Genetically different isolates of Trypanosoma cruzi elicit different infection dynamics in raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana)

Trypanosoma cruzi is a genetically and biologically diverse species. In the current study we determined T. cruzi infection dynamics in two common North American reservoirs, Virginia opossums (Didelphis virginiana) and raccoons (Procyon lotor). Based on previous molecular and culture data from naturally-exposed animals, we hypothesised that raccoons would have a longer patent period than opossums, and raccoons would be competent reservoirs for both genotypes T. cruzi I (TcI) and TcIIa, while opossums would only serve as hosts for TcI. Individuals (n = 2 or 3) of each species were inoculated with 1 × 10⁶ culture-derived T. cruzi trypomastigotes of TcIIa (North American (NA) – raccoon), TcI (NA – opossum), TcIIb (South American – human), or both TcI and TcIIa. Parasitemias in opossums gradually increased and declined rapidly, whereas parasitemias peaked sooner in raccoons and they maintained relatively high parasitemia for 5 weeks. Raccoons became infected with all three T. cruzi strains, while opossums only became infected with TcI and TcIIb. Although opossums were susceptible to TcIIb, infection dynamics were dramatically different compared with TcI. Opossums inoculated with TcIIb seroconverted, but parasitemia duration was short and only detectable by PCR. In addition, raccoons seroconverted sooner (3–7 days post inoculation) than opossums (10 days post inoculation). These data suggest that infection dynamics of various T. cruzi strains can differ considerably in different wildlife hosts.     

5-Nitrofuranes and 5-nitrothiophenes with anti-Trypanosoma cruzi activity and ability to accumulate squalene

Chagas disease represents a serious public health problem in South America. The first line of treatment is Nifurtimox and Benznidazole which generate toxic effects in treated patients. We have recently shown that a number of 5-nitrofuranes possess activity against Trypanosoma cruzi through oxidative stress and inhibition of parasite ergosterol biosynthesis, specifically at the level of squalene epoxidase. Here, we identify new 5-nitrofuranes and the thia-analogues with excellent effects on the viability of T. cruzi and adequate parasite/mammal selectivity indexes. Analysis of the free sterols from parasite incubated, during 120 h, with the compounds showed that some of them accumulated squalene suggesting the squalene epoxidase activity inhibition of the parasite. Nifurtimox was able to accumulate squalene only at lower incubation times. Due to this fact some derivatives were also tested as antifungal agents. Quantitative structure–activity relationship studies were also performed showing relevant features for further new derivatives design. Taken together, the results obtained in the present work point to a more general effect of 5-nitrofuranes and 5-nitrothiophenes in trypanosomatids, opening potential therapeutic possibilities of them for these infectious diseases.     

Molecular characterization and intracellular distribution of the alpha 5 subunit of Trypanosoma cruzi 20S proteasome

Three different monoclonal antibodies were produced against Trypanosona cruzi proteasomes. These antibodies were shown to react with a single 27-kDa band on immunoblots of purified proteasomes. Using a 7E5 monoclonal antibody (IgG1) that recognized the α5 subunit of protozoan protease we have studied the intracellular distribution of the T. cruzi 20S proteasome. Contrary to all cell types described to date, T. cruzi 20S proteasome was found not only in the cytoplasm and nucleus but also in the kinetoplast. As revealed by confocal microscopy, the reactivity of monoclonal antibody 7E5 was highly specific for protozoan proteasome because the antibody recognized only the proteasomes from parasites and not those from the mammalian host in T. cruzi infected cells. These findings were confirmed by immunoblots or immunoprecipitations, followed by chymotrypsin-like activity detection in kinetoplasts isolated by differential centrifugation and sucrose density gradients. Proteasome 20S was present in all T. cruzi stages and only slight differences in terms of relative abundance were found. The potential role of the proteasome in kinetoplast remodeling remains to be determined.     

PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations

PGE₂ involvement in experimental Trypanosoma cruzi infection depends on the lethal capacity of the parasite subpopulation used. Mice acutely infected with non-lethal K98 displayed an enhancement in PGE₂ serum levels during the acute period, while those infected with lethal T. cruzi subpopulations (RA or K98-2) showed levels not different from normal mice. The enhancement detected in K98 group could be related both to an increased number of CD8+ T cell number and to enhanced PGE₂ release per cell by CD8+; values of PGE₂ release by adherent cells were not altered in this group. Treatment with cyclooxygenase inhibitors enhanced mortality rates of mice infected with K98, and administration of 16,16-dimethyl PGE₂ (dPGE) reversed this effect. However, mice infected with RA did not reduce their mortality rates by administration of diverse doses of dPGE. These findings suggest that PGE₂ could play a role in resistance in mice infected with K98.     

The in vivo distribution of Cr-labeled Trypanosoma cruzi in mice

The optimal conditions for labeling Trypanosoma cruzi culture forms with ⁵¹CrO₄²⁻ were determined. Labeled trypanosomes or labeled human red blood cells (RBCs) were injected intravenously into normal C3H(He) female mice and the rate of clearance and organ distribution of the isotope were observed over a 30 h period. It was found that trypanosomes and xenogeneic RBCs were cleared rapidly from the peripheral blood and accumulated primarily in the liver, spleen, lungs and kidneys. A difference was noted in accumulation of trypanosomes and RBCs in these mice.     

Expression in insect cells of active mature cruzipain from Trypanosoma cruzi, containing its C-terminal domain

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the papain family that contains a C-terminal domain in the mature enzyme, in addition to a catalytic moiety homologous to papain and some mammalian cathepsins. The native enzyme is expressed as a complex mixture of isoforms and has not been crystallized. Previous attempts to express recombinant mature cruzipain containing the C-terminal domain have failed. For this reason, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule lacking the C-terminal domain (cruzainΔc) has been determined. We report here the expression of active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 0.2 mg/100 ml of culture supernatant, has an apparent molecular mass similar, and an identical N-terminal sequence, compared with the native enzyme. The expressed protein is able to process itself by self-proteolysis, leaving the isolated C-terminal domain, and has kinetic properties similar to those of native cruzipain, although some differences in substrate specificity were found. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography.     

Attachment of flagellum to the cell body is important to the kinetics of transferrin uptake by Trypanosoma cruzi

The flagellar pocket and the cytostome are surface domains of Trypanosoma cruzi epimastigote involved in acquisition of nutrients. The cytostome is physically connected to the flagellar complex. To investigate if this association plays a role in endocytosis in T. cruzi, the endocytic activity in wild type and gp72 null mutant (flagellum-cell body attachment region is absent) epimastigotes was compared. Both wild type and mutant cells were incubated with transferrin conjugated with Alexa 543 or gold particles over different time periods and thereafter qualitatively and quantitatively analyzed by flow cytometry and transmission electron microscopy. Flow cytometry analysis showed a reduction in transferrin uptake by null mutant after 30 min of incubation. In addition, at this time period, signals detected by fluorescence microscopy were slightly lower in null mutant cells. At lower incubation times, no differences between wild type and mutant epimastigotes could be observed. Quantitative data obtained by morphometric and flow cytometry analysis suggested that the speed of the endocytic process in the null mutant was similar to wild type cells, although null mutants were not able to bind cargo and therefore internalize as much as wild type epimastigotes. Our observations suggest that the physical association between cytostome and the flagellar complex plays a role in endocytosis efficiency by epimastigotes of T. cruzi.     

Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Design,synthesis and biological evaluation of (E)-5-styryl-1,2,4-oxadiazoles as anti-tubercular agents

Cinnamic acid and its derivatives are known for anti-tubercular activity. The present study reports the synthesis of cinnamic acid derivatives via bioisosteric replacement of terminal carboxylic acid with “oxadiazole”. A series of cinnamic acid derivatives (styryl oxadiazoles) were designed and synthesized in good yields by reaction of substituted cinnamic acids (2, 15a-15s) with amidoximes. The synthesized styryl oxadiazoles were evaluated in vitro for anti-tubercular activity against Mycobacterium tuberculosis (Mtb) H37Ra strain. The structure-activity relationship (SAR) study has identified several compounds with mixed anti-tubercular profiles. The compound 32 displayed potent anti-tubercular activity (IC₅₀ = 0.045 µg/mL). Molecular docking studies on mycobacterial enoyl-ACP reductase enzyme corroborated well with the experimental findings providing a platform for structure based hit-to-lead development.     

Energetics of heart mitochondria during acute phase of Trypanosoma cruzi infection in rats

The energetics of heart mitochondria was studied in the acute phase of Trypanosoma cruzi infection in rats. Wistar rats were infected with 2 × 10⁵ trypomastigote forms of the Y strain of T. cruzi, and heart mitochondria and submitochondrial particles isolated after 7 and 25 days of infection. Ultrastructure of mitochondria seemed to be preserved, but cytochrome c levels were significantly depressed. Respiratory control ratios (RCR) were decreased for glutamate and succinate oxidations, as a consequence of inhibition of respiration in state 3 and/or of stimulation of respiration in state 4. Stimulation of hydrolytic activity of FoF1-ATPase by energization of mitochondria was approx. 2-fold higher in relation to controls. Mitochondrial ATP concentration remained constant. In conclusion, during the acute phase of T. cruzi infection in rats there is an energy impairment at the level of heart mitochondria, but their ultrastructure and ATP concentration seem to be preserved; the maintenance of ATP may be due to an adaptative mechanism of the cell which includes inhibition of the hydrolytic activity of FoF1-ATPase.     

Trypanosoma cruzi: Identification of DNA targets of the nuclear periphery coiled-coil protein TcNUP-1

The nuclear lamina is a structure that lines the inner nuclear membrane. In metazoans, lamins are the primary structural components of the nuclear lamina and are involved in several processes. Eukaryotes that lack lamins have distinct proteins with homologous functions. Some years ago, a coiled-coil protein in Trypanosoma brucei, NUP-1, was identified as the major filamentous component of its nuclear lamina. However, its precise role has not been determined. We characterized a homologous protein in Trypanosoma cruzi, TcNUP-1, and identified its in vivo DNA binding sites using a chromatin immunoprecipitation assay. We demonstrate for the first time that TcNUP-1 associates with chromosomal regions containing large non-tandem arrays of genes encoding surface proteins. We therefore suggest that TcNUP-1 is a structural protein that plays an essential role in nuclear organization by anchoring T. cruzi chromosomes to the nuclear envelope.     

Trypanosoma cruzi infection disturbs mitochondrial membrane potential and ROS production rate in cardiomyocytes

In this study, we investigated the role of Trypanosoma cruzi invasion and inflammatory processes in reactive oxygen species (ROS) production in a mouse atrial cardiomyocyte line (HL-1) and primary adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with T. cruzi (Tc) trypomastigotes, Tc lysate (TcTL), or Tc secreted proteins (TcSP) for 0–72 h, and ROS were measured by amplex red assay. Cardiomyocytes infected by T. cruzi (but not those incubated with TcTL or TcSP) exhibited a linear increase in ROS production for 2–48 h postinfection (max 18-fold increase), which was further enhanced by recombinant cytokines (IL-1β, TNF-α, and IFN-γ). We observed no increase in NADPH oxidase, xanthine oxidase, or myeloperoxidase activity, and specific inhibitors of these enzymes did not block the increased rate of ROS production in infected cardiomyocytes. Instead, the mitochondrial membrane potential was perturbed and resulted in inefficient electron transport chain (ETC) activity and enhanced electron leakage and ROS formation in infected cardiomyocytes. HL-1 rho (ρ) cardiomyocytes lacked a functional ETC and exhibited no increase in ROS formation in response to T. cruzi. Together, these results demonstrate that invasion by T. cruzi and an inflammatory milieu affect mitochondrial integrity and contribute to electron transport chain inefficiency and ROS production in cardiomyocytes.     

3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile): A new highly active compound against epimastigote and trypomastigote form of Trypanosoma cruzi

We have synthesized the Morita-Baylis-Hillman adduct (MBHA) 3-hydroxy-2-methylene-3-(4-nitrophenyl)-propanenitrile (3) in quantitative yield and evaluated on Trypanosoma cruzi epimastigote and bloodstream trypomastigote forms. Compound 3 strongly inhibited epimastigote growth, with IC₅₀/72 h of 28.5 μM and also caused intense trypomastigotes lysis, with an IC₅₀/24 h of 25.5 μM. Ultrastructural analysis showed significant morphological changes on both parasite forms treated with 3, including increase of cell volume and rounding of cell body as well as intense intracellular disorganization. Morphological changes indicative of apoptosis, autophagy or necrosis were observed in most affected cells. Docking calculations of 1, 2 and 3 pointed out the possibility of T. cruzi Farnesyl Pyrophosphate Synthase (TcFPPS) enzyme inhibition in 3 mechanism of action.     

Synthesis and molecular docking study of some novel 2,3-disubstituted quinazolin-4(3H)-one derivatives as potent inhibitors of urease

A new series of 2,3-disubstituted quinazolin-4(3H)-one compounds including oxadiazole and furan rings was synthesized. Their inhibitory activities on urease were assessed in vitro. All newly synthesized compounds exhibited potent urease inhibitory activity in the range of IC₅₀ = 1.55 ± 0.07–2.65 ± 0.08 µg/mL, when compared with the standard urease inhibitors such as thiourea (IC₅₀ = 15.08 ± 0.71 µg/mL) and acetohydroxamic acid (IC₅₀ = 21.05 ± 0.96 µg/mL). 2,3-Disubstituted quinazolin-4(3H)-one derivatives containing furan ring (3a-e) were found to be the most active inhibitors when compared with the compounds 2a-e bearing oxadiazole ring. Compound 3a, bearing 4-chloro group on phenyl ring, was found as the most effective inhibitor of urease with the IC₅₀ value of 1.55 ± 0.11 µg/mL. The molecular docking studies of the newly synthesized compounds were performed to identify the probable binding modes in the active site of the Jack bean urease (JBU) enzymes.     

Design,synthesis and molecular docking of substituted 3-hydrazinyl-3-oxo-propanamides as anti-tubercular agents

Based on the anti-mycobacterial activity of various acid hydrazides, a series of substituted 3-hydrazinyl-3-oxo-propanamides has been designed. The target compounds have been synthesized from diethylmalonate using substituted amines and hydrazine hydrate in ethanol. Computational studies and anti-tubercular activity screenings were undertaken to test their inhibitory effect on protein kinase PknB from Mycobacterium tuberculosis. Binding poses of the compounds were energetically favorable and showed good interactions with active site residues. Designed molecules obey the Lipinski’s rule of 5 and gave moderate to good drug likeness score. Among the sixteen compounds (1–16) taken for in silico and in vitro studies, 3 compounds (11, 12 and 15) have shown good binding energies along with exhibiting good anti-tubercular activity and thus may be considered as a good inhibitors of PknB.     

Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence

Virulence of Trypanosoma cruzi depends on a variety of genetic and biochemical factors. It has been proposed that components of the parasites’ antioxidant system may play a key part in this process by pre-adapting the pathogen to the oxidative environment encountered during host cell invasion. Using several isolates (10 strains) belonging to the two major phylogenetic lineages (T. cruzi-I and T. cruzi-II), we investigated whether there was an association between virulence (ranging from highly aggressive to attenuated isolates at the parasitemia and histopathological level) and the antioxidant enzyme content. Antibodies raised against trypanothione synthetase (TcTS), ascorbate peroxidase (TcAPX), mitochondrial and cytosolic tryparedoxin peroxidases (TcMPX and TcCPX) and trypanothione reductase (TcTR) were used to evaluate the antioxidant enzyme levels in epimastigote and metacyclic trypomastigote forms in the T. cruzi strains. Levels of TcCPX, TcMPX and TcTS were shown to increase during differentiation from the non-infective epimastigote to the infective metacyclic trypomastigote stage in all parasite strains examined. Peroxiredoxins were found to be present at higher levels in the metacyclic infective forms of the virulent isolates compared with the attenuated strains. Additionally, an increased resistance of epimastigotes from virulent T. cruzi populations to hydrogen peroxide and peroxynitrite challenge was observed. In mouse infection models, a direct correlation was found between protein levels of TcCPX, TcMPX and TcTS, and the parasitemia elicited by the different isolates studied (Pearson’s coefficient: 0.617, 0.771, 0.499; respectively, P < 0.01). No correlation with parasitemia was found for TcAPX and TcTR proteins in any of the strains analyzed. Our data support that enzymes of the parasite antioxidant armamentarium at the onset of infection represent new virulence factors involved in the establishment of disease.     

Platinum-based complexes of bioactive 3-(5-nitrofuryl)acroleine thiosemicarbazones showing anti-Trypanosoma cruzi activity

Eight new platinum(II) complexes with 3-(5-nitrofuryl)acroleine thiosemicarbazones showing anti-trypanosomal activity were synthesized, characterized and in vitro evaluated. Most of the complexes showed IC₅₀ values in the micromolar range against two different strains of Trypanosoma cruzi, causative agent of Chagas disease (American Trypanosomiasis). In addition, most of the newly developed complexes, together with the analogous platinum 5-nitrofuraldehyde containing thiosemicarbazones previously reported, resulted more active than the reference trypanocidal drug nifurtimox on the infective trypomastigote form of the parasite. Their capacity to produce free radicals that could lead to parasite death was evaluated by ESR experiments in the parasite and by respiration measurements. Compounds were tested for their DNA interaction ability. Results showed that some of the compounds could act as dual inhibitors in the parasite, through production of toxic free radicals and interaction with DNA. All the results were compared with those previously reported for the free ligands, the analogous palladium(II) compounds and the previously reported series of platinum(II) compounds.     

Flow cytometric analysis and microsatellite genotyping reveal extensive DNA content variation in Trypanosoma cruzi populations and expose contrasts between natural and experimental hybrids

Trypanosoma cruzi exhibits remarkable genetic heterogeneity. This is evident at the nucleotide level but also structurally, in the form of karyotypic variation and DNA content differences between strains. Although natural populations of T. cruzi are predominantly clonal, hybrid lineages (TcIId and TcIIe) have been identified and hybridisation has been demonstrated in vitro, raising the possibility that genetic exchange may continue to shape the evolution of this pathogen. The mechanism of genetic exchange identified in the laboratory is unusual, apparently involving fusion of diploid parents followed by genome erosion. We investigated DNA content diversity in natural populations of T. cruzi in the context of its genetic subdivisions by using flow cytometric analysis and multilocus microsatellite genotyping to determine the relative DNA content and estimate the ploidy of 54 cloned isolates. The maximum difference observed was 47.5% between strain Tu18 cl2 (TcIIb) and strain C8 cl1 (TcI), which we estimated to be equivalent to 73 Mb of DNA. Large DNA content differences were identified within and between discrete typing units (DTUs). In particular, the mean DNA content of TcI strains was significantly less than that for TcII strains (P < 0.001). Comparisons of hybrid DTUs TcIId/IIe with corresponding parental DTUs TcIIb/IIc indicated that natural hybrids are predominantly diploid. We also measured the relative DNA content of six in vitro-generated TcI hybrid clones and their parents. In contrast to TcIId/IIe hybrid strains these experimental hybrids comprised populations of sub-tetraploid organisms with mean DNA contents 1.65–1.72 times higher than the parental organisms. The DNA contents of both parents and hybrids were shown to be relatively stable after passage through a mammalian host, heat shock or nutritional stress. The results are discussed in the context of hybridisation mechanisms in both natural and in vitro settings.     

Genotype variation of Trypanosoma cruzi isolates from different Brazilian biomes

Chagas disease is an enzootic disease, in which the flagellate Trypanosoma cruzi infects a large variety of animals. Humans are accidentally infected due to the migration into wild environments. To identify T. cruzi discrete typing units (DTUs), 19 Brazilian isolates from different biomes and hosts were analyzed by PCR amplification of 24Sα rRNA, 18S rRNA and mini-exon gene sequences. The majority of the isolates was classified as TcIIb (TcII) but subtypes TcIIc (TcIII) and TcIId (TcV) were also identified. In addition, in monkeys TcI was detected.