In vitro anti-Leishmania activity of triclabendazole and its synergic effect with amphotericin B.

Introduction: Leishmaniasis is a neglected tropical disease, with approximately 1 million new cases and 30,000 deaths reported every year worldwide. Given the lack of adequate medication for treating leishmaniasis, drug repositioning is essential to save time and money when searching for new therapeutic approaches. This is particularly important given leishmaniasis's status as a neglected disease. Available treatments are still far from being fully effective for treating the different clinical forms of the disease. They are also administered parenterally, making it challenging to ensure complete treatment, and they are extremely toxic, in some cases, causing death. Triclabendazole (TCBZ) is a benzimidazole used to treat fasciolosis in adults and children. It presents a lower toxicity profile than amphotericin B (AmpB) and is administered orally, making it an attractive candidate for treating other parasitoses. The mechanism of action for TCBZ is not yet well understood, although microtubules or polyamines could potentially act as a pharmacological target. TCBZ has already shown antiproliferative activity against T. cruzi, T. brucei, and L. infantum. However, further investigations are still necessary to elucidate the mechanisms of action of TCBZ. Methods: Cytotoxicity assay was performed by MTT assay. Cell inhibition (CI) values were obtained according to the equation CI = (O.D treatment x 100/O.D. negative control). For Infection evaluation, fixated cells were stained with Hoechst and read at Operetta High Content Imaging System (Perkin Elmer). For growth curves, cell culture absorbance was measured daily at 600 nm. For the synergism effect, Fractional Inhibitory Concentrations (FICs) were calculated for the IC50 of the drugs alone or combined. Mitochondrial membrane potential (DYm), cell cycle, and cell death analysis were evaluated by flow cytometry. Reactive oxygen species (ROS) and lipid quantification were also determined by fluorimetry. Treated parasites morphology and ultrastructure were analyzed by electron microscopy. Results: The selectivity index (SI = CC50/IC50) of TCBZ was comparable with AmpB in promastigotes and amastigotes of Leishmania amazonensis. Evaluation of the cell cycle showed an increase of up to 13% of cells concentrated in S and G2, and morphological analysis with scanning electron microscopy showed a high frequency of dividing cells. The ultrastructural analysis demonstrated large cytoplasmic lipid accumulation, which could suggest alterations in lipid metabolism. Combined administration of TCBZ and AmpB demonstrated a synergistic effect in vitro against intracellular amastigote forms with cSFICs of 0.25. Conclusions: Considering that TCBZ has the advantage of being inexpensive and administrated orally, our results suggest that TCBZ, combined with AmpB, is a promising candidate for treating leishmaniasis with reduced toxicity.

Pirahy virus: Identification of a new and potential emerging arbovirus in South Brazil.

Genomic and epidemiological surveillance are paramount for the discovery of new viruses with the potential to cross species barriers. Here, we present a new member of the genus Alphavirus found in Trichoprosopon and Wyeomia mosquitoes, tentatively named Pirahy virus (PIRAV). PIRAV was isolated from mosquito pools collected in a rural area of Piraí do Sul, South Brazil. In vitro assays revealed that PIRAV replicates and causes cytopathic effects in vertebrate cell lines such as Vero E6, SH-SY5Y, BHK-21 and UMNSAH/DF-1. Genomic signature analysis supports these results showing a dinucleotide and codon usage balance compatible with several hosts. Phylogenetic analyses placed PIRAV basal to the Venezuelan equine encephalitis complex. Genome analyses, electron microscopy, and biological characterization show findings that may alert for the emergence of a new arbovirus in South America.

Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins.

Trypanosomatids (order Kinetoplastida), including the human pathogens Trypanosoma cruzi (agent of Chagas disease), Trypanosoma brucei, (African sleeping sickness), and Leishmania (leishmaniasis), affect millions of people and animals globally. T. cruzi is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP) complexes composed of recombinant Cas9 from Staphylococcus aureus (SaCas9), but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9), and in vitro-transcribed single guide RNAs (sgRNAs) results in rapid gene edits in T. cruzi and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of T. cruzi, as well as in T. brucei and Leishmania major RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens.IMPORTANCE Protozoan parasites remain some of the highest-impact human and animal pathogens, with very limited treatment and prevention options. The development of improved therapeutics and vaccines depends on a better understanding of the unique biology of these organisms, and understanding their biology, in turn, requires the ability to track and manipulate the products of genes. In this work, we describe new methods that are available to essentially any laboratory and applicable to any parasite isolate for easily and rapidly editing the genomes of kinetoplastid parasites. We demonstrate that these methods provide the means to quickly assess function, including that of the products of essential genes and potential targets of drugs, and to tag gene products at their endogenous loci. This is all achieved without gene cloning or drug selection. We expect this advance to enable investigations, especially in Trypanosoma cruzi and Leishmania spp., that have eluded investigators for decades.

Structures containing galectin-3 are recruited to the parasitophorous vacuole containing Trypanosoma cruzi in mouse peritoneal macrophages.

Trypanosoma cruzi has a complex life cycle where the infective forms for the vertebrate host are trypomastigotes and amastigotes. Both forms invade and lyse their parasitophorous vacuole (PV) membrane, entering into the cytoplasm of its host cells. Galectin-3 (Gal-3) is a protein abundantly distributed in macrophages and epithelial cells. Previous studies demonstrated that Gal-3 binds to a 45KDa mucin of trypomastigotes surface, enhancing its adhesion to the extracellular matrix and even its entry into cells. Gal-3 has another novel cytoplasmic function recently described: a vacuole lyses marker in intracellular bacteria. Considering (1) the importance of Gal-3 during T. cruzi early infection and (2) the importance of T. cruzi PV lyses for parasite differentiation and replication, this study intended to explore a possible recruitment of structures containing Gal-3 (G3CSs) to T. cruzi PVs. Microscopy analyses showed these G3CSs around PVs after 30 and 90 min of amastigotes and trypomastigotes infection, respectively. This recruitment was specific for T. cruzi PVs since we did not observe the same distribution at macrophages vacuoles containing fluorescent microspheres (FM). Concomitantly, this study intended to analyze the participation of actin cytoskeleton in T. cruzi PV maturation. We observed that actin filaments form a "belt-like" structure around trypomastigotes and amastigotes PVs, also labeled for Gal-3. At the time proposed for PV lysis, we observed an actin disassembling while LAMP-1 was recruited to PVs membrane. However, this pattern was maintained in macrophages derived from Gal-3 knockout mice, revealing that the actin belt structure forms independently from Gal-3. Taken together, these data suggest that G3CSs are recruited to vicinity of T. cruzi PV and that actin filaments localize and remain around T. cruzi PVs until the time of its lysis.

Volutin granules of Eimeria parasites are acidic compartments and have physiological and structural characteristics similar to acidocalcisomes.

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.

Kinetics of pyrophosphate-driven proton uptake by acidocalcisomes of Leptomonas wallacei.

In this work, we show the kinetics of pyrophosphate-driven H+ uptake by acidocalcisomes in digitonin-permeabilized promastigotes of Leptomonas wallacei. The vacuolar proton pyrophosphatase activity was optimal in the pH range of 7.5-8.0, was inhibited by imidiodiphosphate, and was completely dependent on K+ and PPi. H+ was released with the addition of Ca2+, suggesting the presence of a Ca2+/H+ antiport. In addition, X-ray elemental mapping associated with energy-filtering transmission electron microscopy showed that most of the Ca, Na, Mg, P, K, Fe, and Zn were located in acidocalcisomes. L. wallacei immunolabeled with antibodies against Trypanosoma cruzi pyrophosphatase show intense fluorescence in cytoplasmatic organelles of size and distribution similar to the acidocalcisomes. Altogether, the results show that L. wallacei acidocalcisomes possess a H+-pyrophosphatase with characteristics of type I V-H+-PPase. However, we did not find any evidence, either for the presence of H+-ATPases or for Na+/H+ exchangers in these acidocalcisomes.

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

Acidocalcisomes are acidic calcium storage organelles found in several microorganisms. They are characterized by their acidic nature, high electron density, high content of polyphosphates and several cations. Electron microscopy contrast tuned images of Herpetomonas sp. showed the presence of several electron dense organelles ranging from 100 to 300 nm in size. In addition, X-ray element mapping associated with energy-filtering transmission electron microscopy showed that most of the cations, namely Na, Mg, P, K, Fe and Zn, are located in their matrix. Using acridine orange as an indicator dye, a pyrophosphate-driven H+ uptake was measured in cells permeabilized by digitonin. This uptake has an optimal pH of 6.5-6.7 and was inhibited by sodium fluoride (NaF) and imidodiphosphate (IDP), two H+-pyrophosphatase inhibitors. H+ uptake was not promoted by ATP. Addition of 50 microM Ca2+ induced the release of H+, suggesting the presence of a Ca2+/H+ countertransport system in the membranes of the acidic compartments. Na+ was unable to release protons from the organelles. The pyrophosphate-dependent H+ uptake was dependent of ion K+ and inhibited by Na+ Herpetomonas sp. immunolabeled with monoclonal antibodies raised against a Trypanosoma cruzi V-H+-pyrophosphatase shows intense fluorescence in cytoplasmatic organelles of size and distribution similar to the electron-dense vacuoles. Together, these results suggest that the electron dense organelles found in Herpetomonas sp. are homologous to the acidocalcisomes described in other trypanosomatids. They possess a vacuolar H+-pyrophosphatase and a Ca2+/H+ antiport. However, in contrast to the other trypanosomatids so far studied, we were not able to measure any ATP promoted H+ transport in the acidocalcisomes of this parasite.