Differential expression and activity of arginine kinase between the American trypanosomatids Trypanosoma rangeli and Trypanosoma cruzi.

Trypanosoma rangeli is a non-virulent hemoflagellate parasite infecting humans, wild and domestic mammals in Central and Latin America. The share of genotypic, phenotypic, and biological similarities with the virulent, human-infective T. cruzi and T. brucei, allows comparative studies on mechanisms of pathogenesis. In this study, investigation of the T. rangeli Arginine Kinase (TrAK) revealed two highly similar copies of the AK gene in this taxon, and a distinct expression profile and activity between replicative and infective forms. Although TrAK expression seems stable during epimastigotes growth, the enzymatic activity increases during the exponential growth phase and decreases from the stationary phase onwards. No differences were observed in activity or expression levels of TrAK during in vitro differentiation from epimastigotes to infective forms, and no detectable AK expression was observed for blood trypomastigotes. Overexpression of TrAK by T. rangeli showed no effects on the in vitro growth pattern, differentiation to infective forms, or infectivity to mice and triatomines. Although differences in TrAK expression and activity were observed among T. rangeli strains from distinct genetic lineages, our results indicate an up-regulation during parasite replication and putative post-translational myristoylation of this enzyme. We conclude that up-regulation of TrAK activity in epimastigotes appears to improve proliferation fitness, while reduced TrAK expression in blood trypomastigotes may be related to short-term and subpatent parasitemia in mammalian hosts.

Infecting Triatomines with Trypanosomes.

The infection of triatomines with trypanosomes can be performed with different forms of the parasite, and the procedure is important not only for vector-parasite interaction studies but also for maintaining the infectivity of parasite strains, which guarantees more realistic biological and molecular investigations. Here, I describe how to infect the vector Rhodnius prolixus, a model species, with two different species of Trypanosoma.

Modelling triatomine bug population and Trypanosoma rangeli transmission dynamics: Co-feeding, pathogenic effect and linkage with chagas disease.

Trypanosoma rangeli (T. rangeli), a parasite, is not pathogenic to human but pathogenic to some vector species to induce the behavior changes of infected vectors and subsequently impact the transmission dynamics of other diseases such as Chagas disease which shares the same vector species. Here we develop a mathematical model and conduct qualitative analysis for the transmission dynamics of T. rangeli. We incorporate both systemic and co-feeding transmission routes, and account for the pathogenic effect using infection-induced fecundity and fertility change of the triatomine bugs. We derive two thresholds Rv (the triatomine bug basic reproduction number) and R0 (the T. rangeli basic reproduction number) to delineate the dynamical behaviors of the ecological and epidemiological systems. We show that when Rv>1 and R0>1, a unique parasite positive equilibrium E* appears. We find that E* can be unstable and periodic oscillations can be observed where the pathogenic effect plays a significant role. Implications of the qualitative analysis and numerical simulations suggest the need of an integrative vector-borne disease prevention and control strategy when multiple vector-borne diseases are transmitted by the same set of vector species.

Genomic comparison of Trypanosoma conorhini and Trypanosoma rangeli to Trypanosoma cruzi strains of high and low virulence.

BACKGROUND: Trypanosoma conorhini and Trypanosoma rangeli, like Trypanosoma cruzi, are kinetoplastid protist parasites of mammals displaying divergent hosts, geographic ranges and lifestyles. Largely nonpathogenic T. rangeli and T. conorhini represent clades that are phylogenetically closely related to the T. cruzi and T. cruzi-like taxa and provide insights into the evolution of pathogenicity in those parasites. T. rangeli, like T. cruzi is endemic in many Latin American countries, whereas T. conorhini is tropicopolitan. T. rangeli and T. conorhini are exclusively extracellular, while T. cruzi has an intracellular stage in the mammalian host. RESULTS: Here we provide the first comprehensive sequence analysis of T. rangeli AM80 and T. conorhini 025E, and provide a comparison of their genomes to those of T. cruzi G and T. cruzi CL, respectively members of T. cruzi lineages TcI and TcVI. We report de novo assembled genome sequences of the low-virulent T. cruzi G, T. rangeli AM80, and T. conorhini 025E ranging from ~ 21-25 Mbp, with ~ 10,000 to 13,000 genes, and for the highly virulent and hybrid T. cruzi CL we present a ~ 65 Mbp in-house assembled haplotyped genome with ~ 12,500 genes per haplotype. Single copy orthologs of the two T. cruzi strains exhibited ~ 97% amino acid identity, and ~ 78% identity to proteins of T. rangeli or T. conorhini. Proteins of the latter two organisms exhibited ~ 84% identity. T. cruzi CL exhibited the highest heterozygosity. T. rangeli and T. conorhini displayed greater metabolic capabilities for utilization of complex carbohydrates, and contained fewer retrotransposons and multigene family copies, i.e. trans-sialidases, mucins, DGF-1, and MASP, compared to T. cruzi. CONCLUSIONS: Our analyses of the T. rangeli and T. conorhini genomes closely reflected their phylogenetic proximity to the T. cruzi clade, and were largely consistent with their divergent life cycles. Our results provide a greater context for understanding the life cycles, host range expansion, immunity evasion, and pathogenesis of these trypanosomatids.

Effect of temperature and vector nutrition on the development and multiplication of Trypanosoma rangeli in Rhodnius prolixus.

Trypanosoma rangeli is a protozoan parasite that infects mammals and triatomines, causing different levels of pathogenicity in its invertebrate vectors, particularly those from the genus Rhodnius. We have recently shown that temperature can modulate T. rangeli growth during in vitro culture, as well as its in vivo pathogenicity to R. prolixus. In the present study, we investigated colonization of R. prolixus by T. rangeli and assessed the role of temperature and vector nutrition on parasite development and multiplication. We infected nymphs and either assessed parasite density in the first hours after the ingestion of the infected blood or maintained the nymphs for up to 60 days at different temperatures (21, 24, 27, and 30 °C) and under different blood-feeding schedules (either every 15 days, or on day 30 post infection only), with parasite development and multiplication measured on days 15, 30, and 60 post infection. In the first hours after ingesting infected blood, epimastigogenesis not only occurred in the anterior midgut, but a stable parasite population also established in this intestinal region. T. rangeli subsequently colonized all intestinal regions examined, but with fewer parasites being found in the rectum. The number of parasites was only affected by higher temperatures (27 and 30 °C) during the beginning of the infection (15 days post infection). Nutritional status of the vector also had a significant effect on parasite development, as reduced blood-feeding decreased infection rates by approximately 30%.

Temperature and parasite life-history are important modulators of the outcome of Trypanosoma rangeli-Rhodnius prolixus interactions.

Trypanosoma rangeli is a protozoan parasite, which does not cause disease in humans, although it can produce different levels of pathogenicity to triatomines, their invertebrate hosts. We tested whether infection imposed a temperature-dependent cost on triatomine fitness using T. rangeli with different life histories. Parasites cultured only in liver infusion tryptose medium (cultured) and parasites exposed to cyclical passages through mice and triatomines (passaged) were used. We held infected insects at four temperatures between 21 and 30 °C and measured T. rangeli growth in vitro at the same temperatures in parallel. Overall, T. rangeli infection induced negative effects on insect fitness. In the case of cultured infection, parasite effects were temperature-dependent. Intermoult period, mortality rates and ecdysis success were affected in those insects exposed to lower temperatures (21 and 24 °C). For passaged-infected insects, the effects were independent of temperature, intermoult period being prolonged in all infected groups. Trypanosoma rangeli seem to be less tolerant to higher temperatures since cultured-infected insects showed a reduction in the infection rates and passaged-infected insects decreased the salivary gland infection rates in those insects submitted to 30 °C. In vitro growth of T. rangeli was consistent with these results.

Trypanosoma cruzi-Trypanosoma rangeli co-infection ameliorates negative effects of single trypanosome infections in experimentally infected Rhodnius prolixus.

Trypanosoma cruzi, causative agent of Chagas disease, co-infects its triatomine vector with its sister species Trypanosoma rangeli, which shares 60% of its antigens with T. cruzi. Additionally, T. rangeli has been observed to be pathogenic in some of its vector species. Although T. cruzi-T. rangeli co-infections are common, their effect on the vector has rarely been investigated. Therefore, we measured the fitness (survival and reproduction) of triatomine species Rhodnius prolixus infected with just T. cruzi, just T. rangeli, or both T. cruzi and T. rangeli. We found that survival (as estimated by survival probability and hazard ratios) was significantly different between treatments, with the T. cruzi treatment group having lower survival than the co-infected treatment. Reproduction and total fitness estimates in the T. cruzi and T. rangeli treatments were significantly lower than in the co-infected and control groups. The T. cruzi and T. rangeli treatment group fitness estimates were not significantly different from each other. Additionally, co-infected insects appeared to tolerate higher doses of parasites than insects with single-species infections. Our results suggest that T. cruzi-T. rangeli co-infection could ameliorate negative effects of single infections of either parasite on R. prolixus and potentially help it to tolerate higher parasite doses.

What is the 'true' effect of Trypanosoma rangeli on its triatomine bug vector?

The phrase, "T. rangeli is pathogenic to its insect vector," is commonly found in peer-reviewed publications on the matter, such that it has become the orthodox view of this interaction. In a literature survey, we identified over 20 papers with almost the exact phrase and several others alluding to it. The idea is of particular importance in triatomine population dynamics and the study of vector-borne T. cruzi transmission, as it could mean that triatomines infected with T. rangeli have lower fitness than uninfected insects. Trypanosoma rangeli pathogenicity was first observed in a series of studies carried out over fifty years ago using the triatomine species Rhodnius prolixus. However, there are few studies of the effect of T. rangeli on its other vector species, and several of the studies were carried out with R. prolixus under non-physiological conditions. Here, we re-evaluate the published studies that led to the conclusion that T. rangeli is pathogenic to its vector, to determine whether or not this indeed is the "true" effect of T. rangeli on its triatomine vector.

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.

Avaliação da colonização de Trypanosoma cruzi e Trypanosoma rangeli no trato intestinal de Rhodnius prolixus

Os parasitos Trypanosoma rangeli e Trypanosoma cruzi entram no tubo digestivo do vetor através do repasto sanguíneo e o intestino médio anterior (IMA) é o local onde se dão as primeiras interações parasito-hospedeiro. Sendo assim, a colonização do IMA de Rhodnius prolixus por T. cruzi (cepa CL) e T. rangeli (cepa CHOACHI) foi avaliada no presente estudo. Inicialmente, o estabelecimento da infecção em ninfas infectadas com epimastigotas de cultura ou tripomastigotas sanguíneas foi comparativamente avaliado. A avaliação de diferentes parâmetros mostrou que a dinâmica de colonização foi diferente dependendo da forma do parasito utilizada para a infecção em ambas as espécies. Dessa forma, a avaliação da dinâmica de colonização dos parasitos no intestino de insetos infectados foi realizada com tripomastigotas sanguíneas, as formas naturalmente presentes no hospedeiro vertebrado. Além disso, a porção do trato intestinal onde ocorre a diferenciação das formas tripomastigotas para epimastigotas foi determinada para as duas espécies. Para infecções por T. rangeli, a diminuição gradativa no número de tripomastigotas no IMA e o concomitante aparecimento de formas intermediárias e epimastigotas a partir do primeiro dia pós-infecção (pi) indicaram que a sua diferenciação acontece nessa porção do intestino. No caso de infecções por T. cruzi,a presença de apenas formas tripomastigotas e intermediárias no IMA e a presença de formas intermediárias e epimastigotas no intestino médio posterior (IMP) dos insetos sugere que a sua diferenciação ocorre no IMP do vetor.

A quantificação dos parasitos mostrou uma redução de mais de 80% nas populações de T. cruzi nas primeiras 24 horas pi. A primeira hipótese para avaliar esta redução seria a de que os parasitos estariam cruzando rapidamente o IMA e estabelecendo a infecção no IMP dos insetos. Entretanto, o reduzido número de parasitos encontrado no IMP não corroborou a hipótese. Na segunda hipótese, os parasitos poderiam estar aderidos ao epitélio do IMA e por essa razão não teriam sido quantificados nas contagens a fresco. A ausência de parasitos no IMA através da quantificação por qPCR,associada a análise do epitélio intestinal em miscroscópio, sugerem fortemente que o parasito não se adere ao epitélio intestinal do IMA. Finalmente, a possibilidade dos parasitos estarem sendo eliminados do IMA nestas primeiras horas pi foi avaliada. A significativa redução no número de parasitos incubados com diferentes tecidos de R. prolixus, particularmente com o IMA de insetos recém-alimentados, sugere que uma parte significativa da população de T. cruzi que entra no vetor juntamente com o repasto é eliminada por fatores presentes no IMA do inseto. Em conclusão, o presente estudo mostrou que embora T. cruzi e T. rangeli apresentem formas infectivas similares, os parasitos utilizam diferentes estratégias para iniciara infecção nos seus hospedeiros invertebrados.

Avaliação da colonização de Trypanosoma cruzi e Trypanosoma rangeli no trato intestinal de Rhodnius prolixus / Trypanosoma cruzi colonization assessment and Trypanosoma rangeli in the intestinal tract of Rhodnius prolixus

Os parasitos Trypanosoma rangeli e Trypanosoma cruzi entram no tubo digestivo do vetor através do repasto sanguíneo e o intestino médio anterior (IMA) é o local onde se dão as primeiras interações parasito-hospedeiro. Sendo assim, a colonização do IMA de Rhodnius prolixus por T. cruzi (cepa CL) e T. rangeli (cepa CHOACHI) foi avaliada no presente estudo. Inicialmente, o estabelecimento da infecção em ninfas infectadas com epimastigotas de cultura ou tripomastigotas sanguíneas foi comparativamente avaliado. A avaliação de diferentes parâmetros mostrou que a dinâmica de colonização foi diferente dependendo da forma do parasito utilizada para a infecção em ambas as espécies. Dessa forma, a avaliação da dinâmica de colonização dos parasitos no intestino de insetos infectados foi realizada com tripomastigotas sanguíneas, as formas naturalmente presentes no hospedeiro vertebrado. Além disso, a porção do trato intestinal onde ocorre a diferenciação das formas tripomastigotas para epimastigotas foi determinada para as duas espécies. Para infecções por T. rangeli, a diminuição gradativa no número de tripomastigotas no IMA e o concomitante aparecimento de formas intermediárias e epimastigotas a partir do primeiro dia pós-infecção (pi) indicaram que a sua diferenciação acontece nessa porção do intestino. No caso de infecções por T. cruzi,a presença de apenas formas tripomastigotas e intermediárias no IMA e a presença de formas intermediárias e epimastigotas no intestino médio posterior (IMP) dos insetos sugere que a sua diferenciação ocorre no IMP do vetor...

A quantificação dos parasitos mostrou uma redução de mais de 80% nas populações de T. cruzi nas primeiras 24 horas pi. A primeira hipótese para avaliar esta redução seria a de que os parasitos estariam cruzando rapidamente o IMA e estabelecendo a infecção no IMP dos insetos. Entretanto, o reduzido número de parasitos encontrado no IMP não corroborou a hipótese. Na segunda hipótese, os parasitos poderiam estar aderidos ao epitélio do IMA e por essa razão não teriam sido quantificados nas contagens a fresco. A ausência de parasitos no IMA através da quantificação por qPCR,associada a análise do epitélio intestinal em miscroscópio, sugerem fortemente que o parasito não se adere ao epitélio intestinal do IMA. Finalmente, a possibilidade dos parasitos estarem sendo eliminados do IMA nestas primeiras horas pi foi avaliada. A significativa redução no número de parasitos incubados com diferentes tecidos de R. prolixus, particularmente com o IMA de insetos recém-alimentados, sugere que uma parte significativa da população de T. cruzi que entra no vetor juntamente com o repasto é eliminada por fatores presentes no IMA do inseto. Em conclusão, o presente estudo mostrou que embora T. cruzi e T. rangeli apresentem formas infectivas similares, os parasitos utilizam diferentes estratégias para iniciara infecção nos seus hospedeiros invertebrados...

Glycoinositolphospholipids from Trypanosomatids subvert nitric oxide production in Rhodnius prolixus salivary glands.

BACKGROUND: Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands. METHODOLOGY/PRINCIPAL FINDINGS: Here we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities. CONCLUSIONS/SIGNIFICANCE: Taken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.

A standardizable protocol for infection of Rhodnius prolixus with Trypanosoma rangeli, which mimics natural infections and reveals physiological effects of infection upon the insect.

Trypanosoma rangeli is a protozoan parasite that shares hosts - mammals and triatomines - with Trypanosoma cruzi, the etiological agent of Chagas disease. Although T. rangeli is customarily considered to be non-pathogenic to human hosts, it is able to produce pathologies in its invertebrate hosts. However, advances are hindered by a lack of standardization of infection procedures and these pathologies need documentation. To establish a suitable, and standardizable, infection protocol, the duration of the fourth instar was evaluated in nymphs infected by injection into the thorax with different concentrations of parasites, and compared with nymphs infected naturally (i.e. orally). We demonstrate that delays in moult were attributable to the presence of the parasite in the haemolymph (vs. the gut) and propose that the protocol presented here simulates closely natural infections. This methodology was then used for the evaluation of physiological parameters and several hitherto unreported effects of T. rangeli infection on Rhodnius prolixus were revealed. Haemolymph volume was greater in infected than uninfected nymphs but this alteration could not be attributed to water retention, since infected insects lost the same amount of water as controls. However, we found that lipid content and fat body weight were both increased in insects infected by T. rangeli. We propose that this is due to the parasite's sequestration of host blood lipids and carrier proteins. With these findings, we have taken a few first steps to unravelling physiological details of the host-parasite interaction. We suggest future directions towards a fuller understanding of mechanistic and adaptive aspects of triatomine-trypanosomatid interactions.