Antileishmanial Activity of the Estrogen Receptor Modulator Raloxifene

Background

The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis.

Methodology/Principal Findings

Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC₅₀ values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene''s mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC₂(3), rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis – infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden.

Conclusions/Significance

The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death.     

Encapsulation of Living Leishmania Promastigotes in Artificial Lipid Vacuoles

After phagocytosis by mammalian macrophages, promastigote forms of Leishmania parasites settle inside intracellular parasitophorous vacuoles (PVs) in which they transform into amastigote forms and replicate. Here, using a variant of the ‘inverted emulsion’ method, we succeeded in encapsulating living L. amazonensis parasites in giant artificial liposomes that serve as model PVs. We were able to control the size of liposomes, the pH and the composition of their internal volume, and the number of internalized parasites per liposome. L. amazonensis promastigotes encapsulated in liposomes filled with RPMI-Dextran solution at pH 7.5 or 6.5 survived up to 96 h at 24°C. At 37°C and pH 5.5, parasites survived 48h. This method paves the way to identifying certain effectors secreted by the parasite and to unraveling specific mechanisms of fusion between the PV and intracellular vesicles of the host cell. This method will also facilitate the study of the temporal evolution of biophysical properties of the PV during its maturation.     

A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence

Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.     

Leishmanicidal Effect of Synthetic trans-Resveratrol Analogs

Background

Stilbene-based compounds show antitumoral, antioxidant, antihistaminic, anti-inflammatory and antimicrobial activities. Here, we evaluated the effect of the trans-resveratrol analogs, pterostilbene, piceatannol, polydatin and oxyresveratrol, against Leishmania amazonensis.

Methodology/Principal Findings

Our results demonstrated a low murine macrophage cytotoxicity of all four analogs. Moreover, pterostilbene, piceatannol, polydatin and oxyresveratrol showed an anti-L. amazonensis activity with IC₅₀ values of 18 μM, 65 μM, 95 μM and 65 μM for promastigotes, respectively. For intracellular amastigotes, the IC₅₀ values of the analogs were 33.2 μM, 45 μM, 29 μM and 30.5 μM, respectively. Among the analogs assayed only piceatannol altered the cell cycle of the parasite, increasing 5-fold the cells in the Sub-G0 phase and decreasing 1.7-fold the cells in the G0-G1 phase. Piceatannol also changed the parasite mitochondrial membrane potential (ΔΨm) and increased the number of annexin-V positive promastigotes, which suggests incidental death.

Conclusion/Significance

Among the analogs tested, piceatannol, which is a metabolite of resveratrol, was the more promising candidate for future studies regarding treatment of leishmaniasis.     

Leishmania amazonensis Amastigotes Highly Express a Tryparedoxin Peroxidase Isoform That Increases Parasite Resistance to Macrophage Antimicrobial Defenses and Fosters Parasite Virulence

Professional phagocytes generate a myriad of antimicrobial molecules to kill invading microorganisms, of which nitrogen oxides are integral in controlling the obligate intracellular pathogen Leishmania. Although reactive nitrogen species produced by the inducible nitric oxide synthase (iNOS) can promote the clearance of intracellular parasites, some Leishmania species/stages are relatively resistant to iNOS-mediated antimicrobial activity. The underlying mechanism for this resistance remains largely uncharacterized. Here, we show that the amastigote form of L. amazonensis is hyper-resistant to the antimicrobial actions of cytokine-activated murine and human macrophages as compared to its promastigote counterpart. Amastigotes exhibit a marked ability to directly counter the cytotoxicity of peroxynitrite (ONOO⁻), a leishmanicidal oxidant that is generated during infection through the combined enzymatic activities of NADPH oxidase and iNOS. The enhanced antinitrosative defense of amastigotes correlates with the increased expression of a tryparedoxin peroxidase (TXNPx) isoform that is also upregulated in response to iNOS enzymatic activity within infected macrophages. Accordingly, ectopic over-expression of the TXNPx isoform by L. amazonensis promastigotes significantly enhances parasite resistance against ONOO⁻ cytotoxicity. Moreover, TXNPx-overexpressing parasites exhibit greater intra-macrophage survival, and increased parasite growth and lesion development in a murine model of leishmaniasis. Our investigations indicate that TXNPx isoforms contribute to Leishmania''s ability to adapt to and antagonize the hostile microenvironment of cytokine-activated macrophages, and provide a mechanistic explanation for persistent infection in experimental and human leishmaniasis.     

Immucillins Impair Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis Multiplication In Vitro

Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05–500 μM and, when needed, 0.01–50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.     

Antileishmanial activity and trypanothione reductase effects of terpenes from the Amazonian species Croton cajucara Benth (Euphorbiaceae)

BackgroundLeishmaniasis comprises several infectious diseases caused by protozoa parasites of Leishmania genus. In recent years, there has been a growing interest in the therapeutic use of natural products to treat parasitic diseases. Among them Croton cajucara Benth. (Euphorbiaceae) is a plant found in the Amazonian region with a history of safe use in folk medicine.PurposeThe purpose of this study was to investigate the effects of clerodane diterpenes, trans-dehydrocrotonin (DCTN), trans-crotonin (CTN) and acetylaleuritolic acid (AAA) obtained from powdered bark of C. cajucara against promastigotes, axenic and intracellular amastigotes of Leishmania amazonensis. Furthermore, the effects of DCTN and CTN on the trypanotiona reductase enzyme were also investigated. The extraction of the terpenes was carried out as previously reported (Maciel et al., 1998, Maciel et al., 2003).MethodsThe effect of the isolated compounds (DCTN, CTN and AAA) from the bark of C. cajucara was assessed in vitro against promastigotes, axenic amastigotes and intracellular amastigotes of L. amazonensis by counting of remaining parasites in a Neubauer chamber in comparison to pentamidine used as standard drug. The action of natural products on trypanothione reductase was assessed using soluble protein fraction of promastigotes. The assays were performed by incubation with HEPES, EDTA, NADPH and trypanothione disulfide to quantify the NAPH consumption by TryR.ResultsThe results showed very high efficacy, especially of the diterpene DCTN, against promastigotes (IC₅₀ = 6.30 ± 0.06 µg/ml) and axenic amastigotes (IC₅₀ = 19.98 ± 0.05 µg/ml) of L. amazonenesis. The cytotoxic effect of the best active natural product was evaluated on mouse peritoneal infected macrophages (IC₅₀ = 0.47 ± 0.03 µg/ml in 24 h of culture), and the treatment revealed that DCTN never reaches toxic concentrations while reducing the infection and, most importantly, with no toxicity (>100 µg/ml with 0% of macrophage kill) when compared to pentamidine (37.5 µg/ml with 100% of macrophage kill). Furthermore, all of the natural products assayed on the trypanothione reductase enzyme inhibited the enzyme activity compared to the control.ConclusionClerodane diterpenes from C. cajucara showed promising in vitro antileishmanial effects against L. amazonensis, specially the DCTN with no macrophage toxicity up to the assayed concentration. In addition, the action on trypanothione reductase enzyme revealed a possible mechanism of action.     

Constitutive nitric oxide synthase-like enzyme in two species involved in cutaneous and mucocutaneous leishmaniasis

Leishmania is an obligate intracellular parasite that primarily inhabits macrophages. The destruction of the parasite in the host cell is a fundamental mechanism for infection control. In addition, inhibition of the leishmanicidal activity of macrophages seems to be related to the ability of some species to inhibit the production of nitric oxide (NO) by depleting arginine. Some species of Leishmania have the ability to produce NO from a constitutive nitric oxide synthase-like enzyme (cNOS-like). However, the localization of cNOS-like in Leishmania has not been described before. As such, this study was designed to locate cNOS-like enzyme and NO production in promastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. NO production was initially quantified by flow cytometry, which indicated a significant difference in NO production between L. (L.) amazonensis (GMFC = 92.17 +/− 4.6) and L. (V.) braziliensis (GMFC = 18.89 +/− 2.29) (P < 0.05). Analysis of cNOS expression by immunoblotting showed more expression in L. (L.) amazonensis versus L. (V.) braziliensis. Subsequently, cNOS-like immunolabeling was observed in promastigotes in regions near vesicles, the flagellar pocket and mitochondria, and small clusters of particles appeared to be fusing with vesicles suggestive of glycosomes, peroxisome-like-organelles that compartmentalize the glycolytic pathway in trypanosomatid parasites. In addition, confocal microscopy analysis demonstrated colocalization of cNOS-like and GAPDH, a specific marker for glycosomes. Thus, L. (L.) amazonensis produces greater amounts of NO than L. (V.) braziliensis, and both species present the cNOS-like enzyme inside glycosomes.     

Benzaldehyde Thiosemicarbazone Derived from Limonene Complexed with Copper Induced Mitochondrial Dysfunction in Leishmania amazonensis

Background

Leishmaniasis is a major health problem that affects more than 12 million people. Treatment presents several problems, including high toxicity and many adverse effects, leading to the discontinuation of treatment and emergence of resistant strains.

Methodology/Principal Findings

We evaluated the in vitro antileishmanial activity of benzaldehyde thiosemicarbazone derived from limonene complexed with copper, termed BenzCo, against Leishmania amazonensis. BenzCo inhibited the growth of the promastigote and axenic amastigote forms, with IC₅₀ concentrations of 3.8 and 9.5 µM, respectively, with 72 h of incubation. Intracellular amastigotes were inhibited by the compound, with an IC₅₀ of 10.7 µM. BenzCo altered the shape, size, and ultrastructure of the parasites. Mitochondrial membrane depolarization was observed in protozoa treated with BenzCo but caused no alterations in the plasma membrane. Additionally, BenzCo induced lipoperoxidation and the production of mitochondrial superoxide anion radicals in promastigotes and axenic amastigotes of Leishmania amazonensis.

Conclusion/Significance

Our studies indicated that the antileishmania activity of BenzCo might be associated with mitochondrial dysfunction and oxidative damage, leading to parasite death.     

Cyclosporin A Treatment of Leishmania donovani Reveals Stage-Specific Functions of Cyclophilins in Parasite Proliferation and Viability

Background

Cyclosporin A (CsA) has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs) as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development.

Methodology/Principal Findings

Multiple sequence alignment and cluster analysis identified 17 Leishmania CyPs with significant sequence differences to human CyPs, but with highly conserved functional residues implicated in PPIase function and CsA binding. CsA treatment of promastigotes resulted in a dose-dependent inhibition of cell growth with an IC50 between 15 and 20 µM as demonstrated by proliferation assay and cell cycle analysis. Scanning electron microscopy revealed striking morphological changes in CsA treated promastigotes reminiscent to developing amastigotes, suggesting a role for parasite CyPs in Leishmania differentiation. In contrast to promastigotes, CsA was highly toxic to amastigotes with an IC50 between 5 and 10 µM, revealing for the first time a direct lethal effect of CsA on the pathogenic mammalian stage linked to parasite thermotolerance, independent from host CyPs. Structural modeling, enrichment of CsA-binding proteins from parasite extracts by FPLC, and PPIase activity assays revealed direct interaction of the inhibitor with LmaCyP40, a bifunctional cyclophilin with potential co-chaperone function.

Conclusions/Significance

The evolutionary expansion of the Leishmania CyP protein family and the toxicity of CsA on host-free amastigotes suggest important roles of PPIases in parasite biology and implicate Leishmania CyPs in key processes relevant for parasite proliferation and viability. The requirement of Leishmania CyP functions for intracellular parasite survival and their substantial divergence form host CyPs defines these proteins as prime drug targets.     

Interplay of Trypanosome Lytic Factor and innate immune cells in the resolution of cutaneous Leishmania infection

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1^- and lpg5A^-/lpg5B^- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania.     

Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes

Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.     

In vitro effects of triterpenic acids from olive leaf extracts on the mitochondrial membrane potential of promastigote stage of Leishmania spp

Protozoan diseases, such as leishmaniasis, are a cause of considerable morbidity throughout the world, affecting millions every year. In this study, two triterpenic acids (maslinic and oleanolic acids) were isolated from Tunisian olive leaf extracts and their in vitro activity against the promastigotes stage of Leishmania (L.) infantum and Leishmania (L.) amazonensis was investigated. Maslinic acid showed the highest activity with an IC₅₀ of 9.32 ± 1.654 and 12.460 ± 1.25 μg/ml against L. infantum and L. amazonensis, respectively. The mechanism of action of these drugs was investigated by detecting changes in the phosphatidylserine (PS) exposure, the plasma membrane permeability, the mitochondrial membrane potential and the ATP level production in the treated parasites. By using the fluorescent probe SYTOX® Green, both triterpenic acids showed that they produce a time-dependent plasma membrane permeabilization in the treated Leishmania species. In addition, spectrofluorimeteric data revealed the surface exposure of PS in promastigotes. Both molecules reduced the mitochondrial membrane potential and decreased the ATP levels to 15% in parasites treated with IC₉₀ for 24 h. We conclude that the triterpenic acids tested in this study, show potential as future therapeutic alternative against leishmaniasis. Further studies are needed to confirm this.     

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

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

Characterization of a Leishmania stage‐specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulence

Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27 kDa protein‐encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co‐immunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene‐deleted parasites (Ldp27^?/?) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27^?/? parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis.     

Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis,but Not by Leishmania (Viannia) guyanensis

Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.     

Nuphar lutea: In vitro anti-leishmanial activity against Leishmania major promastigotes and amastigotes

Several anti-leishmanial drugs of choice are of plant origin. Many of the available drugs against the disease are toxic and in certain cases parasite drug resistance is developed. The development of new compounds is urgently required.Aims of the studyTo determine the leishmanicidal activity of the Nuphar lutea plant extract against Leishmania major in vitro.Materials and methodsThe leishmanicidal activity of methanolic plant extract against L. major free living promastigotes and intracellular amastigotes was evaluated, using microscopic examinations and the enzymatic XTT assay.ResultsMethanolic extract of N. lutea was highly effective against both Leishmania promastigotes and L. amastigotes (IC₅₀=2±0.12 μg/ml; ID₅₀=0.65±0.023 μg/ml; LD₅₀=2.1±0.096 μg/ml, STI=3.23). The extract at 1.25 μg/ml totally eliminated the intracellular parasites within 3 days of treatment. Also, a synergistic anti-leishmanial activity was demonstrated with N. lutea extract combined with the anti-leishmanial drug – paromomycin. The partially purified N. lutea active component was found to be a thermo-stable alkaloid(s) with no electrical charge and is resistant to boiling and to methanol, dichloromethane and xylene treatment.ConclusionsThe present study suggests that N. lutea might be a potential source of anti-leishmanial compounds.     

In Vitro and In Vivo Miltefosine Susceptibility of a Leishmania amazonensis Isolate from a Patient with Diffuse Cutaneous Leishmaniasis

Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo. This study contributed to establish that the miltefosine transporter is essential for drug activity in L. amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.     

Kharon1 Null Mutants of Leishmania mexicana Are Avirulent in Mice and Exhibit a Cytokinesis Defect within Macrophages

In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δ kharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δ kharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δ kharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δ kharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.     

Leishmania mexicana amazonensis: Surface charge of amastigote and promastigote forms

The surface charge of Leishmania mexicana amazonensis was evaluated by means of the binding of colloidal iron hydroxyde particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, visualizated by electron microscopy and by direct measurements of the electrophoretic mobility of cells suspended in solutions of different pH. The following forms of the parasite were analysed: amastigotes (surrounded or not by the membrane of the endocytic vacuole, isolated from lesions), transitional forms, and infective (5 passages) and noninfective (176 passages) promastigotes. The results obtained indicate that the surface of L. m. amazonensis contains both negatively and positively charged dissociating groups and that changes occur in the surface charge during amastigote-promastigote transformation. Treatment of the parasite with neuraminidase significantly reduced the electrophoretic mobility of the cells. Neuraminidase-treated cells recovered their normal electrophoretic mobility when incubated for 8 hr in fresh culture medium by a process that is inhibited by puromycin.