The glycoforms of a Trypanosoma brucei variant surface glycoprotein and molecular modeling of a glycosylated surface coat

The plasma membrane of the African sleeping sickness parasite Trypanosoma brucei is covered with a dense, protective surface coat. This surface coat is a monolayer of five million variant surface glycoprotein (VSG) dimers that form a macromolecular diffusion barrier. The surface coat protects the parasite from the innate immune system and, through antigenic variation, the specific host immune response. There are several hundred VSG genes per parasite, and they encode glycoproteins that vary in primary amino acid sequence, the number of N-glycosylation sites, and the types of N-linked oligosaccharides and glycosylphosphatidylinositol membrane anchors they contain. In this study, we show that VSG MITat.1.5 is glycosylated at all three potential N-glycosylation sites, and we assign the oligosaccharides present at each site. Using the most abundant oligosaccharides at each site, we construct a molecular model of the glycoprotein to assess the role of N-linked oligosaccharides in the architecture of the surface coat.     

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.     

Expression and function of the Trypanosoma brucei major surface protease (GP63) genes

The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.     

Trypanosoma cruzi and Trypanosoma rangeli: Glutamate dehydrogenases and proteolytic activities

• 1.1. Trypanosoma (Herpetosoma) rangeli contains proteolytic activity with azocasein, casein and BAPA as substrates, and aminopeptidase activity with Arg-BNA as substrate. The respective pH optima were 5.5, 7.0, 8.5 and 7.0.
• 2.2. The effect of the protease inhibitors PMSF, TLCK and trasylol was studied. 0.5 mM TLCK caused considerable inhibition of all these activities, whereas 1 mM PMSF was much less effective. Trasylol (0.14 mg/ml) inhibited the activities with azocasein and Arg-BNA as substrates.
• 3.3. Trypanosoma (Schizotrypanum) cruzi contains very similar proteolytic activities, with some slight differences in pH optima and in response to inhibitors. Thus Trasylol was not effective on any activity, and the activity on Arg-BNA was little sensitive to TLCK.
• 4.4. The levels of these enzymes, and also of the NAD- and NADP-linked glutamate dehydrogenases, were studied in six stocks of T. (Sch.) cruzi and three stocks of T. (H.) rangeli. There was no significant difference in the NAD-gluDH, nor in the proteolytic activity with BAPA. On the other hand, the other enzymes tested presented differences which ranged from about 3-fold for the aminopeptidase to nearly 100-fold for the NADP-gluDH. The electrophoretic behaviour of the latter was identical in all the stocks of both species, thus showing that the difference was only quantitative.
• 5.5. The three stocks of T. (H.) rangeli were more similar to some T. (Sch.) cruzi stocks (Tul 2) than the latter were to each other, thus emphasizing at a biochemical level the similarities between these two species.

     

Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations

Trypanosoma rangeli can infect humans as well as the same domestic and wild animals and triatomine vectors infected by Trypanosoma cruzi in Central and South America. This overlapping distribution complicates the epidemiology of American trypanosomiasis due to the cross-reactivity between T. rangeli and T. cruzi antigens and the presence of conserved DNA sequences in these parasites. We have isolated a T. rangeli-specific DNA repetitive element which is represented in approximately 103 copies per parasite genome and is distributed in several chromosomal bands. The 542-bp nucleotide sequence of this element, named P542, was determined and a PCR assay was standardized for its amplification. The sensitivity of the assay is high, allowing the detection of one tenth of the DNA content of a single parasite. The presence of the P542 element was confirmed in 11 T. rangeli isolates from mammalian hosts and insect vectors originating from several countries in Latin America. Negative amplification was observed with different T. cruzi strains and other trypanosomatids. The potential field application of the P542 PCR assay was investigated in simulated samples containing T. rangeli and/or T. cruzi and intestinal tract and feces of Rhodnius prolixus. Epidemiological studies were conducted in DNA preparations obtained from the digestive tracts of 12 Rhodnius colombiensis insects collected in a sylvatic area in Colombia. Positive amplification of the P542 element was obtained in 9/12 insects. We have also compared in the same samples the diagnostic performance of two PCR assays for the amplification of the variable domain of minicircle kinetoplast DNA (kDNA) and of the large subunit (LSU) of the ribosomal RNA gene of T. cruzi and T. rangeli. Data indicate that the kDNA PCR assay does not allow diagnosis of mixed infections in most insects. On the other hand, the PCR assay of the LSU RNA gene showed lower sensitivity in the detection of T. rangeli than the PCR assay of the P542 element. It is predicted that the use of sensitive detection techniques will indicate that the actual distribution of T. rangeli in America is wider than presumed.     

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.     

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.     

Gene activation and re-expression of a Trypanosoma brucei variant surface glycoprotein

The expression of the Trypanosoma brucei variant surface glycoprotein AnTat 1.1 proceeds by a mechanism that transfers a duplicated gene copy into a new genomic environment, the so-called expression site, where it will be expressed. We have isolated a genomic fragment containing the region spanning the expression site-transposon junction, and the 5' half of the coding sequence. Comparing this DNA segment with its template copy (basic copy) allowed us to identify the exact breaking point and indicated a base sequence which could be involved in initiating the transposition event. Sequencing data also indicated that the co-transposed segment 5' to the coding sequence is 430 bp in length. The extreme 5' end of the mRNA is derived from a region in the expression site not immediately adjacent to the transposed DNA segment. This particular sequence exists in multiple copies in the genome and is common to the mRNA of all variant surface glycoproteins so far analysed.     

Trypanosoma cruzi: identification of a cell surface polysaccharide.

An immunogenic polysaccharide prepared by phenol-water extraction of Trypanosoma cruzi was characterized by polyacrylamide gel electrophoresis and molecular sieve chromatography. The polysaccharide was shown to be a cell surface constituent by adsorption of rabbit anti-polysaccharide serum with live culture forms of the protozoa. The cell surface localization of the antigen was visualized using fluorescein- and ferritin-conjugated antibodies.     

The major surface protein of Leishmania promastigotes is a protease

The major surface protein of Leishmania promastigotes is evolutionarily conserved and is found in isolates of L. donovani, L. major, L. tropica, L. mexicana, and L. braziliensis. The data provided in this communication demonstrate that in L. major this integral membrane protein is a protease, which we now designate promastigote surface protease. The enzyme has an alkaline pH optimum and is active both in its detergent-solubilized form and at the surface of living or fixed promastigotes. A water-soluble form of promastigote surface protease is obtained following digestion with the phospholipase C responsible for the release of the variant surface glycoprotein of Trypanosoma brucei. Possible biological functions of promastigote surface protease during the life cycle of Leishmania parasites are discussed.     

Serine hydroxymethyltransferase activity in Trypanosoma cruzi, Trypanosoma rangeli and American Leishmania spp

Serine hydroxymethyltransferase (SHMT) was studied in several American trypanosomatids, Trypanosoma cruzi epimastigotes displaying, in contrast with T. rangeli, high enzymatic activity. Several Leishmania spp. members, including L. braziliensis, L. mexicana and L. garnhami promastigotes, under identical assay conditions, showed low enzymatic activity. The T. cruzi and leishmanial enzymes presented several different kinetic properties, and thus apparent Km for THF was 0.30 mM for the trypanosomal SHMT vs 0.60 mM for the leishmanial enzyme, while the apparent Km for serine was 0.40 mM for trypanosomal SHMT vs 0.15 mM for leishmanial enzyme. There were significant variations in the specific activity of SHMT between the several different trypanosomatids strains studied, but the meaning of these results is not clear because they showed no correlation either with taxonomy or infectivity.     

Revisiting Trypanosoma rangeli Transmission Involving Susceptible and Non-Susceptible Hosts

Trypanosoma rangeli infects several triatomine and mammal species in South America. Its transmission is known to occur when a healthy insect feeds on an infected mammal or when an infected insect bites a healthy mammal. In the present study we evaluated the classic way of T. rangeli transmission started by the bite of a single infected triatomine, as well as alternative ways of circulation of this parasite among invertebrate hosts. The number of metacyclic trypomastigotes eliminated from salivary glands during a blood meal was quantified for unfed and recently fed nymphs. The quantification showed that ~50,000 parasites can be liberated during a single blood meal. The transmission of T. rangeli from mice to R. prolixus was evaluated using infections started through the bite of a single infected nymph. The mice that served as the blood source for single infected nymphs showed a high percentage of infection and efficiently transmitted the infection to new insects. Parasites were recovered by xenodiagnosis in insects fed on mice with infections that lasted approximately four months. Hemolymphagy and co-feeding were tested to evaluate insect-insect T. rangeli transmission. T. rangeli was not transmitted during hemolymphagy. However, insects that had co-fed on mice with infected conspecifics exhibited infection rates of approximately 80%. Surprisingly, 16% of the recipient nymphs became infected when pigeons were used as hosts. Our results show that T. rangeli is efficiently transmitted between the evaluated hosts. Not only are the insect-mouse-insect transmission rates high, but parasites can also be transmitted between insects while co-feeding on a living host. We show for the first time that birds can be part of the T. rangeli transmission cycle as we proved that insect-insect transmission is feasible during a co-feeding on these hosts.     

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.     

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.