Leishmania major phosphoglycerate kinase transcript and protein stability contributes to differences in isoform expression levels.

Leishmania contains two phosphoglycerate kinase (PGK) genes, PGKB and PGKC, which code for the cytosolic and glycosomal isoforms of the enzyme, respectively. Although differences in PGKB and PGKC transcript and protein levels and isoform activities have been well documented, the mechanisms of control of both transcript and protein abundance have not been described to date. To better understand the regulation of Leishmania PGK expression, we investigated the stabilities of both PGK transcripts using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in combination with transcription and trans-splicing inhibitors. Cells were treated with sinefungin and actinomycin D, and RNA decay kinetics were assessed. In addition, immunoblotting and protein synthesis inhibition by cycloheximide were employed to evaluate protein steady states and degradation. We observed increased stabilities of both PGKB mRNA and protein compared with the glycosomal isoform (PGKC). Our results indicate that both post-transcriptional and post-translational events contribute to the distinct expression levels of the PGKB and PGKC isoforms in Leishmania major.

Mycoleptones A-C and polyketides from the endophyte Mycoleptodiscus indicus.

Three new azaphilones with an unusual methylene bridge, named mycoleptones A, B, and C (2, 4, and 5), were isolated from cultures of Mycoleptodiscus indicus, a fungus associated with the South American medicinal plant Borreria verticillata. Additionally, four known polyketides, austdiol (1), eugenitin (3), 6-methoxyeugenin (6), and 9-hydroxyeugenin (7), were also isolated. The structural characterization of compounds was carried out by nuclear magnetic resonance spectroscopy, high-resolution mass spectrometry, electronic circular dichroism spectroscopy, time-dependent density functional theory calculations, and X-ray crystallography. Compounds 1-9 were weakly active when tested in antileishmanial and cytotoxicity assays.

In vitro leishmanicidal activities of sesquiterpene lactones from Tithonia diversifolia against Leishmania braziliensis promastigotes and amastigotes.

Natural compounds represent a rich and promising source of novel, biologically active chemical entities for treating leishmaniasis. Sesquiterpene lactones are a recognized class of terpenoids with a wide spectrum of biological activities, including activity against Leishmania spp. In this work, a sesquiterpene lactone-rich preparation-a leaf rinse extract (LRE) from Tithonia diversifolia-was tested against promastigote forms of L. braziliensis. The results revealed that the LRE is a rich source of potent leishmanicidal compounds, with an LD50 value 1.5 ± 0.50 µg·mL-1. Therefore, eight sesquiterpene lactones from the LRE were initially investigated against promastigote forms of L. braziliensis. One of them did not present any significant leishmanicidal effect (LD50 > 50 µg·mL-1). Another had a cytotoxic effect against macrophages (4.5 µg·mL-1). The five leishmanicidal compounds with the highest level of selectivity were further evaluated against intracellular parasites (amastigotes) using peritoneal macrophages. Tirotundin 3-O-methyl ether, tagitinin F, and a guaianolide reduced the internalization of parasites after 48 h, in comparison with the negative control. This is the first report on sesquiterpene lactones that have potent leishmanicidal effects on both developmental stages of L. braziliensis.

Metabolomics as a tool to evaluate the toxicity of formulations containing amphotericin B, an antileishmanial drug.

Amphotericin B (AmB) is a drug of choice against life-threatening systemic fungal infections and an alternative therapy for the treatment of all forms of leishmaniasis. It is known that AmB and its conventional formulation cause renal damage; however, the lipid formulations can reduce these effects. The aim of the present study was to identify metabolic changes in mice treated with two different AmB formulations, a nanoemulsion (NE) (lipid system carrier) loaded with AmB and the conventional formulation (C-AmB). For this purpose, metabolic fingerprinting represents a valuable strategy to monitor, in a non-targeted manner, the changes that are at the base of the toxicity mechanism of AmB. Plasma samples of BALB-c mice were collected after treatment with 3 alternate doses of AmB at 1 mg kg-1 administered intravenously and analysed with CE, LC and GC coupled to MS. Blood urea nitrogen (BUN) and plasma creatinine levels were also analysed. Kidney tissue specimens were collected and evaluated. It was not observed that there were any alterations in BUN and creatinine levels as well as in histopathological analysis. Approximately 30 metabolites were identified as potentially related to early C-AmB-induced nephrotoxicity. Disturbances in the arachidonic acid, glycerophospholipid, acylcarnitine and polyunsaturated fatty acid (PUFA) pathways were observed in C-AmB-treated mice. In the AmB-loaded NE group, it was observed that there were fewer metabolic changes, including changes in the plasma levels of cortisol and pyranose. The candidate biomarkers revealed in this study could be useful in the detection of the onset and severity of kidney injury induced by AmB formulations.

Differential Gene Expression and Infection Profiles of Cutaneous and Mucosal Leishmania braziliensis Isolates from the Same Patient.

BACKGROUND: Leishmaniasis is a complex disease in which clinical outcome depends on factors such as parasite species, host genetics and immunity and vector species. In Brazil, Leishmania (Viannia) braziliensis is a major etiological agent of cutaneous (CL) and mucosal leishmaniasis (MCL), a disfiguring form of the disease, which occurs in ~10% of L. braziliensis-infected patients. Thus, clinical isolates from patients with CL and MCL may be a relevant source of information to uncover parasite factors contributing to pathogenesis. In this study, we investigated two pairs of L. (V.) braziliensis isolates from mucosal (LbrM) and cutaneous (LbrC) sites of the same patient to identify factors distinguishing parasites that migrate from those that remain at the primary site of infection. METHODOLOGY/PRINCIPAL FINDINGS: We observed no major genomic divergences among the clinical isolates by molecular karyotype and genomic sequencing. RT-PCR revealed that the isolates lacked Leishmania RNA virus (LRV). However, the isolates exhibited distinct in vivo pathogenesis in BALB/c mice; the LbrC isolates were more virulent than the LbrM isolates. Metabolomic analysis revealed significantly increased levels of 14 metabolites in LbrC parasites and 31 metabolites in LbrM parasites that were mainly related to inflammation and chemotaxis. A proteome comparative analysis revealed the overexpression of LbrPGF2S (prostaglandin f2-alpha synthase) and HSP70 in both LbrC isolates. Overexpression of LbrPGF2S in LbrC and LbrM promastigotes led to an increase in infected macrophages and the number of amastigotes per cell at 24-48 h post-infection (p.i.). CONCLUSIONS/SIGNIFICANCE: Despite sharing high similarity at the genome structure and ploidy levels, the parasites exhibited divergent expressed genomes. The proteome and metabolome results indicated differential profiles between the cutaneous and mucosal isolates, primarily related to inflammation and chemotaxis. BALB/c infection revealed that the cutaneous isolates were more virulent than the mucosal parasites. Furthermore, our data suggest that the LbrPGF2S protein is a candidate to contribute to parasite virulence profiles in the mammalian host.

Synthesis, cytotoxicity and in vitro antileishmanial activity of naphthothiazoles.

The leishmaniasis is a spectral disease caused by the protozoan Leishmania spp., which threatens millions of people worldwide. Current treatments exhibit high toxicity, and there is no vaccine available. The need for new lead compounds with leishmanicidal activity is urgent. Considering that many lead leishmanicidal compounds contain a quinoidal scaffold and the thiazole heterocyclic ring is found in a number of antimicrobial drugs, we proposed a hybridization approach to generate a diverse set of semi-synthetic heterocycles with antileishmanial activity. We found that almost all synthesized compounds demonstrated potent activity against promastigotes of Leishmania (Viannia) braziliensis and reduced the survival index of Leishmania amastigotes in mammalian macrophages. Furthermore, the compounds were not cytotoxic to macrophages at fivefold higher concentrations than the EC50 for promastigotes. All molecules fulfilled Lipinski's Rule of Five, which predicts efficient orally absorption and permeation through biological membranes, the in silico pharmacokinetic profile confirmed these characteristics. The potent and selective activity of semi-synthetic naphthothiazoles against promastigotes and amastigotes reveals that the 2-amino-naphthothiazole ring may represent a scaffold for the design of compounds with leishmanicidal properties and encourage the development of drug formulation and new compounds for further studies in vivo.

Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

Bioactive extracts and chemical constituents of two endophytic strains of Fusarium oxysporum

Ethyl acetate extracts of cultures grown in liquid Czapek and on solid rice media of the fungal endophyte Fusarium oxysporum SS46 isolated from the medicinal plant Smallanthus sonchifolius (Poepp.) H. Rob., Asteraceae, exhibited considerable cytotoxic activity when tested in vitro against human cancer cells. Chromatographic separation yielded anhydrofusarubin (1) and beauvericin (2) that were identified based on their ¹H and 13C NMR data. Compounds 1 and 2 showed the strongest cytotoxic activity against different cancer cell lines. Compound 2 also showed promising activity against Leishmania braziliensis. Hexanic extract of F. oxysporum SS50 grown on solid rice media also afforded a mixture of compounds that displayed cytotoxic activity against different cancer cell lines. Chemical analysis of the mixture of compounds, investigated by gas chromatography-mass spectrometry (GC-MS), showed that there was a predominance of methyl esters of fatty acids and alkanes.