Comparative Analysis of the Secretome and Interactome of Trypanosoma cruzi and Trypanosoma rangeli Reveals Species Specific Immune Response Modulating Proteins.

Chagas disease, a zoonosis caused by the flagellate protozoan Trypanosoma cruzi, is a chronic and systemic parasitic infection that affects ~5-7 million people worldwide, mainly in Latin America. Chagas disease is an emerging public health problem due to the lack of vaccines and effective treatments. According to recent studies, several T. cruzi secreted proteins interact with the human host during cell invasion. Moreover, some comparative studies with T. rangeli, which is non-pathogenic in humans, have been performed to identify proteins directly involved in the pathogenesis of the disease. In this study, we present an integrated analysis of canonical putative secreted proteins (PSPs) from both species. Additionally, we propose an interactome with human host and gene family clusters, and a phylogenetic inference of a selected protein. In total, we identified 322 exclusively PSPs in T. cruzi and 202 in T. rangeli. Among the PSPs identified in T. cruzi, we found several trans-sialidases, mucins, MASPs, proteins with phospholipase 2 domains (PLA2-like), and proteins with Hsp70 domains (Hsp70-like) which have been previously characterized and demonstrated to be related to T. cruzi virulence. PSPs found in T. rangeli were related to protozoan metabolism, specifically carboxylases and phosphatases. Furthermore, we also identified PSPs that may interact with the human immune system, including heat shock and MASP proteins, but in a lower number compared to T. cruzi. Interestingly, we describe a hypothetical hybrid interactome of PSPs which reveals that T. cruzi secreted molecules may be down-regulating IL-17 whilst T. rangeli may enhance the production of IL-15. These results will pave the way for a better understanding of the pathophysiology of Chagas disease and may ultimately lead to the identification of molecular targets, such as key PSPs, that could be used to minimize the health outcomes of Chagas disease by modulating the immune response triggered by T. cruzi infection.

Vaccination of dogs with Trypanosoma rangeli induces antibodies against Trypanosoma cruzi in a rural area of Córdoba, Argentina.

Dogs play a major role in the domestic cycle of Trypanosoma cruzi, acting as reservoirs. In a previous work we have developed a model of vaccination of dogs in captivity with nonpathogenic Trypanosoma rangeli epimastigotes, resulting in the production of protective antibodies against T. cruzi, with dramatic decrease of parasitaemia upon challenge with 100,000 virulent forms of this parasite. The aim of this work was to evaluate the immunogenicity of this vaccine in dogs living in a rural area. Domestic dogs, free from T. cruzi infection, received three immunisations with fixed T. rangeli epimastigotes. Dogs were not challenged with T. cruzi, but they were left in their environment. This immunisation induced antibodies against T. cruzi for more than three years in dogs in their natural habitat, while control dogs remained serologically negative.

Experimental Chagas disease in Balb/c mice previously vaccinated with T. rangeli. II. The innate immune response shows immunological memory: reality or fiction?

Trypanosoma cruzi is a real challenge to the host's immune system, because it requires strong humoral and cellular immune response to remove circulating trypomastigote forms, and to prevent the replication of amastigote forms in tissues, involving many regulator and effector components. This protozoan is responsible for Chagas disease, a major public health problem in Latinamerica. We have developed a model of vaccination with Trypanosoma rangeli, a parasite closely related to T. cruzi, but nonpathogenic to humans, which reduces the infectiousness in three different species of animals, mice, dogs and guinea pigs, against challenge with T. cruzi. In a previous work, we demonstrated that mice vaccinated with T. rangeli showed important soluble mediators that stimulate phagocytic activity versus only infected groups. The aim of this work was to study the innate immune response in mice vaccinated or not with T. rangeli. Different population cells and some soluble mediators (cytokines) in peritoneal fluid and plasma in mice vaccinated-infected and only infected with T. cruzi were studied. In the first hours of challenge vaccinated mice showed an increase of macrophages, NK, granulocytes, and regulation of IL6, IFNγ, TNFα and IL10, with an increase of IL12, with respect to only infected mice. Furthermore an increase was observed of Li T, Li B responsible for adaptative response. Finally the findings showed that the innate immune response plays an important role in vaccinated mice for the early elimination of the parasites, complementary with the adaptative immune response, suggesting that vaccination with T. rangeli modulates the innate response, which develops some kind of immunological memory, recognizing shared antigens with T. cruzi. These results could contribute to the knowledge of new mechanisms which would have an important role in the immune response to Chagas disease.

Can we heal Chagas infection?

We present a model for the parasite-antibody dynamical competition between Trypanosoma rangeli and its antibodies during the acute phase of an infection in a mammal host. The model reproduces experimental data from murine models found in the literature and allows us to demonstrate that a preinfection with T. rangeli induces a temporary protective effect against Chagas disease. As the mammal immune system is able to eliminate a single T. rangeli infection, the host high antibody levels, needed to resist the Chagas infection, are reduced with time, returning the system to the initial healthy state. Our results suggest that a preinfection with T. rangeli could be used to reduce the in-house vectorial parasitemia through repeated vaccination of domestic animals.

Vaccination with Trypanosoma rangeli induces resistance of guinea pigs to virulent Trypanosoma cruzi.

Chagas' disease, endemic in Latin America, is spread in natural environments through animal reservoirs, including marsupials, mice and guinea pigs. Farms breeding guinea pigs for food are located in some Latin-American countries with consequent risk of digestive infection. The aim of this work was to study the effect of vaccination with Trypanosoma rangeli in guinea pigs challenged with Trypanosoma cruzi. Animals were vaccinated with fixated epimastigotes of T. rangeli, emulsified with saponin. Controls received only PBS. Before being challenged with T. cruzi, parasitemia, survival rates and histological studies were performed. The vaccinated guinea pigs revealed significantly lower parasitemia than controls (p<0.0001-0.01) and a discrete lymphomonocytic infiltrate in cardiac and skeletal muscles was present. In the chronic phase, the histological view was normal. In contrast, control group revealed amastigote nests and typical histopathological alterations compatible with chagasic myocarditis, endocarditis and pericarditis. These results, together with previous works in our laboratory, show that T. rangeli induces immunoprotection in three species of animals: mice, guinea pigs and dogs. The development of vaccines for use in animals, like domestic dogs and guinea pigs in captivity, opens up new opportunities for preventive tools, and could reduce the risk of infection with T. cruzi in the community.

Experimental Chagas disease. Innate immune response in Balb/c mice previously vaccinated with Trypanosoma rangeli. I. The macrophage shows immunological memory: Reality or fiction?

Chagas' disease, caused by Trypanosoma cruzi, is a major vector borne health problem in Latin America and an emerging or re-emerging infectious disease in several countries. Immune response to T. cruzi infection is highly complex and involves many components, both regulators and effectors. Although different parasites have been shown to activate different mechanisms of innate immunity, T. cruzi is often able to survive and replicate in its host because they are well adapted to resisting host defences. An experimental model for vaccinating mice with Trypanosoma rangeli, a parasite closely related to T. cruzi, but nonpathogenic to humans, has been designed in our laboratory, showing protection against challenge with T. cruzi infection. The aim of this work was to analyze some mechanisms of the early innate immune response in T. rangeli vaccinated mice challenged with T. cruzi. For this purpose, some interactions were studied between T. cruzi and peritoneal macrophages of mice vaccinated with T. rangeli, infected or not with T. cruzi and the levels of some molecules or soluble mediators which could modify these interactions. The results in vaccinated animals showed a strong innate immune response, where the adherent cells of the vaccinated mice revealed important phagocytic activity, and some soluble mediator (Respiratory Burst: significantly increase, p ≤ 0.03; NO: the levels of vaccinated animals were lower than those of the control group; Arginasa: significantly increase, p ≤ 0.04). The results showed an important role in the early elimination of the parasites and their close relation with the absence of histological lesions that these animals present with regard to the only infected mice. This behaviour reveals that the macrophages act with some type of memory, recognizing the antigens to which they have previously been exposed, in mice were vaccinated with T. rangeli, which shares epitopes with T. cruzi.

Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum.

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

Variable sensitivity to complement-mediated lysis among Trypanosoma rangeli reference strains.

Six reference strains of Trypanosoma rangeli from different days of growth in axenic cultures were assayed for susceptibility to complement-mediated lysis by non-immune guinea-pig serum. Their authenticity was also confirmed by isoenzyme analyses. Parasites were incubated with 25% active or 68°C-inactivated serum (37°C, 30 min) for all tests; thereafter the lysis rates were determined. Highly variable lysis percentages were observed among T. rangeli strains and in the same stock at different growing days. In a few assays, three strains (Macias, R-1625 and Choachi) presented total or very high resistance. The others (H-14, San Agustín and SC-58) were generally most susceptible, and could reach lysis rates as high as Trypanosoma cruzi. After incubation with active sera, the epimastigotes were usually the predominant stages, being followed by spheromastigotes and/or transitional forms. Those stages and trypomastigotes could also be partially susceptible. In four strains, the short epimastigotes were more resistant to lysis than the long ones. Experiments with C3-deficient serum displayed total or partial participation of the alternative-complement pathway in T. rangeli lysis. This study confirmed the variable complement sensitivity of T. rangeli, which can be related to its intraspecific heterogeneity, to the remarkable complexity of its life-cycle stages, and to the methodology employed.

Vaccination with Trypanosoma rangeli modulates the profiles of immunoglobulins and IL-6 at local and systemic levels in the early phase of Trypanosoma cruzi experimental infection.

In America, there are two species of Trypanosoma that can infect humans: Trypanosoma cruzi, which is responsible for Chagas disease and Trypanosoma rangeli, which is not pathogenic. We have developed a model of vaccination in mice with T. rangeli epimastigotes that protects against T. cruzi infection. The goal of this work was to study the pattern of specific immunoglobulins in the peritoneum (the site of infection) and in the sera of mice immunized with T. rangeli before and after challenge with T. cruzi. Additionally, we studied the effects triggered by antigen-antibodies binding and the levels of key cytokines involved in the humoral response, such as IL-4, IL-5 and IL-6. The immunization triggered the production of antibodies reactive with T. cruzi in peritoneal fluid (PF) and in serum, mainly IgG1 and, to a lesser magnitude, IgG2. Only immunized mice developed specific IgG3 antibodies in their peritoneal cavities. Antibodies were able to bind to the surface of the parasites and agglutinate them. Among the cytokines studied, IL-6 was elevated in PF during early infection, with higher levels in non-immunized-infected mice. The results indicate that T. rangeli vaccination against T. cruzi infection triggers a high production of specific IgG isotypes in PF and sera before infection and modulates the levels of IL-6 in PF in the early periods of infection.

Vaccination with Trypanosoma rangeli modulates the profiles of immunoglobulins and IL-6 at local and systemic levels in the early phase of Trypanosoma cruzi experimental infection

In America, there are two species of Trypanosoma that can infect humans: Trypanosoma cruzi, which is responsible for Chagas disease and Trypanosoma rangeli, which is not pathogenic. We have developed a model of vaccination in mice with T. rangeli epimastigotes that protects against T. cruzi infection. The goal of this work was to study the pattern of specific immunoglobulins in the peritoneum (the site of infection) and in the sera of mice immunized with T. rangeli before and after challenge with T. cruzi. Additionally, we studied the effects triggered by antigen-antibodies binding and the levels of key cytokines involved in the humoral response, such as IL-4, IL-5 and IL-6. The immunization triggered the production of antibodies reactive with T. cruzi in peritoneal fluid (PF) and in serum, mainly IgG1 and, to a lesser magnitude, IgG2. Only immunized mice developed specific IgG3 antibodies in their peritoneal cavities. Antibodies were able to bind to the surface of the parasites and agglutinate them. Among the cytokines studied, IL-6 was elevated in PF during early infection, with higher levels in non-immunized-infected mice. The results indicate that T. rangeli vaccination against T. cruzi infection triggers a high production of specific IgG isotypes in PF and sera before infection and modulates the levels of IL-6 in PF in the early periods of infection.