Calcium Uptake and Proton Transport by Acidocalcisomes of Toxoplasma gondii

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca2+/H+ countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles

Green SPIONs as a novel highly selective treatment for leishmaniasis: an in vitro study against Leishmania amazonensis intracellular amastigotes

The main goal of this work was to evaluate the therapeutic potential of green superparamagnetic iron oxide nanoparticles (SPIONs) produced with coconut water for treating cutaneous leishmaniasis caused by Leishmania amazonensis. Optical and electron microscopy techniques were used to evaluate the effects on cell proliferation, infectivity percentage, and ultrastructure. SPIONs were internalized by both parasite stages, randomly distributed in the cytosol and located mainly in membrane-bound compartments. The selectivity index for intracellular amastigotes was more than 240 times higher compared to current drugs used to treat the disease. The synthesized SPIONs showed promising activity against Leishmania and can be considered a strong candidate for a new therapeutic approach for treating leishmaniases

Ultrastructural and Biochemical Alterations Induced by 22,26-Azasterol, a Δ(24(25))-Sterol Methyltransferase Inhibitor, on Promastigote and Amastigote Forms of Leishmania amazonensis

We report on the antiproliferative effects and the ultrastructural and biochemical alterations induced in vitro by 22,26-azasterol, a sterol Δ(24(25))-methyltransferase (24-SMT) inhibitor, on Leishmania amazonensis. When promastigotes and amastigotes were exposed to 100 nM 22,26-azasterol, complete growth arrest and cell lysis ensued after 72 (promastigotes) or 120 (amastigotes) h. Exposure of parasites to this azasterol led to the complete depletion of parasite endogenous sterols (episterol and 5-dehydroepisterol) and their replacement by 24-desalkyl sterols (zymosterol, cholesta-5,7,24-trien-3β-ol, and cholesta-7,24-dien-3β-ol), while 14-methyl-zymosterol and 4,14-dimethyl-zymosterol accumulated as a result of simultaneous incubation of the parasites with 22,26-azasterol and ketoconazole, a known inhibitor of the parasite’s sterol C14-demethylase. These results confirmed that 24-SMT is the primary site of action of the azasterol. Profound changes were also observed in the phospholipid compositions of treated cells, in which a twofold reduction in the content of phosphatidylserine was observed; this was accompanied by a concomitant increase in the content of phosphatidylinositol. Transmission electron microscopy showed that 22,26-azasterol induced marked morphological changes, including mitochondrial swelling, invaginations of the inner mitochondrial membrane, and the appearance of large bodies containing concentric membranes. Other modifications included increases in the numbers of acidocalcisomes, megasomes, and lipid inclusions and the appearance of typical autophagic structures and cell body protrusions toward the flagellar pocket. We conclude that the dramatic alteration of the lipid composition of the parasite’s membranes induced by the drug underlies the ultrastructural alterations that lead to the loss of cell viability and that 24-SMT inhibitors could be useful as selective antileishmanial agents

The cell biology of Leishmania: how to teach using animations.

Submitted by Nuzia Santos ([email protected]) on 2018-11-19T11:35:24Z No. of bitstreams: 1 The Cell Biology of Leishmania.pdf: 827064 bytes, checksum: c0a36de15baadb16d056af055b475f46 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2018-11-19T11:39:57Z (GMT) No. of bitstreams: 1 The Cell Biology of Leishmania.pdf: 827064 bytes, checksum: c0a36de15baadb16d056af055b475f46 (MD5)Made available in DSpace on 2018-11-19T11:39:57Z (GMT). No. of bitstreams: 1 The Cell Biology of Leishmania.pdf: 827064 bytes, checksum: c0a36de15baadb16d056af055b475f46 (MD5) Previous issue date: 2013Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, Brasil/Fundação Cecierj. Rio de Janeiro, RJ, Brasil/Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, BrasilFundação Cecierj. Rio de Janeiro, RJ, Brasil/Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil/Universidade Santa Úrsula. Rio de Janeiro, RJ, BrasilInstituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil/Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil/Universidade Federal do Rio de Janeiro. Núcleo Multidisciplinar de Pesquisa. Rio de Janeiro, RJ, Brasil/Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem. Rio de Janeiro, RJ, BrasilFundação Cecierj. Rio de Janeiro, RJ, Brasil/Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilInstituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil,/Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil/Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem. Rio de Janeiro, RJ, BrasilParasitic protozoa are important agents of human and animal diseases in Brazil and around the world. Protozoan parasites of the Leishmania genus are the causative agent of leishmaniasis, one of the most important neglected tropical diseases as designated by the World Health Organization (WHO). Leishmaniasis affects about 12 million people worldwide and can be divided into the following three main clinical manifestations: cutaneous, mucocutaneous, and visceral leishmaniasi

Alterations on the growth and ultrastructure of <em>Leishmania chagasi </em>induced by squalene synthase inhibitors

Leishmaniasis is an important disease in widely dispersed regions of the world. In South America, visceral leishmaniasis (VL) is mainly caused by Leishmania chagasi. The morbidity associated with the infection is high, and death may occur in some untreated patients. Treatment has been based upon pentavalent antimonial drugs for more than half a century and problems, including development of resistance to antimonials and lack of efficacy against VL/HIV co-infections, have emphasized the need for new drugs. Squalene synthase (SQS) is an essential enzyme for the biosynthesis of protozoal sterol molecules. In this work, nineteen synthetic quinuclidines, potentially inhibitors of SQS, were tested against promastigote forms of L. chagasi and the IC50 values of the compounds were determined. The most active compounds had IC50 values of around 30 nM and induced complete growth arrest and cell lysis at sub-micromolar concentrations. We analyzed the morphological structure of the parasites treated with these compounds by transmission electron microscopy of thin sections. Treated parasites showed significant ultrastructural changes, which varied from discrete alterations to total destruction of the cells, depending on the drug concentration and the time of incubation. One important change observed was a typical swelling of the unique and highly branched mitochondrion, where the inner membrane lost its organization. There was an increase in the number of autophagosomal structures. Changes in the organization of the nuclear chromatin and alterations in the flagellar pocket and flagellar membrane were also observed. Crown Copyright (c) 2007 Published by Elsevier B.V. All rights reserved.</p

Schematic 3D view of the phases of interaction between the <i>Leishmania amazonensis</i> parasite and vertebrate cells (2a), and between the parasite and the sandfly (2b).

<p>(A) Attachment of a promastigote to the macrophage surface. (B) The process of internalization via phagocytosis begins with the formation of pseudopods (C), leading to the formation of the parasitophorous vacuole (PV). In the PV, the promastigote transforms into an amastigote. (D) Recruitment and fusion of host cell lysosomes with the PV takes place. (E) In the PV, amastigotes divide several times. (F–G) Intense multiplication generates several hundreds of amastigotes. (H) The host cell bursts, and the parasites reach the extracellular space. (I) Schematic view of female sandfly showing the digestive tract. (J) During a blood meal, a female sandfly ingests infected macrophages with amastigote forms present in the blood of the vertebrate host. (K) Amastigotes form “nest cells” in the abdominal midgut. (L) Amastigotes transform into procyclic promastigotes. (M) Promastigotes multiply and attach to the midgut epithelium. (N) Parasites migrate toward the anterior midgut, resume replication and start to produce promastigote secretory gel (PSG). (O) Promastigotes transform into infective metacyclic promastigotes. (P) Metacyclic promastigotes infect a new mammalian host via regurgitation during the blood meal. These images are based on micrographs obtained by scanning and transmission electron microscopy and by video microscopy.</p

The life cycle of <i>Leishmania amazonensis</i> (A), and structural organization of the promastigote (B) and amastigote (C) forms.

<p>(A) The female sandfly (1) insect bites an infected mammal during the blood meal. Infected macrophages (2) with amastigote forms. (3) Amastigote form. (4) Amastigotes transform into procyclic promastigotes. (5) Procyclic promastigotes multiply in the midgut. (6) Promastigotes migrate toward the stomodeal valve in the anterior midgut and re-initiate cell division. (7) Promastigotes transform into infective metacyclic promastigotes. (8) The female sandfly releases the metacyclic promastigotes into a new mammalian host via regurgitation during the blood meal. (9) Metacyclic promastigotes. (10) Metacyclic promastigotes infect macrophages. (11) Metacyclic promastigotes transform into amastigotes. (12) Amastigotes attach to the membrane of the parasitophorous vacuole. (13) Amastigotes multiply in the vacuole. (14) Intense amastigote multiplication. (15) Amastigotes burst out of the cell. (16) Amastigote form. (17) An amastigote infects a macrophage. In the central portion of the figure, we added the most important reservoirs involved in the maintenance of the parasite. Schematic 3D representations of the organelles found in the <i>Leishmania</i> promastigote (B) and amastigote (C).</p

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

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.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.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

Novel Azasterols as Potential Agents for Treatment of Leishmaniasis and Trypanosomiasis

This paper describes the design and evaluation of novel azasterols as potential compounds for the treatment of leishmaniasis and other diseases caused by trypanosomatid parasites. Azasterols are a known class of (S)-adenosyl-l-methionine: Δ(24)-sterol methyltransferase(24-SMT) inhibitors in fungi, plants, and some parasitic protozoa. The compounds prepared showed activity at micromolar and nanomolar concentrations when tested against Leishmania spp. and Trypanosoma spp. The enzymatic and sterol composition studies indicated that the most active compounds acted by inhibiting 24-SMT. The role of the free hydroxyl group at position 3 of the sterol nucleus was also probed. When an acetate was attached to the 3β-OH, the compounds did not inhibit the enzyme but had an effect on parasite growth and the levels of sterols in the parasite, suggesting that the acetate group was removed in the organism. Thus, an acetate group on the 3β-OH may have application as a prodrug. However, there may be an additional mode(s) of action for these acetate derivatives. These compounds were shown to have ultrastructural effects on Leishmania amazonensis promastigote membranes, including the plasma membrane, the mitochondrial membrane, and the endoplasmic reticulum. The compounds were also found to be active against the bloodstream form (trypomastigotes) of Trypanosoma brucei rhodesiense, a causative agent of African trypanosomiasis

Quinuclidine Derivatives as Potential Antiparasitics▿

There is an urgent need for the development of new drugs for the treatment of tropical parasitic diseases such as Chagas' disease and leishmaniasis. One potential drug target in the organisms that cause these diseases is sterol biosynthesis. This paper describes the design and synthesis of quinuclidine derivatives as potential inhibitors of a key enzyme in sterol biosynthesis, squalene synthase (SQS). A number of compounds that were inhibitors of the recombinant Leishmania major SQS at submicromolar concentrations were discovered. Some of these compounds were also selective for the parasite enzyme rather than the homologous human enzyme. The compounds inhibited the growth of and sterol biosynthesis in Leishmania parasites. In addition, we identified other quinuclidine derivatives that inhibit the growth of Trypanosoma brucei (the causative organism of human African trypanosomiasis) and Plasmodium falciparum (a causative agent of malaria), but through an unknown mode(s) of action