High-content imaging for automated determination of host-cell infection rate by the intracellular parasite Trypanosoma cruzi

Chagas disease affects 8–11 million people, mostly in Latin America. Sequelae include cardiac, peripheral nervous and/or gastrointestinal disorders, thus placing a large economic and social burden on endemic countries. The pathogenesis and the evolutive pattern of the disease are not fully clarified. Moreover, available drugs are partially effective and toxic, and there is no vaccine. Therefore, there is an urgent need to speed up basic and translational research in the field. Here, we applied automated high-content imaging to generate multiparametric data on a cell-by-cell basis to precisely and quickly determine several parameters associated with in vitro infection of host cell by Trypanosoma cruzi, the causative agent of Chagas disease. Automated and manual quantifications were used to determine the percentage of T. cruzi-infected cells in a 96-well microplate format and the data generated was statistically evaluated. Most importantly, this automated approach can be widely applied for discovery of potential drugs as well as molecular pathway elucidation not only in T. cruzi but also in other human intracellular pathogens.     

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.     

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.     

Trypanosoma cruzi: effect of the infection on the 20S proteasome in non-immune cells

Human infection with the protozoan Trypanosoma cruzi leads to Chagas disease. After 10-20 years of the normal acute phase, this disease develops to a chronic phase characterized mainly by dilated congestive cardiomyopathy. The mechanisms involved in the chronic phase are poorly understood, and it has been suggested that the parasite evades immune surveillance by down regulating the MHC class I antigen processing pathway. Here we analyzed whether composition or expression of the 20S proteasome, the major proteinase responsible for the generation of MHC class I ligands, were altered upon infection of HeLa cells by T. cruzi. Two-dimensional gel electrophoresis and RT-PCR experiments comparing non-infected and infected cells did not show differences between the composition of 20S proteasome or expression of its subunits. However, the proteasome’s trypsin- and chymotrypsin-like activities were 2.5 and 3.6 times higher in infected cells than in non-infected cells. Our results suggest that in vitroT. cruzi infection of human or rat cells do not alter the expression of 20S proteasomal subunits or particle composition, and fails to induce the formation of immunoproteasome. However, a significant increase in the trypsin- and chymotrypsin-like activities of the host proteasome was observed.     

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.     

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: 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.     

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.     

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.     

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.     

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.     

siRNA下调20S蛋白酶体β5亚基抑制立氏立克次体细胞内增殖

[目的]探究宿主20S蛋白酶体β5亚基(proteasome 20S subunit beta 5,PSMB5)对革兰氏阴性专性胞内寄生菌立氏立克次体胞内生长繁殖的影响.[方法]利用小干扰RNA转染Vero和THP-1宿主细胞,设置未转染细胞为空白对照组、无义小干扰RNA转染组为阴性对照组、PSMB5特异性小干扰RNA...     

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.     

Colombian Trypanosoma cruzi major genotypes circulating in patients: Minicircle homologies by cross-hybridization analysis

Here we present compelling evidence of Trypanosoma cruzi genotypes infecting 77 human cases of Chagas disease in Santander Department of Colombia. The patients were clinically studied and classified according to the presence of cardiac symptoms. We describe the distribution of the major T. cruzi genotypes circulating in this area by means of direct PCR analysis of blood samples. PCR was directed to minicircles and amplified DNAs were hybridized using genotype-specific DNA probes. These samples were previously genotyped with miniexon, 24 α rRNA and cytochrome oxidase subunit II (COII) markers. Minicircle DNA analyses were more sensitive than miniexon, 24 α rRNA and CO II genes in detecting infective T. cruzi II (Tc II). Two Tc II genotypes were identified by hybridization using two complementary DNA probes in 27.3% of the patients, with 15.3% using all three markers. These corresponded to 10 cases genotyped only by hybridization. The lineage Tc I, determined by hybridization, was the most prevalent singly or combined with different genotypes (72.7%), and at least three different T. cruzi genotypes were identified. Attempts to find two T. cruzi genotypes Tc I and Tc II in other endemic areas of Colombia revealed that one similar to the most prevalent Tc I genotype was detected in distant geographical areas. A similar Tc II genotype was found in Bolivia and Chile, revealing the great distribution of some ancestral T. cruzi genotypes. We did not detect any association between infective Tc I and Tc II lineages and the severity of the patients’ cardiac symptoms.     

Trypanosoma cruzi: Effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide and iNOS expression in cardiac tissue in the acute phase of infection in mice

Leukotrienes are important mediators of inflammatory responses. In this study, we investigated the effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide (NO) and iNOS expression in cardiac tissue of mice infected with Trypanosoma cruzi, the agent of Chagas’ disease. NO is a key mediator of parasite killing in mice experimentally infected with T. cruzi, and previous studies have suggested that leukotrienes, such as LTB₄, induces NO synthesis in T. cruzi-infected macrophages and plays a relevant role in the killing of parasite in a NO-dependent manner. We therefore investigated whether leukotrienes would have a similar role in vivo in controlling the parasite burden by regulating NO activity. We have made the striking observation that absence of 5-LO-derived leukotrienes results in increased NO and IL-6 production in the plasma with a concomitant decrease in the expression of iNOS in the cardiac tissue on day 12 after T. cruzi infection. These findings indicate that endogenous leukotrienes are important regulators of NO activity in the heart and therefore influence the cardiac parasite burden without exerting a direct action on IL-6 production in the acute phase of infection with T. cruzi.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.