Activation of distinct signal transduction pathways in Trypanosoma cruzi isolates with differential capacity to invade host cells

Mammalian cell invasion by Trypanosoma cruzi requires the activation of signal transduction pathways that result in a Ca(2+) response both in the parasite and the host cell. By using drugs that interfere with the signalling processes, we investigated if the difference in the ability of T. cruzi isolates to invade host cells was associated with the activation of distinct signalling routes in the parasites. Experiments were performed with metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, using the highly invasive isolate CL and the poorly infective isolate G, which belong to distinct phylogenetic lineages. Treatment of parasites with adenylyl cyclase activator forskolin increased the infectivity of the G but not of the CL isolate towards HeLa cells. On the other hand, a specific protein tyrosine kinase inhibitor genistein reduced by approximately 75% the penetration of CL but not of G isolate into HeLa cells. In the CL but not in the G isolate, protein tyrosine kinase mediated the phosphorylation of a 175kDa protein in a manner inducible by a HeLa cell extract. Upon treatment with the phospholipase C inhibitor U73122, or with drugs such as caffeine, which affects Ca(2+) release from inositol-1,4,5-triphosphate-sensitive stores, or thapsigargin, an inhibitor of intracellular Ca(2+) transport ATPases, the infectivity of the CL but not of the G isolate diminished significantly (P<0.005). In both isolates, a combination of ionomycin plus NH(4)Cl or nigericin released Ca(2+) from acidic vacuoles containing a Ca(2+)/H(+) exchange system. This treatment reduced the infectivity of metacyclic forms of the G but not of the CL isolate. Taken together, these data suggest that, for host cell invasion, distinct signalling pathways are activated in metacyclic trypomastigotes of the two isolates, leading to Ca(2+) release from different intracellular compartments.     

Cultivation of Trypanosoma cruzi epimastigotes in low glucose axenic media shifts its competence to differentiate at metacyclic trypomastigotes

This study offers an insight into why Trypanosoma cruzi epimastigotes lose their capacity to differentiate into metacyclic forms, if maintained in culture media long-term through serial passages. The biological and metabolic behaviour of two T. cruzi strains isolated from various origins (human, opossum), and maintained under two schedules (alternate triatomine/mouse passages and serial culture media) were compared. To determine the effect of the environment on the parasites, the epimastigotes were grown under extreme conditions (high and low glucose concentrations), and the glucose consumption, ammonia production and changes in pH, either in one compartment (along the growth curve) or two compartments (induced metacyclogenesis) were compared. The glucose effect on the stages involved in metacyclogenesis at antigenic level was also evaluated. The results indicate that T. cruzi adapts to various environmental conditions and also that the ability of epimastigotes to undergo metacyclogenesis are influenced by the maintenance schedule. Antigenic profile analysis supports the idea that epimastigotes adapted to culture media do not complete their molecular differentiation into the trypomastigote metacyclic stage. These transition forms conserve some degree of gene expression of the epimastigote stage.     

Interaction with host factors exacerbates Trypanosoma cruzi cell invasion capacity upon oral infection

Outbreaks of severe acute Chagas’ disease acquired by oral infection, leading to death in some cases, have occurred in recent years. Using the mouse model, we investigated the basis of such virulence by analyzing a Trypanosoma cruzi isolate, SC, from a patient with severe acute clinical symptoms, who was infected by oral route. It has previously been shown that, upon oral inoculation into mice, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium by engaging the stage-specific surface glycoprotein gp82, whereas the surface molecule gp90 functions as a down-modulator of cell invasion. We found that, when orally inoculated into mice, metacyclic forms of the SC isolate, which express high levels of gp90, produced high parasitemias and high mortality, in sharp contrast with the reduced infectivity in vitro. Upon recovery from the mouse stomach 1 h after oral inoculation, the gp90 molecule of the parasites was completely degraded, and their entry into HeLa cells, as well as into Caco-2 cells, was increased. The gp82 molecule was more resistant to digestive action of the gastric juice. Host cell invasion of SC isolate metacyclic trypomastigotes was augmented in the presence of gastric mucin. No alteration in infectivity was observed in T. cruzi strains CL and G which were used as references and which express gp90 molecules resistant to degradation by gastric juice. Taken together, our findings suggest that the exacerbation of T. cruzi infectivity, such as observed upon interaction of the SC isolate with the mouse stomach components, may be responsible for the severity of acute Chagas’ disease that has been reported in outbreaks of oral T. cruzi infection.     

Novel strategy in Trypanosoma cruzi cell invasion: implication of cholesterol and host cell microdomains

Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligatory intracellular parasite in the mammalian host. In order to invade a wide variety of mammalian cells, T. cruzi engages parasite components that are differentially expressed among strains and infective forms. Because the identification of putative protein receptors has been particularly challenging, we investigated whether cholesterol and membrane rafts, sterol- and sphingolipid-enriched membrane domains, could be general host surface components involved in invasion of metacyclic trypomastigotes and extracellular amastigotes of two parasite strains with distinct infectivities. HeLa or Vero cells treated with methyl-beta-cyclodextrin (MbetaCD) are less susceptible to invasion by both infective forms, and the effect was dose-dependent for trypomastigote but not amastigote invasion. Moreover, treatment of parasites with MbetaCD only inhibited trypomastigote invasion. Filipin labeling confirmed that host cell cholesterol concentrated at the invasion sites. Binding of a cholera toxin B subunit (CTX-B) to ganglioside GM1, a marker of membrane rafts, inhibited parasite infection. Cell labeling with CTX-B conjugated to fluorescein isothiocyanate revealed that not only cholesterol but also GM1 is implicated in parasite entry. These findings thus indicate that microdomains present in mammalian cell membranes, that are enriched in cholesterol and GM1, are involved in invasion by T. cruzi infective forms.     

Bioluminescent imaging of Trypanosoma cruzi infection

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.     

Regulation of Trypanosoma cruzi invasion of nonphagocytic cells by the endocytically active GTPases dynamin, Rab5, and Rab7

During invasion of nonphagocytic cells by Trypanosoma cruzi (T. cruzi), host cell lysosomes are recruited to the plasma membrane attachment site followed by lysosomal enzyme secretion. The membrane trafficking events involved in invasion have not been delineated. We demonstrate here that T. cruzi invasion of nonphagocytic cells was completely abolished by overexpression of a dominant negative mutant of dynamin. Likewise, overexpression of a dominant negative mutant of Rab5, the rate-limiting GTPase for endocytosis, resulted in reduced infection rates compared with cells expressing Rab5 wild-type. Moreover, cells expressing the activated mutant of Rab5 experienced higher infection rates. A similar pattern was also observed when Rab7-transfected cells were examined. Confocal microscopy experiments showed that parasites colocalized with green fluorescent protein-Rab5-positive early endosomes after 5 min of invasion. These data clearly indicate that newly forming T. cruzi phagosomes first interact with an early endosomal compartment and subsequently with other late component markers before lysosomal interaction occurs.     

Trypanosoma cruzi: Serum levels of nitric oxide and expression of inducible nitric oxide synthase in myocardium and spleen of dogs in the acute stage of infection with metacyclic or blood trypomastigotes

The participation of nitric oxide (NO) in the control of blood parasitemia and parasitism during the acute phase of infection in dogs inoculated with blood trypomastigotes (BT) or metacyclic trypomastigotes (MT group) of Berenice-78 Trypanosoma cruzi strain has been evaluated. Animals of the MT group (n = 4) presented increased levels of serum NO throughout the infection when compared with the BT (n = 4) or control (n = 4) groups, and a delay in parasitemia peak compared with the BT group. In spleen fragments, tissue parasitism was not observed but the MT group presented larger areas associated with inducible NO synthase (iNOS) in relation to BT and control groups. Heart fragments of MT-infected animals exhibited comparatively low tissue parasitism and high iNOS expression, while animals of the BT group presented high inflammatory infiltrate, high tissue parasitism and low iNOS expression. These results indicate that the source of inoculum can interfere with the development of the acute phase of Chagas disease, and may also trigger a distinct parasite-host interaction during this phase.     

Acidification modulates the traffic of Trypanosoma cruzi trypomastigotes in Vero cells harbouring Coxiella burnetii vacuoles

We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella burnetii. When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin A1 and related drugs, increased invasion of both metacyclic and tissue-culture trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. The higher tissue-culture trypomastigote hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2 cells facilitate the escape of both forms. Endosomal-lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of C. burnetii vacuolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells.     

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.     

Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids

Trypanosoma cruzi, the causative agent of Chagas disease, has at least two principal intraspecific subdivisions, T. cruzi I (TCI) and T. cruzi II (TCII), the latter containing up to five subgroups (a-e). Whilst it is known that TCI predominates from the Amazon basin northwards and TCII to the South, where the disease is considered to be clinically more severe, the precise clinical and evolutionary significance of these divisions remains enigmatic. Here, we present compelling evidence of an association between TCI and opossums (Didelphis), and TCII and armadillos, on the basis of key new findings from the Paraguayan Chaco region, together with a comprehensive analysis of historical data. We suggest that the distinct arboreal and terrestrial ecologies, respectively, of these mammal hosts provide a persuasive explanation for the extant T. cruzi intraspecific diversity in South America, and for separate origins of Chagas disease in northern South America and in the southern cone countries.     

TcRho1 of Trypanosoma cruzi: role in metacyclogenesis and cellular localization

Here we have investigated the function of TcRho1, a Rho family orthologue from the parasite Trypanosoma cruzi. We have selected parasites overexpressing wild-type TcRho1 and a truncated form of TcRho1 (TcRho1-DeltaCaaX) which is unable to undergo farnesylation and supposed to interfere with recruitment of Rho effectors to membranes. TcRho1 protein was localized at the anterior region of wild-type and TcRho1 overexpressing epimastigotes, suggesting association with the Golgi apparatus. Accordingly, parasites overexpressing TcRho1-DeltaCaaX presented cytoplasmic fluorescence. To address the function of TcRho1 during differentiation, from epimastigotes to trypomastigotes, we submitted parasites overexpressing the above-cited lineages to metacyclogenesis assays. Parasites overexpressing TcRho1-DeltaCaaX generated a discrete number of metacyclic trypomastigotes when compared with other lineages. Strikingly, TcRho1-DeltaCaaX cells died synchronously during the process of metacyclogenesis.     

Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina

Genetic diversity of Trypanosoma cruzi populations and parasite transmission dynamics have been well documented throughout the Americas, but few studies have been conducted in the Gran Chaco ecoregion, one of the most highly endemic areas for Chagas disease, caused by T. cruzi. In this study, we assessed the distribution of T. cruzi lineages (identified by PCR strategies) in Triatoma infestans, domestic dogs, cats, humans and sylvatic mammals from two neighbouring rural areas with different histories of transmission and vector control in northern Argentina. Lineage II predominated amongst the 99 isolates characterised and lineage I amongst the six isolates obtained from sylvatic mammals. T. cruzi lineage IIe predominated in domestic habitats; it was found in 87% of 54 isolates from Tr. infestans, in 82% of 33 isolates from dogs, and in the four cats found infected. Domestic and sylvatic cycles overlapped in the study area in the late 1980s, when intense domestic transmission occurred, and still overlap marginally. The introduction of T. cruzi from sylvatic into domestic habitats is likely to occur very rarely in the current epidemiological context. The household distribution of T. cruzi lineages showed that Tr. infestans, dogs and cats from a given house compound shared the same parasite lineage in most cases. Based on molecular evidence, this result lends further support to the importance of dogs and cats as domestic reservoir hosts of T. cruzi. We believe that in Argentina, this is the first time that lineage IIc has been isolated from naturally infected domestic dogs and Tr. infestans.     

Trypanosoma cruzi reinfections in mice determine the severity of cardiac damage

In two murine models we studied Trypanosoma cruzi reinfection in the acute and chronic phase of experimental Chagas' disease in order to elucidate the relevance of reinfections in determining the variability of cardiac symptoms and the irreversible cardiac damage. They were followed for 120 and 600 days post infection (p.i.) for the acute and chronic model, respectively. Reinfected mice reached higher parasitaemia levels than infected mice. The survival was 33 and 21% in the chronic phase for mice reinfected in the acute phase and 13% for mice reinfected in the chronic stage at the end of the experiments. Sixty-six percent of the infected group presented electrocardiographic abnormalities (heart frequency, prolonged PQ segment or QRS complex) in the chronic stage whereas 100% of the reinfected animals exhibited electric cardiac dysfunction since 90 and 390 days p.i. for the acute and chronic reinfected model, respectively (P<0.01). Heart histopathological studies showed fibrosis and necrosis areas and mononuclear infiltrates supporting the view that parasite persistence is a major factor in continuing the tissue inflammation. This work shows that T. cruzi reinfections could be related to the variability and severity of the clinical course of Chagas' disease and that parasite persistence is involved in exacerbation of the disease.     

Platelet-activating factor-like activity isolated from Trypanosoma cruzi

Platelet-activating factor is a phospholipid mediator that exhibits a wide variety of physiological and pathophysiological effects, including induction of inflammatory response, chemotaxis and cellular differentiation. Trypanosoma cruzi, the etiological agent of Chagas' disease, is transmitted by triatomine insects and while in the triatomine midgut the parasite differentiates from a non-infective epimastigote stage into the pathogenic trypomastigote metacyclic form. We have previously demonstrated that platelet activating factor triggers in vitro cell differentiation of T. cruzi. Here we show a platelet activating factor-like activity isolated from lipid extract of T. cruzi epimastigotes incubated in the presence of [14C]acetate. Trypanosoma cruzi-platelet activating factor-like lipid induced the aggregation of rabbit platelets, which was prevented by platelet activating factor-acetylhydrolase. Mouse macrophage infection by T. cruzi was stimulated when epimastigotes were kept for 5 days in the presence of T. cruzi-platelet activating factor, before interacting with the macrophages. The differentiation of epimastigotes into metacyclic trypomastigotes was also triggered by T. cruzi-platelet activating factor. These effects were abrogated by a platelet activating factor antagonist, WEB 2086. Polyclonal antibody raised against mouse platelet activating factor receptor showed labelling for T. cruzi epimastigotes using immunoblotting and immunofluorescence assays. These data suggest that T. cruzi contain the components of an autocrine platelet activating factor-like ligand-receptor system that modulates cell differentiation towards the infectious stage.     

Trypanosoma cruzi: variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis

Nineteen Trypanosoma cruzi stocks, most of them of wild origin, and four Trypanosoma rangeli stocks from Colombia were analysed by molecular karyotype analysis with cloned DNA cruzipain as the probe. Another 27 cloned stocks of T. cruzi from different geographic areas of South America were used as reference for T. cruzi lineages. Phenetic analysis of chromosome size polymorphism demonstrated a great variability of Colombian T. cruzi stocks, suggesting that most belong to lineage I, although two of them belong to lineage II. The 2 lineage II T. cruzi, 17 T. cruzi lineage I, and 3 T. rangeli stocks from Colombia were studied further by Southern blot analysis with a panel of kinetoplast DNA minicircle probes. Hybridisation results indicate that the two T. cruzi II stocks are genetically distant from each other and from T. cruzi lineages IIb, IId, and IIe from Chile. Finally, T. cruzi minicircle probes do not cross-hybridise in any stringency condition tested with T. rangeli minicircles, a clear indication that these parasites can be easily distinguished by this method.     

Molecular characterization of ribonucleoproteic antigens containing repeated amino acid sequences from Trypanosoma cruzi

Trypanosoma cruzi expresses several proteins containing antigenic amino acid repeats. Here we characterized TcRpL7a and TcRBP28, which carry similar repeat motifs and share homology to the eukaryotic L7a ribosomal protein and to a Trypanosoma brucei RNA binding protein, respectively. Analyses of the full length and truncated recombinant TcRpL7a showed that the humoral response of patients with Chagas disease is directed towards its repetitive domain. Sequence analyses of distinct copies of TcRpL7a genes present in the genome of six T. cruzi strains indicate that the number of repeats is higher in proteins from T. cruzi II than T. cruzi I strains. A serum panel of 59 T. cruzi infected patients showed that 73% reacted with TcRpL7a, 71% reacted with TcRBP28 and 80% reacted with 1:1 mixture of both antigens. Synthetic peptides harboring the TcRpL7a repeat motif reacted with 46% of the serum samples. Antibodies raised against both antigens identified equivalent amounts of the native proteins in all three stages of the parasite life cycle. Analyses of subcellular fractions indicated that TcRBP28 is present in the cytoplasm whereas TcRpL7a co-fractionates with polysomes. Confirming their predicted cellular localization, GFP fusions showed that, whereas GFP::TcRBP28 localizes in the cytoplasm, GFP::TcRpL7a accumulates in the nucleus, where ribosome biogenesis occurs.     

Trypanosoma cruzi: effects of social stress in Calomys callosus a natural reservoir of infection

Social environment can represent a major source of stress affecting cortisol and/or corticosterone levels, thereby altering the immune response. We have investigated the effects of social isolation on the development of Trypanosoma cruzi infection in female Calomys callosus, a natural reservoir of this protozoan parasite. Animals were divided in groups of five animals each. The animals of one group were kept together in a single cage. In a second group, four females were kept together in a cage with one male. In the final group, five individuals were kept isolated in private cages. The isolated animals showed body weight reduction, decreased numbers of peritoneal macrophages, lower global leucocytes counts, smaller lytic antibody percentage and a significantly higher level of blood parasites compared to the other animals. Their behavior was also altered. They were more aggressive than grouped females, or females exposed to the presence of a male. These results suggest that isolation creates a distinct social behavior in which immunity is impaired and pathogenesis is enhanced.     

A conserved domain of the gp85/trans-sialidase family activates host cell extracellular signal-regulated kinase and facilitates Trypanosoma cruzi infection

Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.