A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi

Miranda, Cristian Gabriel; Solana, María Elisa; Curto, Maria de Los Angeles; Lammel, E.; Schijman, Alejandro G.; Soto, Catalina Dirney Alba

doi:10.1016/j.actatropica.2015.08.004

Cited by 22 publications

(14 citation statements)

References 40 publications

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“…This T. cruzi isolate wil be referred to as VD strain. For experimental purposes, VD strain bloodstream trypomastigotes were purified from heparinized mice blood as previously described [10].…”

Section: Strain Isolationmentioning

confidence: 99%

Molecular and biological characterization of a highly pathogenic Trypanosoma cruzi strain isolated from a patient with congenital infection

Gulin

Bisio

Rocco

et al. 2018

Experimental Parasitology

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Although many Trypanosoma cruzi (T. cruzi) strains isolated from a wide range of hosts have been characterized, there is a lack of information about biological features from vertically transmitted strains. We describe the molecular and biological characteristics of the T. cruzi VD strain isolated from a congenital Chagas disease patient. The VD strain was typified as DTU TcVI; in vitro sensitivity to nifurtimox (NFX) and beznidazole (BZ) were 2.88 μM and 6.19 μM respectively, while inhibitory concentrations for intracellular amastigotes were 0.24 μM for BZ, and 0.66 μM for NFX. Biological behavior of VD strain was studied in a mouse model of acute infection, resulting in high levels of parasitemia and mortality with a rapid clearence of bloodstream trypomastigotes when treated with BZ or NFX, preventing mortality and reducing parasitic load and intensity of inflammatory infiltrate in skeletal and cardiac muscle. Treatment-induced parasitological cure, evaluated after immunossupression were 41% and 35% for BZ and NFX treatment respectively, suggesting a partial response to these drugs in elimination of parasite burden. This exhaustive characterization of this T. cruzi strain provides the basis for inclusion of this strain in a panel of reference strains for drug screening and adds a new valuable tool for the study of experimental T. cruzi infection.

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Section: Strain Isolationmentioning

confidence: 99%

Molecular and biological characterization of a highly pathogenic Trypanosoma cruzi strain isolated from a patient with congenital infection

Gulin

Bisio

Rocco

et al. 2018

Experimental Parasitology

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“…Purification of bloodstream trypomastigotes was essentially carried out by a Ficoll gradient with a swinging bucket rotor, essentially as previously described [ 55 ]. Bloodstream RA trypomastigotes used for EV purification, were either purified as stated above or by swimming (2 x 40 min at 37°C), essentially as described by Miranda et al (2015) [ 56 ]. Briefly, heparinized blood was diluted with 3 volumes of PBS and, after centrifuged at 300 x g for 5 min, the sample was incubated for 40 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

The protein family TcTASV-C is a novel Trypanosoma cruzi virulence factor secreted in extracellular vesicles by trypomastigotes and highly expressed in bloodstream forms

et al. 2018

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TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime—protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes.

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“…Trypomastigotes (1 × 10 4 per well) were cultured in a 96 well-plate (final volume 200 μl) in RPMI medium supplemented with 10% FBS at 37°C in 5% CO 2 atmosphere. After 24 hours motile parasites were counted in hemocytometer chamber under the light microscope (Miranda et al, 2015 ). Results were expressed as % viability of trypomastigotes at 20 μM of the hits.…”

Section: Methodsmentioning

confidence: 99%

Cascade Ligand- and Structure-Based Virtual Screening to Identify New Trypanocidal Compounds Inhibiting Putrescine Uptake

Alberca

Sbaraglini

Morales

et al. 2018

Front. Cell. Infect. Microbiol.

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Chagas disease is a neglected tropical disease endemic to Latin America, though migratory movements have recently spread it to other regions. Here, we have applied a cascade virtual screening campaign combining ligand- and structure-based methods. In order to find novel inhibitors of putrescine uptake in Trypanosoma cruzi, an ensemble of linear ligand-based classifiers obtained by has been applied as initial screening filter, followed by docking into a homology model of the putrescine permease TcPAT12. 1,000 individual linear classifiers were inferred from a balanced dataset. Subsequently, different schemes were tested to combine the individual classifiers: MIN operator, average ranking, average score, average voting, with MIN operator leading to the best performance. The homology model was based on the arginine/agmatine antiporter (AdiC) from Escherichia coli as template. It showed 64% coverage of the entire query sequence and it was selected based on the normalized Discrete Optimized Protein Energy parameter and the GA341 score. The modeled structure had 96% in the allowed area of Ramachandran's plot, and none of the residues located in non-allowed regions were involved in the active site of the transporter. Positivity Predictive Value surfaces were applied to optimize the score thresholds to be used in the ligand-based virtual screening step: for that purpose Positivity Predictive Value was charted as a function of putative yields of active in the range 0.001–0.010 and the Se/Sp ratio. With a focus on drug repositioning opportunities, DrugBank and Sweetlead databases were subjected to screening. Among 8 hits, cinnarizine, a drug frequently prescribed for motion sickness and balance disorder, was tested against T. cruzi epimastigotes and amastigotes, confirming its trypanocidal effects and its inhibitory effects on putrescine uptake. Furthermore, clofazimine, an antibiotic with already proven trypanocidal effects, also displayed inhibitory effects on putrescine uptake. Two other hits, meclizine and butoconazole, also displayed trypanocidal effects (in the case of meclizine, against both epimastigotes and amastigotes), without inhibiting putrescine uptake.

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A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi

Cited by 22 publications

References 40 publications

Molecular and biological characterization of a highly pathogenic Trypanosoma cruzi strain isolated from a patient with congenital infection

Molecular and biological characterization of a highly pathogenic Trypanosoma cruzi strain isolated from a patient with congenital infection

The protein family TcTASV-C is a novel Trypanosoma cruzi virulence factor secreted in extracellular vesicles by trypomastigotes and highly expressed in bloodstream forms

Cascade Ligand- and Structure-Based Virtual Screening to Identify New Trypanocidal Compounds Inhibiting Putrescine Uptake

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