Sialylation of N-Carbohydrate Chains of Glycoproteins with Trans-Sialidase from Trypanosoma cruzi

2,3-Sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosoma cruzi is reported. Trans-sialidase (160 kDa, pI 5.35–5.65) and its catalytic fragment (70 kDa, pI 6.0–6.3) were isolated from T. ruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained its activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarin-labeled oligosaccharides with various numbers of antennas and for 2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.     

Antigenic significance of a Trypanosoma rangeli sialidase

The Trypanosoma rangeli-secreted sialidase was purified by bovine submaxillary gland mucin-sepharose affinity chromatography. In immunoblotting analysis, antibodies raised against this molecule recognized polypeptides of 73 kDa in T. rangeli medium supernatant (TrSialr) and of 70 kDa in the cell lysates of T. rangeli (TrSials) and T. cruzi (TcSialL) epimastigotes. TrSialr, TrSials, and TcSialL were subjected to proteolytic cleavage with papain; the resultant peptide pattern displayed differences in the immunoblotting profiles. TrSials was purified by immunoprecipitation, and this protein band was recognized by sera from T. cruzi-infected chronic mice and Chagas' disease patients. In contrast, TrSialr was not recognized by these sera. The antibodies from the infected mice also recognized a band of 70 kDa present in the medium. These preliminary observations imply that the released and somatic sialidases are partially different molecules, with probably different biological roles. The related proteins recognized in T. rangeli and T. cruzi epimastigotes share many antigenic characteristics but have some structural differences, probably related to their function in the parasitic cell. On the basis of the strong antigenicity of TrSials, this molecule is proposed as the antigen for the detection of antibodies arising during T. cruzi infection.     

Trypanosoma saimirii Rodhain, a junior synonym of Trypanosoma rangeli Tejera

In the present study, we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to compare polypeptides of trypanosomes isolated by hemoculture of squirrel monkeys displaying Trypanosoma saimirii blood trypomastigotes, with other trypanosomes that infect primates to evaluate the validity of T. saimirii. The polypeptide profiles of trypanosomes isolated directly from squirrel monkeys or after their passage in mice were identical to those of 3 standard strains of T. rangeli, but they were distinct from those of T. cruzi, T. conorhini, and T. minasense. These results strengthen previous morphological and biological findings by Rodhain on trypanosomes of the squirrel monkey and lead to the conclusion that T. saimirii is indeed a junior synonym of T. rangeli.     

Genes for cysteine proteinases from Trypanosoma rangeli

Abstract PCR amplification of genomic DNA from the American trypanosome, Trypanosoma rangeli , using as primers oligonucleotides derived from the gene of cruzipain, the major cysteine proteinase (CP) from Trypanosoma cruzi , allowed the production of a probe which was used to obtain three clones encoding a CP with 70% overall identity with cruzipain. The genes are organized in tandem, with a monomere size of approximately 2 kbp, located on two chromosomes which, in some parasite isolates, have a high molecular mass (higher than 5.7 Mbp), and in others are much smaller (about 500 kbp). The low expression of this CP at the protein level correlates well with the low level of specific mRNA found in Northern blots.     

Trypanosoma rangeli: characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity

In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.     

Action of Trypanosoma rangeli in infections with virulent Trypanosoma cruzi populations

In experimental murine infections with Trypanosoma rangeli it has been observed development immune response to Trypanosoma cruzi. The aim of the present work was to analyze the result of antigenic stimuli and the protective effect with T. rangeli in T. cruzi infections. Mice groups immunized with metacyclic trypomastigotes of T. rangeli (Choach -2V strain), derived from haemolymph and salivary gland and reinfected with T. cruzi virulent populations (Tulahuen strain, SA strain and Dm28c clone) from infected in vitro cells, showed decrease severity of disease outcomes, low parasitemia levels and 100% survival of all mice immunized, in comparison with groups infected only with T. cruzi populations, which demonstrated tissue affection, high parasitemia levels and the death of all animals. The above mentioned data contribute to understand the biological behaviour of T. cruzi and T. rangeli and their interaction with vertebrate host.     

Purification and partial characterization of a cysteine proteinase from Trypanosoma rangeli

Abstract Epimastigotes of the American Trypanosome Trypanosoma rangeli contain a very low cysteine proteinase (CP) activity. The enzyme was purified to homogeneity by affinity chromatography on ConA-Sepharose and Cystatin-Sepharose. This CP had a similar apparent molecular mass and an identical N-terminal sequence (15 amino acids) as compared with cruzipain from Trypanosoma cruzi ; cross-reacted immunologically with the latter enzyme, it was inhibited by E-64 and TLCK, but not by PMSF, o-phenanthroline or Pepstatin, and was able to use the same substrates, although with different order of effectiveness and optimum pH.     

Action of pyrazolopyrimidine derivatives on Trypanosoma rangeli culture forms

The capacity of 54 different pyrazolo(3,4-d)- or pyrazolo(4,3-d)pyrimidine derivatives to inhibit the multiplication of Trypanosoma rangeli culture forms was evaluated. Among pyrazolo(3,4-d)pyrimidines, 14 derivatives showed trypanostatic activity, 4-aminopyrazolo-(3,4-d)pyrimidine (APP) being the most active, with 4-hydroxypyrazolo(3,4-d)pyrimidine (HPP) lacking trypanostatic activity. 7-Hydroxy-3-beta-D-ribofuranosylpyrazolo(4,3-d)pyrimidine (FoB) was as active as 7-amino-3-beta-D-ribofuranosylpyrazolo(4,3-d)pyrimidine (FoA), both compounds being five-fold less inhibitory than APP. It can be concluded that, regarding T. rangeli, the chemical analogy to hypoxanthine or inosine of pyrazolo(3,4-d)- and pyrazolo(4,3-d)pyrimidine, respectively, is not absolutely critical, as different modifications on the heterocyclic ring did not abolish the inhibitory activity of these compounds.     

Trypanosoma rangeli interactions within the vector Rhodnius prolixus: a mini review

This article is an integrative mini review of the research on the interactions between Trypanosoma rangeli and the insect vector, Rhodnius prolixus. Special attention is given to the interactions of these parasites with the gut environment, gut walls, with hemolymph invasion, hemocytes, hemocyte microaggregations, prophenoloxidase-activating system, superoxide, and nitric acid generation and eicosanoid pathways. We described factors affecting vectorial capacity and suggested that T. rangeli may modulate the hemocoelic invasion and the survival of the parasites by overcoming the cellular and humoral defense reactions of the insect vector at different physiological events. The mechanisms of these interactions and their significance for parasite transmission are discussed.     

The taxonomic position and evolutionary relationships of Trypanosoma rangeli.

This paper presents a re-evaluation of the taxonomic position and evolutionary relationships of Trypanosoma (Herpetosoma) rangeli based on the phylogenetic analysis of ssrRNA sequences of 64 Trypanosoma species and comparison of mini-exon sequences. All five isolates of T. rangeli grouped together in a clade containing Trypanosoma (Schizotrypanum) cruzi and a range of closely related trypanosome species from bats [Trypanosoma (Schizotrypanum) dionisii, Trypanosoma (Schizotrypanum) vespertilionis] and other South American mammals [Trypanosoma (Herpetosoma) leeuwenhoeki, Trypanosoma (Megatrypanum) minasense, Trypanosoma (Megatrypanum) conorhini] and an as yet unidentified species of trypanosome from an Australian kangaroo. Significantly T. rangeli failed to group with (a) species of subgenus Herpetosoma, other than those which are probably synonyms of T. rangeli, or (b) species transmitted via the salivarian route, although either of these outcomes would have been more consistent with the current taxonomic and biological status of T. rangeli. We propose that use of the names Herpetosoma and Megatrypanum should be discontinued, since these subgenera are clearly polyphyletic and lack evolutionary and taxonomic relevance. We hypothesise that T. rangeli and T. cruzi represent a group of mammalian trypanosomes which completed their early evolution and diversification in South America.     

Gene identification and comparative molecular modeling of a Trypanosoma rangeli major surface protease

Trypanosoma rangeli is a hemoflagellate parasite which is able to infect humans. Distinct from Trypanosoma cruzi, the causative agent of Chagas disease, T. rangeli is non-pathogenic to the vertebrate host. The manner by which the T. rangeli interacts with the host is still unknown, but it certainly depends on the surface molecules. Major surface proteins (MSP) are GPI-anchored, zinc-dependent metalloproteases present in the surface of all trypanosomatids studied so far, which are implicated as virulence factors in pathogenic trypanosomatids, such as Leishmania spp and T. cruzi. The aims of this work were to generate the complete sequence of a T. rangeli MSP (TrMSP) gene and to determine the 3D-structure of the predicted protein by homology modeling. The plasmid bearing a complete copy of a TrMSP gene was completely sequenced and the predicted protein was modeled using Modeller software. Results indicate that TrMSP open reading frame (ORF) codes for a predicted 588 amino acid protein and shows all elements required for its posttranslational processing. Multiple sequence alignment of TrMSP with other trypanosomatids’ MSPs showed an extensive conservation of the N-terminal and central regions and a more divergent C-terminal region. Leishmania major MSP (LmMSP), which had its crystal structure previously determined, has an overall 35 % identity with TrMSP. This identity allowed the comparative molecular modeling of TrMSP, which demonstrated a high degree of structural conservation between MSPs from other trypanosomatids (TrypMSPs). All modeled MSPs have a conserved folding pattern, apart from structural divergences in the C-domain and discrete differences of charge and topology in the catalytic cleft, and present the same geometry of the canonical HEXXH zinc-binding motif. The determination of surface charges of the molecules revealed that TrMSP is a predominantly positive protein, whereas LmMSP and Trypanosoma cruzi MSP (TcMSP) are negative proteins, suggesting that substrates recognized by TcMSP and LmMSP could not interact with TrMSP. Moreover, the comparison between TrMSP and TcMSP protein sequences has revealed 45 non-neutral amino acid substitutions, which can be further assessed through protein engineering. The characteristics of TrMSP could explain, at least in part, the lack of pathogenicity of T. rangeli to humans and point to the necessity of identifying the biological targets of this enzyme.

Vaccination with epimastigotes of different strains of Trypanosoma rangeli protects mice against Trypanosoma cruzi infection

In our laboratory, we have developed a model of vaccination in mice with Trypanosoma rangeli, a non-pathogenic parasite that shares many antigens with Trypanosoma cruzi. The vaccinated mice were protected against infection with virulent T. cruzi. The goal of the present work was to study the protective activity of strains of T. rangeli of different origin, with the aim of analysing whether this protective capacity is a common feature of T. rangeli. BALB/c mice were vaccinated with live or fixed epimastigotes of two T. rangeli strains, Choachi and SC-58. Vaccinated (VM) and control mice (CM) were infected with virulent T. cruzi, Tulahuen strain. The results showed that the levels of parasitemia of VM, vaccinated with the two strains of T. rangeli were significantly lower than those developed in CM. The survival rate of VM was higher than that CM. Histological studies revealed many amastigote nests and severe inflammatory infiltrates in the heart and skeletal muscles of CM, whereas in the VM only moderate lymphomonocytic infiltrates were detected. Altogether, the results of the present work as well as previous studies show that the antigens involved in the protection induced by T. rangeli are expressed in different strains of this parasite. These findings could prove useful in vaccine preparation.     

Laboratory maintenance of Trypanosoma (Herpetosoma) rangeli Tejera, 1920

Two laboratory maintenance systems of Trypanosoma rangeli were compared. The maintenance by weekly subinoculations in Tobie's culture medium and the intrafemoral inoculation of Rhodnius prolixus with cultured flagellates, resulted in loss of infectivity of the metacyclic salivarian trypomastigotes for mice, ten months after maintenance in culture. With the system of cyclical passes through culture-Rhodnius-mouse-culture-Rhodnius, the infectivity of the metacyclic trypomastigotes for mice, was maintained during the three years of the experiment. The number and percentage of metacyclic trypomastigotes formed in the salivary glands of R. prolixus, previously inoculated intrafemorally or intracoelomically with culture forms of T. rangeli, did not show correlation with the inoculated dose, however the inoculated quantity demonstrated a direct relation with the mortality rate of the insects. The results indicate that T. rangeli requires an adequate maintenance system, so that under experimental condition the biological characteristics, normally expressed under natural conditions, are conserved.     

Sialylation of N-carbohydrate chains of glycoproteins with immobilized trans-sialidase from Trypanosoma cruzi

Alpha2,3-sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosoma cruzi is reported. Trans-sialidase (160 kDa, pI 5.35-5.65) and its catalytic fragment (70 kDa, pI 6.0-6.3) were isolated from T. cruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarine-labeled oligosaccharides with various numbers of antennas and for alpha2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.     

Differential Distribution of Genes Encoding the Virulence Factor Trans-Sialidase along Trypanosoma cruzi Discrete Typing Units

Trypanosoma cruzi the agent of Chagas disease is a monophyletic but heterogeneous group conformed by several Discrete Typing Units (DTUs) named TcI to TcVI characterized by genetic markers. The trans-sialidase (TS) is a virulence factor involved in cell invasion and pathogenesis that is differentially expressed in aggressive and less virulent parasite stocks. Genes encoding TS-related proteins are included in a large family divided in several groups but only one of them contains TS genes. Two closely related genes differing in a T/C transition encode the enzymatically active TS (aTS) and a lectin-like TS (iTS). We quantified the aTS/iTS genes from TcII and TcVI aggressive and TcI low virulent strains and found variable aTS number (1–32) per haploid genome. In spite of being low TS enzyme-expressers, TcI strains carry 28–32 aTS gene copies. The intriguing absence of iTS genes in TcI strains together with the presence of aTS/iTS in TcII and TcVI strains (virulent) were observed. Moreover, after sequencing aTS/iTS from 38 isolates collected along the Americas encompassing all DTUs, the persistent absence of the iTS gene in TcI, TcIII and TcIV was found. In addition, the sequence clustering together with T/C transition analysis correlated to DTUs of T. cruzi. The consistence of TS results with both evolutionary genome models proposed for T. cruzi, namely the “Two Hybridization” and the “Three Ancestor” was discussed and reviewed to fit present findings. Parasite stocks to attempt genetic KO or to assay the involvement of iTS in parasite biology and virulence are finally available.     

Trypanosoma rangeli: discrimination from Trypanosoma cruzi based on a variable domain from the large subunit ribosomal RNA gene

306-314. Three synthetic oligonucleotides corresponding to sequences within the D7a divergent domain of the large subunit ribosomal RNA gene have been used to amplify the total DNA of Trypanosoma rangeli and Trypanosoma cruzi, two morphologically similar protozoa with overlapping geographical distribution and hosts. The two organisms may be distinguished by the electrophoretic mobilities of their respective amplification products. For T. rangeli a 210-bp product was obtained. The presence of this fragment was confirmed in 14 T. rangeli strains. For T. cruzi two possible amplification products were originated: a 265-bp DNA fragment for strains typed as lineage 1 and a 250-bp fragment for lineage 2 strains. Eleven unidentified trypanosome stocks, recently isolated from Amazonian vectors, could be discriminated using the proposed assay. The potential field application of multiplex PCR was further demonstrated by identification of the two parasite species in samples containing intestinal tract and feces of triatomines. In the present study we have also amplified the D7a domain of several trypanosomatids employing primers complementary to the conserved flanking regions. Size and sequence polymorphisms were observed, indicating that this region could also be explored as a target for specific detection of other members of the Trypanosomatidae family.