynamics of Leishmania differentiation: deconvoluting signals and identifying phosphorylation trends.

Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of diseases in humans, cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosomes of mammalian macrophages. Although many of the molecular mechanisms of development inside macrophages remain a mystery, the development of a host-free system that simulates phagolysosome conditions (37 °C and pH 5.5) has provided new insights into these processes. The time course of promastigote-to-amastigote differentiation can be divided into four morphologically distinct phases: I, signal perception (0-5 h after exposure); II, movement cessation and aggregation (5-10 h); III, amastigote morphogenesis (10-24 h); and IV, maturation (24-120 h). Transcriptomic and proteomic analyses have indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Recent phosphoproteomic analysis revealed extensive differences in phosphorylation between promastigotes and amastigotes and identified stage-specific phosphorylation motifs. We hypothesized that the differentiation signal activates a phosphorylation pathway that initiates Leishmania transformation, and here we used isobaric tags for relative and absolute quantitation to interrogate the dynamics of changes in the phosphorylation profile during Leishmania donovani promastigote-to-amastigote differentiation. Analysis of 163 phosphopeptides (from 106 proteins) revealed six distinct kinetic profiles; with increases in phosphorylation predominated during phases I and III, whereas phases II and IV were characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signal-specific; 37 °C and pH 5.5), but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and phosphatases also showed changes in phosphorylation during differentiation. This work constitutes the first genome-scale interrogation of phosphorylation dynamics in a parasitic protozoa, revealing the outline of a signaling pathway during Leishmania differentiation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (identifier PXD000671). Data can be viewed using ProteinPilot™ software.

The streamlined genome of Phytomonas spp. relative to human pathogenic kinetoplastids reveals a parasite tailored for plants.

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.

Leishmania panamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival.

OBJECTIVES: Treatment failure is multifactorial. Despite the importance of host cell drug transporters and metabolizing enzymes in the accumulation, distribution and metabolism of drugs targeting intracellular pathogens, their impact on the efficacy of antileishmanials is unknown. We examined the contribution of pharmacologically relevant determinants in human macrophages in the antimony-mediated killing of intracellular Leishmania panamensis and its relationship with the outcome of treatment with meglumine antimoniate. METHODS: Patients with cutaneous leishmaniasis who failed (n = 8) or responded (n =8) to treatment were recruited. Gene expression profiling of pharmacological determinants in primary macrophages was evaluated by quantitative RT-PCR and correlated to the drug-mediated intracellular parasite killing. Functional validation was conducted through short hairpin RNA gene knockdown. RESULTS: Survival of L. panamensis after exposure to antimonials was significantly higher in macrophages from patients who failed treatment. Sixteen macrophage drug-response genes were modulated by infection and exposure to meglumine antimoniate. Correlation analyses of gene expression and intracellular parasite survival revealed the involvement of host cell metallothionein-2A and ABCB6 in the survival of Leishmania during exposure to antimonials. ABCB6 was functionally validated as a transporter of antimonial compounds localized in both the cell and phagolysosomal membranes of macrophages, revealing a novel mechanism of host cell-mediated regulation of intracellular drug exposure and parasite survival within phagocytes. CONCLUSIONS: These results provide insight into host cell mechanisms regulating the intracellular exposure of Leishmania to antimonials and variations among individuals that impact parasite survival. Understanding of host cell determinants of intracellular pharmacokinetics/pharmacodynamics opens new avenues to improved drug efficacy for intracellular pathogens.