Leishmania mexicana can utilize amino acids as major carbon sources in macrophages but not in animal models

Leishmania parasites target macrophages in their mammalian hosts and proliferate within the mature phagolysosome compartment of these cells. Intracellular amastigote stages are dependent on sugars as a major carbon source in vivo, but retain the capacity to utilize other carbon sources. To investigate whether amastigotes can switch to using other carbon sources, we have screened for suppressor strains of the L. mexicana Δlmxgt1‐3 mutant which lacks the major glucose transporters LmxGT1‐3. We identified a novel suppressor line (Δlmxgt1‐3^s2) that has restored growth in rich culture medium and virulence in ex vivo infected macrophages, but failed to induce lesions in mice. Δlmxgt1‐3^s2 amastigotes had lower rates of glucose utilization than the parental line and primarily catabolized non‐essential amino acids. The increased mitochondrial metabolism of this line was associated with elevated levels of intracellular reactive oxygen species, as well as increased sensitivity to inhibitors of the tricarboxylic acid (TCA) cycle, including nitric oxide. These results suggest that hardwired sugar addiction of Leishmania amastigotes contributes to the intrinsic resistance of this stage to macrophage microbicidal processes in vivo, and that these stages have limited capacity to switch to using other carbon sources.     

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.     

In Vivo and In Vitro Localization of Leishmania within Macrophage Phagolysosomes: Use of Colloidal Gold as a Lysosomal Label1

The interaction of Leishmania with lysosomes within macrophages in vivo has been investigated. Lysosomes labeled with colloidal gold in vivo fused with phagocytic vacuoles containing Leishmania amastigotes within the macrophages of infected footpad tissue of BALB/c mice. This localization of Leishmania within macrophage phagolysosomes in vivo is the first confirmation for any obligate intracellulaire protozoon that parasite-lysosome interactions in vitro occur in vivo.     

Trypanosome Lytic Factor,an Antimicrobial High-Density Lipoprotein,Ameliorates Leishmania Infection

Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.     

Proteophosophoglycans Regurgitated by Leishmania-Infected Sand Flies Target the L-Arginine Metabolism of Host Macrophages to Promote Parasite Survival

All natural Leishmania infections start in the skin; however, little is known of the contribution made by the sand fly vector to the earliest events in mammalian infection, especially in inflamed skin that can rapidly kill invading parasites. During transmission sand flies regurgitate a proteophosphoglycan gel synthesized by the parasites inside the fly midgut, termed promastigote secretory gel (PSG). Regurgitated PSG can exacerbate cutaneous leishmaniasis. Here, we show that the amount of Leishmania mexicana PSG regurgitated by Lutzomyia longipalpis sand flies is proportional to the size of its original midgut infection and the number of parasites transmitted. Furthermore, PSG could exacerbate cutaneous L. mexicana infection for a wide range of doses (10–10,000 parasites) and enhance infection by as early as 48 hours in inflamed dermal air pouches. This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours. Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth. The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo. Thus, PSG is an essential component of the infectious sand fly bite for the early establishment of Leishmania in skin, which should be considered when designing and screening therapies against leishmaniasis.     

Characterization of Metabolically Quiescent Leishmania Parasites in Murine Lesions Using Heavy Water Labeling

Information on the growth rate and metabolism of microbial pathogens that cause long-term chronic infections is limited, reflecting the absence of suitable tools for measuring these parameters in vivo. Here, we have measured the replication and physiological state of Leishmania mexicana parasites in murine inflammatory lesions using ²H₂O labeling. Infected BALB/c mice were labeled with ²H₂O for up to 4 months, and the turnover of parasite DNA, RNA, protein and membrane lipids estimated from the rate of deuterium enrichment in constituent pentose sugars, amino acids, and fatty acids, respectively. We show that the replication rate of parasite stages in these tissues is very slow (doubling time of ~12 days), but remarkably constant throughout lesion development. Lesion parasites also exhibit markedly lower rates of RNA synthesis, protein turnover and membrane lipid synthesis than parasite stages isolated from ex vivo infected macrophages or cultured in vitro, suggesting that formation of lesions induces parasites to enter a semi-quiescent physiological state. Significantly, the determined parasite growth rate accounts for the overall increase in parasite burden indicating that parasite death and turnover of infected host cells in these lesions is minimal. We propose that the Leishmania response to lesion formation is an important adaptive strategy that minimizes macrophage activation, providing a permissive environment that supports progressive expansion of parasite burden. This labeling approach can be used to measure the dynamics of other host-microbe interactions in situ.     

Leishmania major intracellular survival is not altered in SHP-1 deficient mev or CD45-/- mice

Protozoan parasites of the genus Leishmania escape from the immune response by interfering with signal transduction pathways of its host cell, the macrophage, thereby establishing permissive conditions for intracellular survival. Inhibition of macrophage activation after Leishmania infection has been suggested to require activation of the host cell phosphatase SHP-1. However, by utilizing infections of SHP-1 deficient (me^v) and CD45 null mutant mice or macrophages, we provide evidence that intracellular survival of Leishmania major is not generally dependent on these cellular phosphatases.     

Evidence that intracellular stages of Leishmania major utilize amino sugars as a major carbon source

Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Δgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Δgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Δgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.     

Leishmania infection inhibits macrophage motility by altering F‐actin dynamics and the expression of adhesion complex proteins

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self‐healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis‐infected macrophages also show reduced directional migration in response to the chemokine MCP‐1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F‐actin turnover frequency in L. amazonensis‐infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane–extracellular matrix interactions.     

Localization and induction of the A2 virulence factor in Leishmania: evidence that A2 is a stress response protein

Leishmaniasis is a vector‐borne infectious disease with a wide range of pathologies depending on the species of Leishmania. Leishmania parasites are transmitted by the sand fly vector as promastigotes; within the mammalian host, Leishmania parasites differentiate into amastigotes and replicate in macrophages. The A2 protein from Leishmania donovani is expressed predominantly in amastigotes and therefore likely plays a role in survival in the mammalian host. In the present study, we have determined that the A2 protein colocalized with the Leishmania endoplasmic reticulum binding protein, BiP, was induced by stress and complexed with BiP following heat shock. The A2 gene in Leishmania major is a non‐expressed pseudogene, and we present evidence that ectopic expression of a transfected A2 gene in L. major enhanced its viability following heat shock. A2 may therefore play a role in protecting L. donovani from stress associated with infection in visceral organs, including the fever typically associated with visceral leishmaniasis. Interestingly, when comparing A2 protein localization, we also observed that the Leishmania secreted acid phosphatase SAcP protein was transported out of the parasite‐containing phagolysosome and was located throughout the macrophage cytoplasm in vesicles, providing the first example of a secreted Leishmania‐derived protein exiting the parasite‐containing phagolysosome.     

Changes in macrophage membrane properties during early Leishmania amazonensis infection differ from those observed during established infection and are partially explained by phagocytosis

Understanding the impact of intracellular pathogens on the behavior of their host cells is key to designing new interventions. We are interested in how Leishmania alters the electrical function of the plasma membrane of the macrophage it infects. The specific question addressed here is the impact of Leishmania infection on macrophage membrane properties during the first 12 h post-infection. A decrease of 29% in macrophage membrane capacitance at 3 h post-infection indicates that the phagolysosome membrane is donated on entry by the macrophage plasma membrane. Macrophage membrane potential depolarized during the first 12 h post-infection, which associated with a decreased inward potassium current density, changed in inward rectifier conductance and increased outward potassium current density. Decreased membrane capacitance and membrane potential, with no changes in ion current density, were found in macrophages after phagocytosis of latex beads. Therefore we suggest that the macrophage membrane changes observed during early Leishmania infection appear to be associated with the phagocytic and activation processes.     

LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure,Is Involved in the Infectivity and Pathogenicity of Leishmania

Leishmaniasis is a neglected disease produced by the intracellular protozoan parasite Leishmania. In the present study, we show that LABCG2, a new ATP-binding cassette half-transporter (ABCG subfamily) from Leishmania, is involved in parasite virulence. Down-regulation of LABCG2 function upon expression of an inactive mutant version of this half-transporter (LABCG2^K/M) is shown to reduce the translocation of short-chain analogues of phosphatidylserine (PS). This dominant-negative phenotype is specific for the headgroup of the phospholipid, as the movement of phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin is not affected. In addition, promastigotes expressing LABCG2^K/M expose less endogenous PS in the stationary phase than control parasites. Transient exposure of PS at the outer leaflet of the plasma membrane is known to be one of the mechanisms used by Leishmania to infect macrophages and to silence their immune response. Stationary phase/metacyclic promastigotes expressing LABCG2^K/M are less infective for macrophages and show decreased pathogenesis in a mouse model of cutaneous leishmaniasis. Thus, mice infected with parasites expressing LABCG2^K/M did not develop any lesion and showed significantly lower inflammation and parasite burden than mice infected with control parasites. Our results indicate that LABCG2 function is required for the externalization of PS in Leishmania promastigotes, a process that is involved in the virulence of the parasite.     

Phenotypic Characterization of a Leishmania donovani Cyclophilin 40 Null Mutant

Protozoan parasites of the genus Leishmania adapt to their arthropod and vertebrate hosts through the development of defined life cycle stages. Stage differentiation is triggered by environmental stress factors and has been linked to parasite chaperone activities. Using a null mutant approach we previously revealed important, nonredundant functions of the cochaperone cyclophilin 40 in L. donovani‐infected macrophages. Here, we characterized in more detail the virulence defect of cyp40?/? null mutants. In vitro viability assays, infection tests using macrophages, and mixed infection experiments ruled out a defect of cyp40?/? parasites in resistance to oxidative and hydrolytic stresses encountered inside the host cell phagolysosome. Investigation of the CyP40‐dependent proteome by quantitative 2D‐DiGE analysis revealed up regulation of various stress proteins in the null mutant, presumably a response to compensate for the lack of CyP40. Applying transmission electron microscopy we showed accumulation of vesicular structures in the flagellar pocket of cyp40?/? parasites that we related to a significant increase in exosome production, a phenomenon previously linked to the parasite stress response. Together these data suggest that cyp40?/? parasites experience important intrinsic homeostatic stress that likely abrogates parasite viability during intracellular infection.     

Apoptotic-like Leishmania exploit the host′s autophagy machinery to reduce T-cell-mediated parasite elimination

Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed—in contrast to viable parasites—that apoptotic-like parasites enter an LC3⁺, autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4⁺ T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells´ autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis.     

Bet-hedging antimicrobial strategies in macrophage phagosome acidification drive the dynamics of Cryptococcus neoformans intracellular escape mechanisms

The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.     

In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites

Background

The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro.

Methodology/Principal Findings

We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance.

Conclusions/Significance

Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation.