Dual Host-Intracellular Parasite Transcriptome of Enucleated Cells Hosting Leishmania amazonensis: Control of Half-Life of Host Cell Transcripts by the Parasite.

Enucleated cells or cytoplasts (cells whose nucleus is removed in vitro) represent an unexplored biological model for intracellular infection studies due to the abrupt interruption of nuclear processing and new RNA synthesis by the host cell in response to pathogen entry. Using enucleated fibroblasts hosting the protozoan parasite Leishmania amazonensis, we demonstrate that parasite multiplication and biogenesis of large parasitophorous vacuoles in which parasites multiply are independent of the host cell nucleus. Dual RNA sequencing of both host cytoplast and intracellular parasite transcripts identified host transcripts that are more preserved or degraded upon interaction with parasites and also parasite genes that are differentially expressed when hosted by nucleated or enucleated cells. Cytoplasts are suitable host cells, which persist in culture for more than 72 h and display functional enrichment of transcripts related to mitochondrial functions and mRNA translation. Crosstalk between nucleated host de novo gene expression in response to intracellular parasitism and the parasite gene expression to counteract or benefit from these host responses induces a parasite transcriptional profile favoring parasite multiplication and aerobic respiration, and a host-parasite transcriptional landscape enriched in host cell metabolic functions related to NAD, fatty acid, and glycolytic metabolism. Conversely, interruption of host nucleus-parasite cross talk by infection of enucleated cells generates a host-parasite transcriptional landscape in which cytoplast transcripts are enriched in phagolysosome-related pathway, prosurvival, and SerpinB-mediated immunomodulation. In addition, predictive in silico analyses indicated that parasite transcript products secreted within cytoplasts interact with host transcript products conserving the host V-ATPase proton translocation function and glutamine/proline metabolism. The collective evidence indicates parasite-mediated control of host cell transcripts half-life that is beneficial to parasite intracellular multiplication and escape from host immune responses. These findings will contribute to improved drug targeting and serve as database for L. amazonensis-host cell interactions.

Protective Cellular Immune Response Induction for Cutaneous Leishmaniasis by a New Immunochemotherapy Schedule.

The palladacycle complex DPPE 1.2 was previously shown to inhibit Leishmania (Leishmania) amazonensis infection in vitro and in vivo. The present study aimed to evaluate the effect of DPPE 1.2 associated with a recombinant cysteine proteinase, rLdccys1, and the adjuvant Propionibacterium acnes on L. (L.) amazonensis infection in two mouse strains, BALB/c, and C57BL/6. Treatment with this association potentiated the leishmanicidal effect of DPPE 1.2 resulting in a reduction of parasite load in both strains of mice which was higher compared to that found in groups treated with either DPPE 1.2 alone or associated with P. acnes or rLdccys1. The reduction of parasite load in both mice strains was followed by immunomodulation mediated by an increase of memory CD4+ and CD8+ T lymphocytes, IFN-γ levels and reduction of active TGF-ß in treated animals. No infection relapse was observed 1 month after the end of treatment in mice which received DPPE 1.2 associated with rLdccys1 or rLdccys1 plus P. acnes in comparison to that exhibited by animals treated with DPPE 1.2 alone. Evaluation of serum levels of AST, ALT, urea, and creatinine showed no alterations among treated groups, indicating that this treatment schedule did not induce hepato or nephrotoxicity. These data indicate the potential use of this association as a therapeutic alternative for cutaneous leishmaniasis caused by L. (L) amazonensis.

Leishmanicidal and Immunomodulatory Activities of the Palladacycle Complex DPPE 1.1, a Potential Candidate for Treatment of Cutaneous Leishmaniasis.

The present study focused on the activity of the palladacycle complex DPPE 1.1 on Leishmania (Leishmania) amazonensis. Promastigotes of L. (L.) amazonensis were destroyed in vitro by nanomolar concentrations of DPPE 1.1, whereas intracellular amastigotes were killed at drug concentrations fivefold less toxic than those harmful to macrophages. L. (L.) amazonensis-infected BALB/c mice were treated by intralesional injection of DPPE 1.1. Animals treated with 3.5 and 7.0 mg/kg of DPPE 1.1 showed a significant decrease of foot lesion sizes and a parasite load reduction of 93 and 99%, respectively, when compared to untreated controls. Furthermore, DPPE 1.1 was non-toxic to treated animals. The cathepsin B activity of L. (L.) amazonensis amastigotes was inhibited by DPPE 1.1 as demonstrated spectrofluorometrically by use of a specific fluorogenic substrate. Analysis of T-cells populations in mice treated with DPPE 1.1 and untreated controls was performed by fluorescence-activated cell sorter (FACS). IFN-γ was measured in supernatants of lymphocytes from popliteal and inguinal lymph nodes isolated from treated and untreated mice and stimulated with L. (L.) amazonensis amastigotes extract and active TGF-ß was evaluated in supernatants of foot lesions; both dosages were carried out by means of a double-sandwich ELISA assay. A significant increase of TCD4+ and TCD8+ lymphocytes and IFN-γ secretion was displayed in mice treated with DPPE 1.1 compared to untreated animals, whereas a significant reduction of active TGF-ß was observed in treated mice. These findings open perspectives for further investment in DPPE 1.1 as an alternative option for the chemotherapy of cutaneous leishmaniasis.

Treatment of Leishmania (Leishmania) Amazonensis-Infected Mice with a Combination of a Palladacycle Complex and Heat-Killed Propionibacterium acnes Triggers Protective Cellular Immune Responses.

Palladacycle complex DPPE 1.2 was previously reported to inhibit the in vitro and in vivo infection by Leishmania (Leishmania) amazonensis. The aim of the present study was to compare the effect of DPPE 1.2, in association with heat-killed Propionibacterium acnes, on L. (L.) amazonensis infection in two mouse strains, BALB/c and C57BL/6, and to evaluate the immune responses of the treated animals. Foot lesions of L. (L.) amazonensis-infected mice were injected with DPPE 1.2 alone, or associated with P. acnes as an adjuvant. Analysis of T-cell populations in the treated mice and in untreated controls was performed by FACS. Detection of IFN-γ-secreting lymphocytes was carried out by an ELISPOT assay and active TGF-ß was measured by means of a double-sandwich ELISA test. The treatment with DPPE 1.2 resulted in a significant reduction of foot lesion sizes and parasite burdens in both mouse strains, and the lowest parasite burden was found in mice treated with DPPE 1.2 plus P. acnes. Mice treated with DPPE 1.2 alone displayed a significant increase of TCD4+ and TCD8+ lymphocytes and IFN-γ secretion which were significantly higher in animals treated with DPPE 1.2 plus P. acnes. A significant reduction of active TGF-ß was observed in mice treated with DPPE 1.2 alone or associated with P. acnes. Moreover, DPPE 1.2 associated to P. acnes was non-toxic to treated animals. The destruction of L. (L.) amazonensis by DPPE 1.2 was followed by host inflammatory responses which were exacerbated when the palladacycle complex was associated with P. acnes.

Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates.

In this study we investigated the peptidase activity in Leishmania (L.) amazonensis live amastigote by confocal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fluorophore inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and serine peptidases. It was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA, Z-RR-MCA) or inhibitors (E64, TLCK) containing positively charged groups. The peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA substrates and inhibitors in the pH range 4.5-9.0. The effects of temperature and different salts were also included in this study. The hydrolytic activities of supernatants on Z-FR-MCA clearly indicate the presence of different cysteine peptidases that adapted to work in different environment conditions. Intact Leishmania cells incorporated Z-RR-MCA, the hydrolysis of which was inhibited only by TLCK indicating the presence of at least one serine peptidase. The pH profile of Z-RR-MCA hydrolysis by amastigotes and promastigotes lysate supernatants, and the hydrolysis time course of the FRET peptide Abz-AGRRRAQ-EDDnp at RA bond, followed by removal of the two C-termini R to yield Abz-AGR-OH that is a unique characteristic of oligopeptidase B, indicate its presence in the parasite.

Natural products from Garcinia brasiliensis as Leishmania protease inhibitors.

The infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which cause renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro Leishmania protease inhibition activity of extracts (hexanic, ethyl-acetate, and ethanolic) and fukugetin, a bioflavonoid purified from the ethyl-acetate extract of the pericarp of the fruit of Garcinia brasiliensis, a tree native to Brazilian forests. The isolated compound was characterized by using spectral analyses with nuclear magnetic resonance, mass spectroscopy, ultraviolet, and infrared techniques. The ethyl-acetate extract and the compound fukugetin showed significant activity as inhibitors of Leishmania's proteases, with mean (±SD) IC(50) (50% inhibition concentration of protease activity) values of 15.0±1.3 µg/mL and 3.2±0.5 µM/mL, respectively, characterizing a bioguided assay. In addition, this isolated compound showed no activity against promastigote and amastigote forms of L. (L.) amazonensis and mammalian cells. These results suggest that fukugetin is a potent protease inhibitor of L. (L.) amazonensis and does not cause toxicity in mammalian or Leishmania cells in vitro. This study provides new perspectives on the development of novel drugs that have leishmanicidal activity obtained from natural products and that target the parasite's proteases.

Tamoxifen as a potential antileishmanial agent: efficacy in the treatment of Leishmania braziliensis and Leishmania chagasi infections.

OBJECTIVES: The aim of this study was to evaluate the efficacy of tamoxifen in vivo in experimental models of cutaneous (CL) and visceral leishmaniasis (VL) caused by Leishmania braziliensis and Leishmania chagasi, respectively. METHODS: Drug activity was assessed against intracellular amastigotes by treating infected macrophage cultures and evaluating the number of infected cells. In vivo efficacy of tamoxifen was tested in L. braziliensis-infected BALB/c mice and in L. chagasi-infected hamsters. Treatment with 20 mg/kg/day tamoxifen was administered for 15 days by the intraperitoneal route. Efficacy was evaluated through measurements of lesion size, parasite burden at the lesion site or liver and spleen and survival rate. RESULTS: Tamoxifen killed L. braziliensis and L. chagasi intracellular amastigotes with 50% inhibitory concentrations (IC(50)) of 1.9 +/- 0.2 and 2.4 +/- 0.3 microM, respectively. Treatment of L. braziliensis-infected mice with tamoxifen resulted in significant reductions in lesion size and 99% decrease in parasite burden, compared with mock-treated controls. L. chagasi-infected hamsters treated with tamoxifen showed significant reductions in liver parasite load expressed as Leishman-Donovan units and 95% to 98% reduction in spleen parasite burden. All animals treated with tamoxifen survived while 100% of the mock-treated animals had died by 11 weeks after the interruption of treatment. CONCLUSIONS: Tamoxifen is effective in the treatment of CL and VL in rodent models.

Ultrastructural and cytochemical identification of megasome in Leishmania (Leishmania) chagasi.

The present work showed the presence of a megasome in Leishmania (Leishmania) chagasi amastigotes. Transmission electron microscopy analysis of ultrathin serial sections and three-dimensional reconstruction allowed visualization of large structures in amastigote forms of L. (L.) chagasi and a multivesicular tubule-lysosome structure in metacyclic promastigotes. Morphometric data showed that the relative volume occupied by the megasome and the multivesicular tubule (MVT)-lysosome structures was about 5% and 3.2%, respectively, in amastigotes and promastigotes of L. (L.) chagasi. Further characterization of the megasome in L. (L.) chagasi amastigotes was carried out by immunolabeling of cysteine proteinase, whereas the lysosomal content of amastigotes and promastigotes was confirmed by arylsulfatase cytochemistry.