In vitro differentiation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigotes using a biphasic medium.

The pathogen Trypanosoma cruzi differentiates from epimastigotes (E) into infective metacyclic trypomastigotes (MTs) to invade the mammalian cell. This process, called metacyclogenesis, is mimicked in vitro by nutrient starvation or incubation with minimal media. Here, we describe an alternative protocol for metacyclogenesis by incubating E forms in a biphasic medium supplemented with human blood. Although time consuming, this procedure yields fully differentiated MTs without the presence of intermediate forms, even for cultures that have been maintained as E for years.

Immunomodulatory, trypanocide, and antioxidant properties of essential oil fractions of Lippia alba (Verbenaceae).

BACKGROUND: Parasite persistence, exacerbated and sustained immune response, and continuous oxidative stress have been described to contribute to the development of the cardiac manifestations in Chronic Chagas Disease. Nevertheless, there are no efficient therapies to resolve the Trypanosoma cruzi infection and prevent the disease progression. Interestingly, trypanocide, antioxidant, and immunodulatory properties have been reported separately for some major terpenes, as citral (neral plus geranial), limonene, and caryophyllene oxide, presents in essential oils (EO) extracted from two chemotypes (Citral and Carvone) of Lippia alba. The aim of this study was to obtain L. alba essential oil fractions enriched with the aforementioned bioactive terpenes and to evaluate the impact of these therapies on trypanocide, oxidative stress, mitochondrial bioenergetics, genotoxicity, and inflammatory markers on T. cruzi-infected macrophages. METHODS: T. cruzi-infected J774A.1 macrophage were treated with limonene-enriched (ACT1) and citral/caryophyllene oxide-enriched (ACT2) essential oils fractions derived from Carvone and Citral-L. alba chemotypes, respectively. RESULTS: ACT1 (IC50 = 45 ± 1.7 µg/mL) and ACT2 (IC50 = 80 ± 1.9 µg/mL) exhibit similar trypanocidal effects to Benznidazole (BZN) (IC50 = 48 ± 2.5 µg/mL), against amastigotes. Synergistic antiparasitic activity was observed when ACT1 was combined with BZN (∑FIC = 0.52 ± 0.13 µg/mL) or ACT2 (∑FIC = 0.46 ± 1.7 µg/mL). ACT1 also decreased the oxidative stress, mitochondrial metabolism, and genotoxicity of the therapies. The ACT1 + ACT2 and ACT1 + BZN experimental treatments reduced the pro-inflammatory cytokines (IFN-γ, IL-2, and TNF-α) and increased the anti-inflammatory cytokines (IL-4 and IL-10). CONCLUSION: Due to its highly trypanocidal and immunomodulatory properties, ACT1 (whether alone or in combination with BZN or ACT2) represents a promising L. alba essential oil fraction for further studies in drug development towards the Chagas disease control.

Tubastatin A, a deacetylase inhibitor, as a tool to study the division, cell cycle and microtubule cytoskeleton of trypanosomatids.

Trypanosoma cruzi is a protozoan of great medical interest since it is the causative agent of Chagas disease, an endemic condition in Latin America. This parasite undergoes epigenetic events, such as phosphorylation, methylation and acetylation, which play a role in several cellular processes including replication, transcription and gene expression. Histone deacetylases (HDAC) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. Tubastatin A (TST) is a specific HDAC6 inhibitor that affects cell growth and promotes structural modifications in cancer cells and parasites. In the present study, we demonstrated that T. cruzi epimastigote cell proliferation and viability are reduced after 72 h of TST treatment. The results obtained through different microscopy methodologies suggest that this inhibitor impairs the polymerization dynamics of cytoskeleton microtubules, generating protozoa displaying atypical morphology and cellular patterns that include polynucleated parasites. Furthermore, the microtubules of treated protozoa were more intensely acetylated, especially at the anterior portion of the cell body. A cell cycle analysis demonstrated an increase in the number of trypanosomatids in the G2/M phase. Together, our results suggest that TST should be explored as a tool to study trypanosomatid cell biology, including microtubule cytoskeleton dynamics, and as an antiparasitic drug.

Apoptosis-like cell death upon kinetoplastid induction by compounds isolated from the brown algae Dictyota spiralis.

BACKGROUND: The in vitro activity of the brown seaweed Dictyota spiralis against both Leishmania amazonensis and Trypanosoma cruzi was evaluated in a previous study. Processing by bio-guided fractionation resulted in the isolation of three active compounds, classified as diterpenes. In the present study, we performed several assays to detect clinical features associated to cell death in L. amazonensis and T. cruzi with the aim to elucidate the mechanism of action of these compounds on parasitic cells. METHODS: The aims of the experiments were to detect and evaluate specific events involved in apoptosis-like cell death in the kinetoplastid, including DNA condensation, accumulation of reactive oxygen species and changes in ATP concentration, cell permeability and mitochondrial membrane potential, respectively, in treated cells. RESULTS: The results demonstrated that the three isolated diterpenes could inhibit the tested parasites by inducing an apoptosis-like cell death. CONCLUSIONS: These results encourage further investigation on the isolated compounds as potential drug candidates against both L. amazonensis and T. cruzi.

Quantitative proteomics and phosphoproteomics of Trypanosoma cruzi epimastigote cell cycle.

The protozoan Trypanosoma cruzi is the causative agent of the neglected infectious illness Chagas disease. During its life cycle it differentiates into replicative and non-replicative life stages. So far, T. cruzi cell division has been investigated by transcriptomics but not by proteomics approaches. Here we show the first quantitative proteome analysis of T. cruzi cell division. T. cruzi epimastigote cultures were subject to synchronization with hydroxyurea and harvested at different time points. Analysis by flow cytometry, bright field and fluorescence microscopy indicated that samples collected at 0 h, 2 h, 6 h and 14 h overrepresented G1, G1-S, S and M cell cycle phases, respectively. After trypsin digestion of these samples, the resulting peptides were labelled with iTRAQ and subjected to LC-MS/MS. Also, iTRAQ-labelled phosphopeptides were enriched with TiO2 to access the phosphoproteome. Overall, 597 protein groups and 94 phosphopeptides presented regulation with the most remarkable variation in abundance at 6 h (S-phase). Comparison of our proteomic data to previous transcriptome-wise analysis of epimastigote cell cycle showed 16 sequence entries in common, with the highest mRNA/protein correlation observed in transcripts with peak abundance in G1-phase. Our data revealed regulated proteins and phosphopeptides which play important roles in the control of cell division in other organisms and some of them were previously detected in the nucleus or associated with T. cruzi chromatin.

Nucleosome landscape reflects phenotypic differences in Trypanosoma cruzi life forms.

Trypanosoma cruzi alternates between replicative and nonreplicative life forms, accompanied by a shift in global transcription levels and by changes in the nuclear architecture, the chromatin proteome and histone posttranslational modifications. To gain further insights into the epigenetic regulation that accompanies life form changes, we performed genome-wide high-resolution nucleosome mapping using two T. cruzi life forms (epimastigotes and cellular trypomastigotes). By combining a powerful pipeline that allowed us to faithfully compare nucleosome positioning and occupancy, more than 125 thousand nucleosomes were mapped, and approximately 20% of them differed between replicative and nonreplicative forms. The nonreplicative forms have less dynamic nucleosomes, possibly reflecting their lower global transcription levels and DNA replication arrest. However, dynamic nucleosomes are enriched at nonreplicative regulatory transcription initiation regions and at multigenic family members, which are associated with infective-stage and virulence factors. Strikingly, dynamic nucleosome regions are associated with GO terms related to nuclear division, translation, gene regulation and metabolism and, notably, associated with transcripts with different expression levels among life forms. Finally, the nucleosome landscape reflects the steady-state transcription expression: more abundant genes have a more deeply nucleosome-depleted region at putative 5' splice sites, likely associated with trans-splicing efficiency. Taken together, our results indicate that chromatin architecture, defined primarily by nucleosome positioning and occupancy, reflects the phenotypic differences found among T. cruzi life forms despite the lack of a canonical transcriptional control context.

Starvation and pH stress conditions induced mitochondrial dysfunction, ROS production and autophagy in Trypanosoma cruzi epimastigotes.

Chagas disease is a neglected illness endemic in Latin America that mainly affects rural populations. The etiological agent of Chagas disease is the protozoan Trypanosoma cruzi, which has three different parasite stages and a dixenous life cycle that includes colonization of the vertebrate and invertebrate hosts. During its life cycle, T. cruzi is subjected to stress conditions, including variations in nutrient availability and pH, which impact parasite survival and differentiation. The plasticity of mitochondrial function in trypanosomatids has been defined as mitochondrial activity related to substrate availability. Thus, mitochondrial remodeling and autophagy, which is a constitutive cellular process of turnover and recycling of cellular components, may constitute a response to the nutritional and pH stress in the host. To assess these processes, epimastigotes were subjected to acidic, alkaline, and nutritional stress conditions, and mitochondrial function and its influence on the autophagic process were evaluated. Our data demonstrated that the three stress conditions affected the mitochondrial structure, inducing organelle swelling and impaired oxidative phosphorylation. Stressed epimastigotes produced increased ROS levels and overexpressed antioxidant enzymes. The stress conditions resulted in an increase in the number of autophagosomes and exacerbated the expression of different autophagy-related genes (Atgs). A correlation between mitochondrial dysfunction and autophagic phenotypes was also observed. After 24 h, acid stress and nutritional deprivation induced metacyclogenesis phenotypes (mitochondrial remodeling and autophagy). On the other hand, alkaline stress was transient due to insect blood feeding and culminated in an increase in autophagic flux as a survival mechanism.

Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis.

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoans Leishmania tarentolae and Trypanosoma cruzi mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

New perspectives for hydrogen peroxide in the amastigogenesis of Trypanosoma cruzi in vitro.

Trypanosoma cruzi has a complex life cycle involving four life stages: the replicative epimastigotes and metacyclic trypomastigotes in the invertebrate host digestive tract, and intracellular amastigotes and bloodstream trypomastigotes in the mammalian host. Trypomastigotes can invade any nucleated cell, including macrophages, which produce ROS that enhance intracellular infection. However, how ROS modulate T. cruzi infection in the mammalian cell remains unclear. Therefore, the present work aimed to investigate the role of ROS during the stimulation of amastigogenesis in vitro. Our results showed that H2O2 improves the differentiation process in vitro and that it was impaired by Peg-Catalase. However, the antioxidants GSH and NAC had no influence on induced amastigogenesis, which suggests the specificity of H2O2 to increase intracellular differentiation. Amastigogenesis physiologically occurs in low pH, thus we investigated whether parasites are able to produce ROS during amastigogenesis. Interestingly, after 60 min of differentiation induction in vitro, we observed an increase in H2O2 production, which was inhibited by the mitochondrial-targeted antioxidant, mitoTEMPO and Cyclosporine A (a mitochondrial permeability transition pore -mPTP- inhibitor), suggesting mitochondrion as a H2O2 source. Indeed, quantitative real time (qPCR) showed an increase of the mitochondrial superoxide dismutase (FeSODA) gene expression after 60 min of induced amastigogenesis, reinforcing the hypothesis of mitochondrial ROS induction during intracellular differentiation of T. cruzi. The reduction of cellular respiration and the decreased ΔΨm observed during amastigogenesis can explain the increased mitochondrial ROS through mPTP opening. In conclusion, our results suggest that H2O2 is involved in the amastigogenesis of T. cruzi.

Mammea type coumarins isolated from Calophyllum brasiliense induced apoptotic cell death of Trypanosoma cruzi through mitochondrial dysfunction, ROS production and cell cycle alterations.

Chagas Disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi which affects 6-8 million people, mostly in Latin America. The medical treatment is based on two nitroimidazole compounds, which have limited effectiveness in the chronic phase of the disease and produce several adverse effects; consequently, there is an urgent need to develop new, safe, and effective drugs. Previous reports had shown that natural coumarins, especially mammea A/BA isolated from the tropical tree Calophyllum brasiliense, is a promissory molecule for developing new drugs, due to its potent activity, higher than benznidazole, selectivity, and its low toxicity in mice. However, its mode of action is still unknown. In the present work, we evaluated the mechanism of action of the coumarin mammea A/BA (93.6%), isolated from the tropical tree C. brasiliense on Querétaro strain (Tc1) of T. cruzi. This compound was tested in vitro on epimastigotes and trypomastigotes of T. cruzi for intracellular esterase activity, plasma membrane integrity, phosphatidylserine exposure, ROS production, mitochondrial membrane potential, caspase-like activity, DNA integrity, cell cycle and autophagy. Mammea A/BA showed a 50% lethal concentration (LC50) of 85.8 and 36.9 µM for epimastigotes and trypomastigotes respectively. It affected intracellular esterase activity, produced important plasma membrane damage and induced phosphatidylserine exposure. An increase in reactive oxygen species (ROS) and decrease in mitochondrial membrane potential were detected. Caspase-like activity was present in both parasite forms producing DNA integrity damage. This compound also induced a cell cycle arrest in the G1 phase and the presence of autophagy vacuoles. The above data suggest that mammea A/BA induce cell death of T. cruzi by autophagy and apoptosis-like phenomena and support our suggestion that mammea A/BA could be a promising molecule for the development of new drugs to treat Chagas Disease.

ELISA saliva for trypanosoma cruzi antibody detection: An alternative for serological surveys in endemic regions

Chagas is a neglected disease endemic in Latin America. Vector transmission control had been aggressively performed. Recent entomological surveillance in Brazil has revealed natural infection rates ranging from 0.40% to 0.52%. Although serological surveys are complex to develop, they are important for disease control. In this study, we validated the use of saliva in ELISA commercial kits with a cohort of 100 patients with Chagas disease followed at Hospital das Clinicas in São Paulo, Brazil, and 50 healthy controls. Five ELISA kits for detecting antibodies against Trypanosoma cruzi were tested. The best discrimination between Chagas patients and controls was observed with the Wiener kit, which yielded a sensitivity of 97% and a specificity of 100%. Our findings reveal that the use of saliva may be an alternative to large-scale screening surveys in detecting T. cruzi antibodies; it is a noninvasive sample collection method potentially key to large-scale screening in children

Isolation of Glycosomes from Trypanosoma cruzi.

Glycosomes are peroxisome-related organelles of trypanosomatids in which the glycolytic and some other metabolic pathways are compartmentalized. We describe here two methods for the purification of glycosomes from Trypanosoma cruzi for preparative purposes, differential and isopycnic centrifugation. These are two techniques that allow the separation of different cellular compartments based on their different physicochemical characteristics. The first type of centrifugation is a rapid method that does not require large inputs and allows for fractions enriched in specific cell compartments to be obtained. The second type of centrifugation is a more elaborate method, but enables highly purified cellular compartments to be isolated. The success in obtaining these purified, intact organelles critically depends on using an appropriate method for controlled rupture of the cells.

Measurement of Energy States of the Trypanosomatid Mitochondrion.

The evaluation of mitochondrial functionality is critical to interpret most biological data at the (eukaryotic) cellular level. For example, metabolism, cell cycle, epigenetic regulation, cell death mechanisms, autophagy, differentiation, and response redox imbalance are dependent on the mitochondrial state. In case of parasitic organisms, such as trypanosomatids, it is very often important to have information on mitochondrial functionality in order to assess the mechanisms of actions of drugs being proposed for therapy. In this chapter we present a set of methods that together allow to evaluate with some precision the mitochondrial functionality in Trypanosoma cruzi and Trypanosoma brucei. We discuss how to determine O2 consumption, mitochondrial inner membrane potential, ATP production, and the endogenous production of reactive oxygen species.

The Influence of Recombinational Processes to Induce Dormancy in Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects around 8 million people worldwide. Chagas disease can be divided into two stages: an acute stage with high parasitemia followed by a low parasitemia chronic stage. Recently, the importance of dormancy concerning drug resistance in T. cruzi amastigotes has been shown. Here, we quantify the percentage of dormant parasites from different T. cruzi DTUs during their replicative epimastigote and amastigote stages. For this study, cells of T. cruzi CL Brener (DTU TcVI); Bug (DTU TcV); Y (DTU TcII); and Dm28c (DTU TcI) were used. In order to determine the proliferation rate and percentage of dormancy in epimastigotes, fluorescent-labeled cells were collected every 24 h for flow cytometer analysis, and cells showing maximum fluorescence after 144 h of growth were considered dormant. For the quantification of dormant amastigotes, fluorescent-labeled trypomastigotes were used for infection of LLC-MK2 cells. The number of amastigotes per infected LLC-MK2 cell was determined, and those parasites that presented fluorescent staining after 96 h of infection were considered dormant. A higher number of dormant cells was observed in hybrid strains when compared to non-hybrid strains for both epimastigote and amastigote forms. In order to investigate, the involvement of homologous recombination in the determination of dormancy in T. cruzi, we treated CL Brener cells with gamma radiation, which generates DNA lesions repaired by this process. Interestingly, the dormancy percentage was increased in gamma-irradiated cells. Since, we have previously shown that naturally-occurring hybrid T. cruzi strains present higher transcription of RAD51-a key gene in recombination process -we also measured the percentage of dormant cells from T. cruzi clone CL Brener harboring single knockout for RAD51. Our results showed a significative reduction of dormant cells in this T. cruzi CL Brener RAD51 mutant, evidencing a role of homologous recombination in the process of dormancy in this parasite. Altogether, our data suggest the existence of an adaptive difference between T. cruzi strains to generate dormant cells, and that homologous recombination may be important for dormancy in this parasite.

Nuclear export of replication protein A in the nonreplicative infective forms of Trypanosoma cruzi.

Replication protein A (RPA), a heterotrimeric complex, is the major single-stranded DNA binding protein in eukaryotes. Recently, we characterized RPA from Trypanosoma cruzi, showing that it is involved in DNA replication and DNA damage response in this organism. Better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms was observed in TcRPA-2 subunit heterozygous knockout cells, suggesting that RPA is involved in this process. Here, we show that RPA cellular localization changes during the T. cruzi life cycle, with RPA being detected only in the cytoplasm of the metacyclic and bloodstream trypomastigotes. We also identify a nuclear export signal (NES) in the trypanosomatid RPA-2 subunit. Mutations in the negatively charged residues of RPA-2 NES impair the differentiation process, suggesting that RPA exportation affects parasite differentiation into infective forms.

Extracellular vesicles isolated from Trypanosoma cruzi affect early parasite migration in the gut of Rhodnius prolixus but not in Triatoma infestans.

The protozoan Trypanosoma cruzi has the ability to spontaneously secrete extracellular vesicles (EVs). In this paper, T. cruzi EVs derived from epimastigote forms were evaluated during interaction with triatomine bugs Rhodnius prolixus and Triatoma infestans. T. cruzi EVs were purified and artificially offered to the insects prior to infection with epimastigote forms. No effect of EVs was detected in the parasite counts in the guts of both vectors after 49-50 days. On the other hand, pre-feeding with EVs delayed parasite migration to rectum only in the gut in R. prolixus after 21-22 days. Those data suggest a possible role of T. cruzi EVs during the earlier events of infection in the invertebrate host.

Autophagic elimination of Trypanosoma cruzi in the presence of metals.

Trypanosoma cruzi is an obligate intracellular parasite transmitted to vertebrate hosts by blood-sucking insects. Molecules present in parasites and mammalian cells allow the recognition and parasite internalization. Metallic ions play an essential role in the establishment and maintenance of host-parasite interaction. However, little is known about how parasites handle with essential and nonessential metal quotas. This study aimed to investigate the influence of metal ions on the biological processes of T. cruzi infected cells. Infected cells were incubated with ZnCl2, CdCl2, and HgCl2 for 12 h and labeled with different specific dyes to investigate the cellular events related to intracellular parasite death and elimination. Infected host cells and parasite's mitochondria underwent functional and structural disorders, in addition to parasite's DNA condensation and pH decrease on host cells, which led to parasite death. Further investigations suggested that lysosomes were involved in pH decrease and the double membrane of the endoplasmic reticulum formed vacuoles surrounding damaged parasites, which indicate the occurrence of autophagy for parasite elimination. In conclusion, low concentrations of nonessential and essential metals cause a series of damage to Trypanosoma cruzi organelles, leading to its loss of viability, death, and elimination, with no removal of the host cells.

Cytotaxonomy of Trypanosoma cruzi (Chagas, 1909): Differentiation of T. cruzi I (TcI) and T. cruzi II (TcII) Genotypes Using Cytogenetic Markers.

Chagas disease is a public health problem caused by the Trypanosoma cruzi, and the T. cruzi I (TcI) and T. cruzi II (TcII) groups are considered important genotypes from the clinical point of view. Currently, the groups need to be molecularly analyzed for their identification; thus, we cytogenetically analyzed these groups with the objective of developing more accessible techniques for the characterization of these parasites. TcI and TcII groups were differentiated by nucleus characterization with lacto-acetic orcein (TcI-nucleus with positive heteropycnosis and TcII-nucleus with negative heteropycnosis), emphasizing the importance of the application of this technique for epidemiological and clinical studies of Chagas disease.

Trypanosoma cruzi actins: Expression analysis of actin 2.

The genome of Trypanosoma cruzi encodes for an expanded number of actins, myosins and actin binding proteins compared to Trypanosoma brucei or Leishmania spp. In T. cruzi only the expression of actin 1 (i.e. conventional actin) and profilin, an actin binding protein, has been described. In this work, the expression of a kinetoplastid-specific actin, named actin 2 (TcAct2; TriTryp Gene ID: TcCLB.507129.10) was characterized in different developmental stages of T. cruzi. With the aid of a polyclonal antibody, we showed that TcAct2 is expressed throughout the life cycle of the parasite. Detergent fractionation of epimastigote extracts showed that this protein is cytosolic and is not associated with membrane or cytoskeletal fractions. The protein is localized along the cellular body and the flagellum in all parasite stages with a fine granular pattern and does not co-localize with actin 1. 2DE-immunoblotting studies demonstrated the presence of several variants of each actin. We also demonstrate that TcAct1 and TcAct2 have distinct subcellular distributions suggesting differential functions in this organism. The search of TcAct2 orthologues in the TriTrypDB, allowed the identification of this gene in other trypanosomatids, all of them restricted to the stercorarian clade. In addition, TcAct2 was also identified in the closely related non-trypanosomatid species Bodo saltans. Our findings are consistent with the appearance of a complex actin system early in the evolution of kinetoplastids.

Transmigration of Trypanosoma cruzi Trypomastigotes through 3D Spheroids Mimicking Host Tissues.

While cellular invasion by T. cruzi trypomastigotes and intracellular amastigote replication are well-characterized events that have been described by using 2D monolayer cultures, other relevant parasite-host interactions, like the dynamics of tissue invasiveness, cannot be captured using monolayer cultures. Spheroids constitute a valuable three-dimensional (3D) culture system because they mimic the microarchitecture of tissues and provide an environment similar to the encountered in natural infections, which includes the presence of extracellular matrix as well as 3D cell-cell interactions. In this work, we describe a protocol for studying transmigration of T. cruzi trypomastigotes into 3D spheroids. In the experimental setup, cells and parasites are labelled with two fluorescent dyes, allowing their visualization by confocal microscopy. We also describe the general procedure and setting of the confocal microscope and downstream applications for acquisition and reconstruction of 3D images. This model was employed to analyze the transmigration of trypomastigotes from the highly virulent and pantropic RA T. cruzi strain. Of course, other aspects encountered by T. cruzi in the mammalian host environment can be studied with this methodology.