Trypanosoma cruzi: single cell live imaging inside infected tissues

Although imaging the live Trypanosoma cruzi parasite is a routine technique in most laboratories, identification of the parasite in infected tissues and organs has been hindered by their intrinsic opaque nature. We describe a simple method for in vivo observation of live single‐cell Trypanosoma cruzi parasites inside mammalian host tissues. BALB/c or C57BL/6 mice infected with DsRed‐CL or GFP‐G trypomastigotes had their organs removed and sectioned with surgical blades. Ex vivo organ sections were observed under confocal microscopy. For the first time, this procedure enabled imaging of individual amastigotes, intermediate forms and motile trypomastigotes within infected tissues of mammalian hosts.     

Biochemical Requirements for Intracellular Invasion by Trypanosoma cruzi: Protein Synthesis1

The effects of irreversible inhibition of protein synthesis by pactamycin in either infective forms of Trypanosoma cruzi or mammalian host cells on cellular invasion by this human pathogen were investigated. Treatment of bloodstream forms of T. cruzi with pactamycin markedly reduced their ability to bind either fibroblast-like cells of monkey origin or myoblasts of rat origin. The number of amastigote forms that could be established intracellularly was also significantly decreased with respect to control values obtained when mock-treated (medium alone) trypomastigotes were incubated with the cells. Pactamycin treatment also reduced the infectivity of T. cruzi trypomastigotes for mice as evidenced by both significantly reduced parasitemia levels and mortality rates when compared with those of control mice infected with mock-treated parasites. Inhibition of protein synthesis in the host cells neither prevented cell infection by untreated trypomastigotes nor altered the percentages of infected cells or the magnitude of the infection in vitro. These results indicate that protein synthesis is a requirement for cell invasion by T. cruzi and that the parasite can establish itself and replicate within cells relying on its own protein synthesis ability.     

Exacerbation of Experimental Trypanosoma cruzi Infection in Mice by Concomitant Malaria*

SYNOPSIS. The effect of malaria on the chronic phase of Chagas’disease was investigated in mice. The animals were given Plasmodium berghei-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y, CL and Gilmar). In all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas’disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curve of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phase with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Interaction between cFLIPL and Itch,a ubiquitin ligase,is obstructed in Trypanosoma cruzi‐infected human cells

Death receptor‐mediated host cell apoptosis, a defense strategy for elimination by the immune system of parasite‐infected cells, is inhibited by Trypanosoma cruzi, the causative agent of Chagas' disease. It has previously been reported by us that, in infected cells, T. cruzi upregulates and exploits cFLIP_L, a mammalian inhibitor of death receptor signaling. Here it is shown that ubiquitination of cFLIP_L, leading to proteasomal degradation, is inhibited in parasite‐infected cells. The extent of expression of Itch, a protein thought to be an ubiquitin ligase for cFLIP_L, was found to be equivalent in T. cruzi‐infected and in uninfected cells. However, co‐immunoprecipitation analysis showed that the interaction between cFLIP_L and Itch is strongly inhibited in T. cruzi‐infected cells. This unique parasite strategy, which has not been reported in any other pathogen‐infected cells, may allow the host cell to accumulate cFLIP_L, eventually resulting in the inhibition of apoptosis of T. cruzi‐infected cells.     

Plasmodium berghei sporozoites in nonreplicative vacuole are eliminated by a PI3P‐mediated autophagy‐independent pathway

The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium‐associated autophagy‐related (PAAR) response. This is characterised by a long‐lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3‐phosphate (PI3P)‐labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P‐labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3‐labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites.     

Inefficient Complement System Clearance of Trypanosoma cruzi Metacyclic Trypomastigotes Enables Resistant Strains to Invade Eukaryotic Cells

The complement system is the main arm of the vertebrate innate immune system against pathogen infection. For the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, subverting the complement system and invading the host cells is crucial to succeed in infection. However, little attention has focused on whether the complement system can effectively control T. cruzi infection. To address this question, we decided to analyse: 1) which complement pathways are activated by T. cruzi using strains isolated from different hosts, 2) the capacity of these strains to resist the complement-mediated killing at nearly physiological conditions, and 3) whether the complement system could limit or control T. cruzi invasion of eukaryotic cells. The complement activating molecules C1q, C3, mannan-binding lectin and ficolins bound to all strains analysed; however, C3b and C4b deposition assays revealed that T. cruzi activates mainly the lectin and alternative complement pathways in non-immune human serum. Strikingly, we detected that metacyclic trypomastigotes of some T. cruzi strains were highly susceptible to complement-mediated killing in non-immune serum, while other strains were resistant. Furthermore, the rate of parasite invasion in eukaryotic cells was decreased by non-immune serum. Altogether, these results establish that the complement system recognizes T. cruzi metacyclic trypomastigotes, resulting in killing of susceptible strains. The complement system, therefore, acts as a physiological barrier which resistant strains have to evade for successful host infection.     

A comparative assessment of mitochondrial function in epimastigotes and bloodstream trypomastigotes of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Trypanosoma cruzi is a hemoflagellate protozoan that causes Chagas’ disease. The life cycle of T. cruzi is complex and involves different evolutive forms that have to encounter different environmental conditions provided by the host. Herein, we performed a functional assessment of mitochondrial metabolism in the following two distinct evolutive forms of T. cruzi: the insect stage epimastigote and the freshly isolated bloodstream trypomastigote. We observed that in comparison to epimastigotes, bloodstream trypomastigotes facilitate the entry of electrons into the electron transport chain by increasing complex II-III activity. Interestingly, cytochrome c oxidase (CCO) activity and the expression of CCO subunit IV were reduced in bloodstream forms, creating an “electron bottleneck” that favored an increase in electron leakage and H₂O₂ formation. We propose that the oxidative preconditioning provided by this mechanism confers protection to bloodstream trypomastigotes against the host immune system. In this scenario, mitochondrial remodeling during the T. cruzi life cycle may represent a key metabolic adaptation for parasite survival in different hosts.     

A Monoclonal Antibody to Alpha Tubulin Recognizes Host Cell and Trypanosoma cruzi Tubulins1

ABSTRACT. A mouse monoclonal anti-α-tubulin antibody was used to investigate the disposition of the cytoskeletal microtubules of three tissue culture cell lines–J774 macrophages, BSC-1, and Vero cells–infected with the Brazil strain of Trypanosoma cruzi. Indirect immunofluorescence light microscopy was used to demonstrate the antigenic response in host cells and parasites, simultaneously. In all morphotypes of T. cruzi, the monoclonal antibody reacted with all subpopulations of microtubules, inclusively, the subpellicular, flagellar, cytopharyngeal, and mitotic. The host cell cytoskeletal microtubule framework was revealed and the redistribution and destruction of the microtubular lattice in response to parasite infection over a 120 h period recorded. Our results show that after the initial inoculation of tissue cultures with trypomastigotes, the parasites penetrate the cells and locate in the perinuclear region of the cell where they multiply. The number and distribution of host cell microtubules were altered during the infection. The normal radial distribution of microtubules extending from the center of the cell to the periphery was destroyed. The remaining microtubules were observed at the periphery encircling, but well removed from the proliferating parasites. The complete transformation of the parasites was monitored throughout the infection with the end result being the liberation of parasites and the near complete destruction of the microtubular framework of the host cell. A residual population of dividing spheromastigotes was observed in cells liberating trypomastigotes. Colloidal gold labeling of thin sections as seen in the electron microscope affirmed the specificity of our monoclonal antibody to all subpopulations of microtubules in T. cruzi.     

The MASP Family of Trypanosoma cruzi: Changes in Gene Expression and Antigenic Profile during the Acute Phase of Experimental Infection

Background

Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host–parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms.

Methods

By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice.

Findings and Conclusions

We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.     

Attenuation of Parasite cAMP Levels in T. cruzi-Host Cell Membrane Interactions In Vitro

Previous investigations have shown that the adhesion of T. cruzi plasma membrane vesicles (PMV) to monolayers of host cell myoblasts and to immobilized heart muscle sarcolemma membranes (PAM) on polyaerylamide beads is mediated by the interaction of T. cruzi attachment sites with the muscarinic cholinergic and β-adrenergic receptors of the host cell membrane. It has also been shown that this interaction is blunted by the specific antagonists of the mammalian receptors atropine and propranol, respectively. In the studies reported here, PAM also rapidly attached to swimming T. cruzi trypomastigotes in a complex, concentration-dependent fashion and binding isotherms showed that the equilibrium between free and bound PAM is rapidly reached within 2 minutes of incubation in physiologically balanced salt solutions. In this time frame, trypomastigote cAMP levels are significantly reduced from steady state values within 30 seconds of the addition of PAM in a buffer system containing a diesterase inhibitor. Maximal attenuation of cAMP levels was measured between 1 and 2 minutes of the addition of PAM to T. cruzi trypomastigotes. The degree of cAMP level attenuation was reduced by blocking PAM attachment with either atropine or propranol. On the basis of these results we propose that a likely pathway for the negative parasite signal generated upon adhesion of host muscle cell membranes to the surface of the flagellates is from the parasite's surface attachment sites directly to a Pertussis toxin sensitive inhibitory protein G_i, thereby blunting adenyl cyclase activity and cAMP formation.     

All <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> developmental forms present lysosome-related organelles

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidified by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have different size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.     

Cryopreservation of Trypanosoma cruzi Bloodstream Forms*

SYNOPSIS. Recovery rates of T. cruzi bloodstream forms subjected to several methods of cryopreservation in liquid nitrogen and at -73 C are reported. Inoculations of animals with cryopreserved and nonpreserved trypomastigotes revealed that prolonged storage at -196 C apparently did not change the biologic characteristics of different T. cruzi strains. The reproducibility and consistency of results suggest that “cryobanks'’or “reference centers'’may be established.     

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.     

Trypanosoma cruzi: Histochemical Characterization of Parasitized Skeletal Muscle Fibers1

C57B1/6 mice were infected with Brasil strain Trypanosoma cruzi trypomastigotes. The leg muscles of the mice were serial-sectioned with a cryostat, and individual fibers were classified histochemically as type I or type II on the basis of succinic dehydrogenase or adenosine triphosphatase activity. Although markedly more type II fibers were present in the leg muscles, the percentage of infected type I fibers was nearly five-fold higher than type II. This is the first demonstration of a preferential in vivo distribution of T. cruzi in muscle fibers based upon muscle type.     

Trypanosoma cruzi: desferrioxamine decreases mortality and parasitemia in infected mice through a trypanostatic effect

Desferrioxamine (DFO) is a potent iron chelator that is also known to modulate inflammation and act as an efficient antioxidant under normal conditions and under oxidative stress. Many in vitro and in vivo studies have shown the efficacy of DFO in the treatment of viral, bacterial and protozoan infections. DFO is known to reduce the intensity of Trypanosoma cruzi infections in mice even during a course of therapy that is not effective in maintaining anaemia or low iron levels. To further clarify these findings, we investigated the action of DFO on mouse T. cruzi infection outcomes and the direct impact of DFO on parasites.Infected animals treated with DFO (5 mg/animal/day) for 35 days, beginning 14 days prior to infection, presented lower parasitemia and lower cumulative mortality rate. No significant effect was observed on iron metabolism markers, erythrograms, leukograms or lymphocyte subsets.In the rapid method for testing in vivo T. cruzi susceptibility, DFO also induced lower parasitemia.In regard to its direct impact on parasites, DFO slightly inhibited the growth of amastigotes and trypomastigotes in fibroblast culture. Trypan blue staining showed no effects of DFO on parasite viability, and only minor apoptosis in trypomastigotes was observed. Nevertheless, a clear decrease in parasite mobility was detected.In conclusion, the beneficial actions of DFO on mice T. cruzi infection seem to be independent of host iron metabolism and free of significant haematological side effects. Through direct action on the parasite, DFO has more effective trypanostatic than trypanocidal properties.     

T lymphocytes migrate upstream after completing the leukocyte adhesion cascade

The leukocyte adhesion cascade is of critical importance for both the maintenance of immune homeostasis and the ability of immune cells to perform effector functions. Here, we present data showing CD4⁺ T cells migrate upstream (against the direction of flow) after completing the leukocyte adhesion cascade on surfaces displaying either ICAM-1 or ICAM-1 and VCAM-1, but migrate downstream on surfaces displaying only VCAM-1. Cells completing the cascade on HUVECs initially migrate upstream before reverting to more random migration, partly caused by transmigration of cells migrating against the flow. Furthermore, cells migrating upstream transmigrate faster than cells migrating downstream. On HUVECs, blocking interactions between LFA-1 and ICAM-1 resulted in downstream migration and slower transmigration. These results further suggest a possible physiological role for upstream migration in vivo.     

Location and expression kinetics of Tc24 in different life stages of Trypanosoma cruzi

Tc24-C4, a modified recombinant flagellar calcium-binding protein of Trypanosoma cruzi, is under development as a therapeutic subunit vaccine candidate to prevent or delay progression of chronic Chagasic cardiomyopathy. When combined with Toll-like receptor agonists, Tc24-C4 immunization reduces parasitemia, parasites in cardiac tissue, and cardiac fibrosis and inflammation in animal models. To support further research on the vaccine candidate and its mechanism of action, murine monoclonal antibodies (mAbs) against Tc24-C4 were generated. Here, we report new findings made with mAb Tc24-C4/884 that detects Tc24-WT and Tc24-C4, as well as native Tc24 in T. cruzi on ELISA, western blots, and different imaging techniques. Surprisingly, detection of Tc24 by Tc24-C/884 in fixed T. cruzi trypomastigotes required permeabilization of the parasite, revealing that Tc24 is not exposed on the surface of T. cruzi, making a direct role of antibodies in the induced protection after Tc24-C4 immunization less likely. We further observed that after immunostaining T. cruzi–infected cells with mAb Tc24-C4/884, the expression of Tc24 decreases significantly when T. cruzi trypomastigotes enter host cells and transform into amastigotes. However, Tc24 is then upregulated in association with parasite flagellar growth linked to re-transformation into the trypomastigote form, prior to host cellular escape. These observations are discussed in the context of potential mechanisms of vaccine immunity.     

Therapeutical effects of vaccine from Trypanosoma cruzi amastigote surface protein 2 by simultaneous inoculation with live parasites

The aim of this study was to evaluate the efficacy of vaccine using replication-deficient human recombinant Type 5 replication-defective adenoviruses (AdHu5) carrying sequences of the amastigote surface protein 2 (ASP2) (AdASP2) in mice infected with the Trypanosoma cruzi ( T cruzi) Y strain. A total of 16 A/Sn mice female were distributed into four groups, as follows (n = 4 per group): Group 1 – Control Group (CTRL); Group 2 – Infected Group (TC): animals were infected by subcutaneous route with 150 bloodstream trypomastigotes of T cruzi Y strain; Group 3 – Immunized Group (AdASP-2): animals were immunized by intramuscular injection (im) route with 50 µL of AdSP-2 (2 × 10 ⁸ plaque forming units [pfu]/cam) at day 0; Group 4-Immunized and Infected Group (AdASP-2+TC): animals were immunized by im route with 50 µL of ASP-2 (2 × 10 ⁸ pfu/cam) and infected by T cruzi at the same day (day 0). It was observed a significant decrease of nests in the group that was immunized with AdASP-2 and infected on the same day. Tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) gene expressions showed a significant increase in the AdASP-2+TC group when compared to TC group, but it was noted that Cyclooxygenase-2 (Cox-2) was increased in TC group when compared to AdASP-2+TC group. Increase of matrix metalloproteinases-2 (MMP-2) and decrease of MMP-9 immunoexpression in the AdASP-2+TC group was noticed as well. Oxidative DNA damage was present in myocardium for AdASP-2+TC group as a result of 8-hydroxydeoxyguanosine immunoexpression. Taken together, our results highlighted an increased oxidative stress, MMP-2 activity and inflammatory host response promoted by AdASP-2 against T cruzi infection.