Joseane Lima Prado Godinho

Biology of Human Pathogenic Trypanosomatids: Epidemiology, Lifecycle and Ultrastructure

Leishmania and Trypanosoma belong to the Trypanosomatidae family and cause important human infections such as leishmaniasis, Chagas disease, and sleeping sickness. Leishmaniasis, caused by protozoa belonging to Leishmania, affects about 12 million people worldwide and can present different clinical manifestations, i.e., visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), and post-kala-azar dermal leishmaniasis (PKDL). Chagas disease, also known as American trypanosomiasis, is caused by Trypanosoma cruzi and is mainly prevalent in Latin America but is increasingly occurring in the United States, Canada, and Europe. Sleeping sickness or human African trypanosomiasis (HAT), caused by two sub-species of Trypanosoma brucei (i.e., T. b. rhodesiense and T. b. gambiense), occurs only in sub-Saharan Africa countries. These pathogenic trypanosomatids alternate between invertebrate and vertebrate hosts throughout their lifecycles, and different developmental stages can live inside the host cells and circulate in the bloodstream or in the insect gut. Trypanosomatids have a classical eukaryotic ultrastructural organization with some of the same main organelles found in mammalian host cells, while also containing special structures and organelles that are absent in other eukaryotic organisms. For example, the mitochondrion is ramified and contains a region known as the kinetoplast, which houses the mitochondrial DNA. Also, the glycosomes are specialized peroxisomes containing glycolytic pathway enzymes. Moreover, a layer of subpellicular microtubules confers mechanic rigidity to the cell. Some of these structures have been investigated to determine their function and identify potential enzymes and metabolic pathways that may constitute targets for new chemotherapeutic drugs.

Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

Background The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. Methodology/Principal Findings Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10–2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. Conclusions This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

Glycosome turnover in Leishmania major is mediated by autophagy

Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasites autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ∼20 glycosomes per cell, whereas amastigotes contain ∼10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ∼15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes.

A novel alkyl phosphocholine-dinitroaniline hybrid molecule exhibits biological activity in vitro against Leishmania amazonensis

Parasitic protozoa of the Leishmania genus cause leishmaniasis, an important complex of tropical diseases that affect about 12 million people around the world. The drugs used to treat leishmaniasis are pentavalent antimonials, miltefosine, amphotericin B and pentamidine. In the present study, we evaluated the effect of a novel alkyl phosphocholine-dinitroaniline hybrid molecule, TC95, against Leishmania amazonensis promastigotes and intracellular amastigotes. Antiproliferative assays indicated that TC95 is a potent inhibitor of promastigotes and intracellular amastigotes with IC50 values of 2.6 and 1.2 μM, respectively. Fluorescence microscopy with anti-α-tubulin antibody revealed changes in the cytoskeleton, whilst scanning electron microscopy showed alterations in the shape, plasma membrane, length of the flagellum, and cell cycle. Flow cytometry confirmed the cell cycle arrest mainly in G1 phase, however a significant population appeared in sub G0/G1 and super-G2. The alterations in the plasma membrane integrity were confirmed by fluorometric analysis using Sytox Blue. Transmission electron microscopy also revealed an accumulation of lipid bodies, confirmed by fluorescence microscopy and fluorometric analysis using Nile Red. Important lesions were also observed in organelles such as mitochondrion, endoplasmic reticulum and Golgi complex. In summary, our study suggests that TC95, an alkyl phosphocholine-trifluralin hybrid molecule, is a promising novel compound against L. amazonensis.

Efficacy of miltefosine treatment in Leishmania amazonensis-infected BALB/c mice

Leishmaniasis is one of the most serious worldwide diseases caused by protozoan parasites of the Leishmania genus, affecting millions of people around the world. All currently available treatments present severe toxic side effects, require long-term compliance, cause serious side effects and are uncomfortable for patients. Leishmania amazonensis, a species endemic to Brazil, causes severe localised or diffuse skin lesions in humans. Owing to the unsatisfactory nature of the currently available chemotherapies, new approaches have been assessed for improved therapeutic intervention strategies against leishmaniasis. Miltefosine is an alkylphospholipid analogue that exhibits potent activity against the different clinical manifestations of leishmaniasis. Thus, the aim of this study was to investigate the long-term efficacy of miltefosine in BALB/c mice infected with L. amazonensis owing to the lack of a profound study demonstrating its dose-dependent and long-term effects. It was observed that animals treated with 20-50 mg/kg/day of miltefosine exhibited a significant dose-dependent reduction in lesion size; furthermore, in mice receiving higher doses, lesions disappeared after the end of treatment. To confirm a possible parasitological cure, mice up to 250 days after the end of treatment were analysed. No lesions or presence of parasite DNA were found in mice treated with 30, 40 and 50 mg/kg/day of miltefosine. In summary, these results show that miltefosine may be used to treat cutaneous leishmaniasis caused by L. amazonensis, alone or as combination therapy.

Trypanosoma cruzi Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions.

Trypanosoma cruzi epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes.

Voacamine alters Leishmania ultrastructure and kills parasite by poisoning unusual bi-subunit topoisomerase IB

ABSTRACT Indole alkaloids possess a large spectrum of biological activities including anti‐protozoal action. Here we report for the first time that voacamine, isolated from the plant Tabernaemontana coronaria, is an antiprotozoal agent effective against a large array of trypanosomatid parasites including Indian strain of Leishmania donovani and Brazilian strains of Leishmania amazonensis and Trypanosoma cruzi. It inhibits the relaxation activity of topoisomerase IB of L. donovani (LdTop1B) and stabilizes the cleavable complex. Voacamine is probably the first LdTop1B‐specific poison to act uncompetitively. It has no impact on human topoisomerase I and II up to 200 &mgr;M concentrations. The study also provides a thorough insight into ultrastructural alterations induced in three kinetoplastid parasites by a specific inhibitor of LdTop1B. Voacamine is also effective against intracellular amastigotes of different drug unresponsive field isolates of Leishmania donovani obtained from endemic zones of India severely affected with visceral leishmaniasis. Most importantly, this is the first report demonstrating the efficacy of a compound to reduce the burden of drug resistant parasites, unresponsive to SAG, amphotericin B and miltefosine, in experimental BALB/c mice model of visceral leishmaniasis. The findings cumulatively provide a strong evidence that voacamine can be a promising drug candidate against trypanosomatid infections.

Isobenzofuranone derivative JVPH3, an inhibitor of L. donovani topoisomerase II, disrupts mitochondrial architecture in trypanosomatid parasites

Kinetoplast DNA (kDNA) bearing unusual mitochondrion of trypanosomatid parasites offers a new paradigm in chemotherapy modality. Topoisomerase II of Leishmania donovani (LdTopII), a key enzyme associated with kDNA replication, is emerging as a potential drug target. However, mode of action of LdTopII targeted compounds in the parasites at sub-cellular level remains largely unknown. Previously, we reported that an isobenzofuranone derivative, namely 3,5-bis(4-chlorophenyl)-7-hydroxyisobenzofuran-1(3H)-one (JVPH3), targets LdTopII and induces apoptosis-like cell death in L. donovani. Here, we elucidate the phenotypic changes and the events occurring at sub-cellular level caused by JVPH3 in L. donovani. In addition, we have evaluated the cytotoxicity and ultrastructural alterations caused by JVPH3 in two brazilian trypanosomatid pathogens viz. L. amazonensis and Trypanosoma cruzi. Despite killing these parasites, JVPH3 caused significantly different phenotypes in L. donovani and L. amazonensis. More than 90% population of parasites showed altered morphology. Mitochondrion was a major target organelle subsequently causing kinetoplast network disorganization in Leishmania. Altered mitochondrial architecture was evident in 75–80% Leishmania population being investigated. Quantification of mitochondrial function using JC-1 fluorophore to measure a possible mitochondrial membrane depolarization further confirmed the mitochondrion as an essential target of the JVPH3 corroborating with the phenotype observed by electron microscopy. However, the impact of JVPH3 was lesser on T. cruzi than Leishmania. The molecule caused mitochondrial alteration in 40% population of the epimastigotes being investigated. To our knowledge, this is the first report to evaluate the proliferation pattern and ultrastructural alterations caused in Brazilian kinetoplastid pathogens by a synthetic LdTopII inhibitor previously established to have promising in vivo activity against Indian strain of L. donovani.

In vitro antileishmanial activity of ravuconazole, a triazole antifungal drug, as a potential treatment for leishmaniasis

Objectives Leishmaniasis, one of the most significant neglected diseases around the world, is caused by protozoan parasites of the Leishmania genus. Nowadays, the available aetiological treatments for leishmaniasis have variable effectiveness and several problems such as serious side effects, toxicity, high cost and an increasing number of resistance cases. Thus, there is an urgent need for safe, oral and cost-effective drugs for leishmaniases. Previously, our group has shown the effect of the ergosterol biosynthesis inhibitors on Leishmania amazonensis. Herein, we showed the effect of ravuconazole against L. amazonensis; ravuconazole is a second-generation triazole antifungal drug that has good bioavailability after oral administration and a long terminal half-life in humans, a broad activity spectrum, high effectiveness in treatment of mycosis and negligible side effects. Methods Several methodologies were used: cell culture, fluorescence and electron microscopy, high-resolution capillary GC coupled with MS, fluorimetry and flow cytometry. Results Our results showed that ravuconazole was able to inhibit the proliferation of L. amazonensis promastigotes and intracellular amastigotes in vitro, with single-digit to sub-micromolar IC50 values, causing several alterations in the morphology, ultrastructure, cell viability and physiology of the parasites. The mitochondrion was significantly affected by the treatment, resulting in a collapse of the mitochondrial transmembrane potential that consequently led to inhibition of ATP production, combined with an increase in reactive oxygen species and mitochondrial superoxide production; by transmission electron microscopy, the organelle displayed a completely altered ultrastructure. The treatment changed the lipid profile, showing a profound depletion of the 14-desmethyl endogenous sterol pool. Conclusions These results suggest that ravuconazole could be an alternative option for the treatment of leishmaniasis.