Limited antigenic variation in the Trypanosoma cruzi candidate vaccine antigen TSA-1.

Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is one of the most important neglected tropical diseases in the Western Hemisphere. The toxicities and limited efficacies of current antitrypanosomal drugs have prompted a search for alternative technologies such as a therapeutic vaccine comprised of T. cruzi antigens, including a recombinant antigen encoding the N-terminal 65 kDa portion of Trypomastigote surface antigen-1 (TSA-1). With at least six known genetically distinct T. cruzi lineages, variability between the different lineages poses a unique challenge for the development of broadly effective therapeutic vaccine. The variability across the major lineages in the current vaccine candidate antigen TSA-1 has not previously been addressed. To assess the variation in TSA-1, we cloned and sequenced TSA-1 from several different T. cruzi strains representing three of the most clinically relevant lineages. Analysis of the different alleles showed limited variation in TSA-1 across the different strains and fit with the current theory for the evolution of the different lineages. Additionally, minimal variation in known antigenic epitopes for the HLA-A 02 allele suggests that interlineage variation in TSA-1 would not impair the range and efficacy of a vaccine containing TSA-1.

Identification of genes encoding hypothetical proteins in open-reading frame expressed sequence tags from mammalian stages of Trypanosoma cruzi.

Approximately 50% of the predicted protein-coding genes of the Trypanosoma cruzi CL Brener strain are annotated as hypothetical or conserved hypothetical proteins. To further characterize these genes, we generated 1161 open-reading frame expressed sequence tags (ORESTES) from the mammalian stages of the VL10 human strain. Sequence clustering resulted in 435 clusters, consisting of 339 singletons and 96 contigs. Significant matches to the T. cruzi predicted gene database were found for ~94% contigs and ~69% singletons. These included genes encoding surface proteins, known to be intensely expressed in the parasite mammalian stages and implicated in host cell invasion and/or immune evasion mechanisms. Among 151 contigs and singletons with similarity to predicted hypothetical protein-coding genes and conserved hypothetical protein-coding genes, 83% showed no match with T. cruzi EST and/or proteome databases. These ORESTES are the first experimental evidence that the corresponding genes are in fact transcribed. Sequences with no significant match were searched against several T. cruzi and National Center for Biotechnology Information non-redundant sequence databases. The ORESTES analysis indicated that 124 predicted conserved hypothetical protein-coding genes and 27 predicted hypothetical protein-coding genes annotated in the CL Brener genome are transcribed in the VL10 mammalian stages. Six ORESTES annotated as hypothetical protein-coding genes showing no match to EST and/or proteome databases were confirmed by Northern blot in VL10. The generation of this set of ORESTES complements the T. cruzi genome annotation and suggests new stage-regulated genes encoding hypothetical proteins.

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI.

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

Haplotype distribution of five nuclear genes based on network genealogies and Bayesian inference indicates that Trypanosoma cruzi hybrid strains are polyphyletic.

Chagas disease is still a major public health problem in Latin America. Its causative agent, Trypanosoma cruzi, can be typed into three major groups, T. cruzi I, T. cruzi II and hybrids. These groups each have specific genetic characteristics and epidemiological distributions. Several highly virulent strains are found in the hybrid group; their origin is still a matter of debate. The null hypothesis is that the hybrids are of polyphyletic origin, evolving independently from various hybridization events. The alternative hypothesis is that all extant hybrid strains originated from a single hybridization event. We sequenced both alleles of genes encoding EF-1alpha, actin and SSU rDNA of 26 T. cruzi strains and DHFR-TS and TR of 12 strains. This information was used for network genealogy analysis and Bayesian phylogenies. We found T. cruzi I and T. cruzi II to be monophyletic and that all hybrids had different combinations of T. cruzi I and T. cruzi II haplotypes plus hybrid-specific haplotypes. Bootstrap values (networks) and posterior probabilities (Bayesian phylogenies) of clades supporting the monophyly of hybrids were far below the 95% confidence interval, indicating that the hybrid group is polyphyletic. We hypothesize that T. cruzi I and T. cruzi II are two different species and that the hybrids are extant representatives of independent events of genome hybridization, which sporadically have sufficient fitness to impact on the epidemiology of Chagas disease.

Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Ribosomal RNA genes in Bacillus subtilis. Evidence for a cotranscription mechanism.

The analysis of the transcriptional mechanism of the ribosomal RNA genes in Bacillus subtilis was undertaken by a study of the rRNA chain elongation in the presence of rifampicin. The residual RNA synthesis after the addition of rifampicin and [3H] uridine to exponentially growing cells has shown that 56% of the radioactivity incorporated into total RNA belongs to the unstable fraction and 44% to the fraction containing mature rRNA and tRNA. Such study allowed an estimation of the half-life of messenger RNAs as being approximately 2 min. The analysis of the transcription pattern of the ribosomal RNA genes, as measured by the amount of radioactivity found in the ribosomal subunits, was complicated by a contamination of the 30 S subunits by 50 S subunits. A contamination of approximately 15% was estimated by polyacrylamide gel electrophoresis and competitive hybridization. The ratios of incorporated radioactivity at zero time when drug and label were concomitantly added ranged between 5.4-6.0, after correction for this contamination. The decay of the 23 S rRNA followed a straight line which became parabolic in its final portion. These results, and theoretical considerations on the lag of rifampicin action and on the variance of the specific activity of the nucleotide pool at the very early times of the experimental observation, favor the interpretation that the 16 and 23 S rRNA genes in B. subtilis belong to the same transcriptional unit, being cotranscribed, in that order, by the same molecule of RNA polymerase. The transcriptional times of the 16 and 23 S rRNA genes were estimated as being 30 and 60 s, respectively.

Characterization of an adenylyl cyclase activity in particulate preparations from epimastigote forms of Trypanosoma cruzi.

Particulate preparations from epimastigote forms of Trypanosoma cruzi contain an adenylyl cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) which could be stored at --20 degree C and resisted 5 cycles of freezing and thawing over 10 days without significant loss of activity. The enzyme reaction strictly required Mn2+, had a pH optimum of 7.7 and was not inhibited or stimulated by NaF. Particles prepared in the presence of 10 mM Mn2+ or Mg2+ were 3--4 times more active than particles prepared in the absence of these cations. However, Mg2+ could not substitute for Mn2+ during enzyme assay nor did it enhance activity in the presence of saturating concentrations of Mn2+. The binary complex Mn - ATP2- was shown to be the true substrate for the adenylyl cyclase and free ATP was highly inhibitory. Plots of enzyme activity against equimolar concentrations of ATP - Mn gave sigmoid curves with n values in Hill plots ranging between 1.5 and 2.0. Excess Mn2+ activated the cyclase catalyzed reaction at low but not at high concentrations of ATP - Mn. In the presence of an excess of 1 mM Mn2+, which transforms 97% of the added ATP to productive Mn - ATP2- complex, the substrate saturation curve assumed a Michaelian pattern with an apparent Km =0.2 mM.

Purification of an adenylyl cyclase-containing plasma membrane fraction from Trypanosoma cruzi.

A fraction containing plasma membrane fragments has been purified from epimastigote forms of Trypanosoma cruzi. Cells were broken by sonic vibration under well defined conditions and membranes were isolated by differential centrifugation and equilibrium centrifugation in sucrose gradients. The co-purification (approximately 10-fold) of adenylyl cyclase and plasma membrane-bound radioactive iodine is highly suggestive of the localization of this enzyme in the plasma membrane of T. cruzi. Determination of succinate cytochrome c reductase and glucose-6-phosphatase activities, as well as of total amounts of DNA and RNA in the purified fraction, indicates a negligible contamination from other cellular organelles. The co-purification of acid phosphatase activity with bound labeled iodine and adenylyl cyclase was taken as circumstantial evidence that part of this enzyme also belongs to the plasma membrane of T. cruzi. Conventional electron miscroscopy and freeze-fracture images of this fraction are consistent with a highly enriched plasma membrane preparation.

Sensitive detection and strain classification of Trypanosoma cruzi by amplification of a ribosomal RNA sequence.

A sequence of about 100 bp of the 24S alpha ribosomal RNA was investigated for sensitive detection of Trypanosoma cruzi. It was shown that the target sequence is specific for this parasite and no cross-reactivity was observed with different species of pathogenic Leishmania, two strains of Trypanosoma rangeli or human RNA. Amplification of the sequence was obtained by reverse transcription coupled to polymerase chain reaction. Following this procedure the equivalent to 0.1% of the nucleic acid content of a single parasite cell could be detected either by ethidium staining or blot hybridization. The distribution of the target sequence in sixteen strains of T. cruzi was investigated. Positive amplification was obtained for all samples employing the same oligonucleotides as primers. However, amplified fragments of 125 bp were obtained in eight strains, while fragments of 110 bp were detected in the remaining eight isolates. No amplification of both classes of fragments has been detected in any of the strains examined. Dimorphism in the target region was confirmed by hybridization to specific internal probes and sequencing, allowing the division of T. cruzi strains in two groups. It is proposed that sensitive parasite detection could be achieved by rRNA amplification followed by hybridization to two probes derived from the target sequences of both groups of T. cruzi strains. Furthermore, the sequence dimorphism found in this sequence opens the perspective of strain typing simultaneous with parasite detection.

cAMP phosphodiesterase and activator protein of mammalian cAMP phosphodiesterase from Trypanosoma cruzi.

Epimastigote forms of Trypanosoma cruzi contain a soluble cAMP phosphodiesterase. Optimal activity was found at pH 8.0 and in the presence of 5 mM Mn2+. Other cations were less efficient and did not give rise to an additional stimulation when added in the presence of optimal concentrations of Mn2+. The enzyme is not Ca2+ dependent. The apparent Km of the enzyme for the substrate is 40 microM and no kinetic evidence for the existence of two enzymes has been found. Theophylline and caffein did not inhibit the T. cruzi cAMP phosphodiesterase. The enzyme activity does not change during cell growth suggesting that the fluctuation observed in the levels of cAMP are largely a response to variations in adenylyl cyclase activity. The intracellular concentrations of cAMP ranged between 0.04--0.15 microM. No evidence that the T. cruzi cAMP phosphodiesterase is regulated by an endogenous activator could be found. However, T. cruzi contains a heat-stable, low molecular weight, non-dialysable protein that activates mammalian cAMP phosphodiesterase in the presence of Ca2+. The properties so far studied of such an activator suggest that it might be equivalent to other Ca2+-dependent regulators described in vertebrate and invertebrate species.

Galactofuranose-containing glycoconjugates of epimastigote and trypomastigote forms of Trypanosoma cruzi.

Antiserum to LPPG, a lipopeptidophosphoglycan originally described on the surface of Trypanosoma cruzi epimastigotes of the Y strain, and antibodies to furanoic galactose (galf) were obtained in rabbits. A micromethod for the extraction and purification of LPPG from a limited amount of parasites is described. Analysis by Western blots of the purified glycoconjugate probed with both antisera confirmed the presence of galf-containing LPPG-like molecules in 10 different strains and clones of T. cruzi. An analogous approach indicated that trypomastigotes also contain LPPG-like components. Quantitation experiments allowed to calculate an average value of 1.0 x 10(7) LPPG molecules per epimastigote cell and 0.16 x 10(7) LPPG-like molecules per trypomastigote cell. Immunoelectron microscopy has shown a homogenous distribution of LPPG on the surface of epimastigotes. The trypomastigote population, however, is highly heterogenous with no more than 15% of the parasites being labeled by the anti-LPPG serum. Intense labeling has also been found in vesicles inside the epimastigote and trypomastigote forms. The distribution of galf epitopes among glycoconjugates of epimastigotes and trypomastigotes was further investigated. It was shown that galf units in epimastigotes are bound to low molecular mass compounds which co-migrate with LPPG whereas in trypomastigotes they have been found in both low molecular mass LPPG-like molecules and glycoproteins of 80-90 kDa. Direct chemical evidence for the presence of galf residues in the N-linked oligosaccharide chains of these surface glycoproteins has been obtained. Finally, the natural antigenicity of LPPG and galf in chronic Chagas' disease was investigated. It was found that all chronic chagasic sera investigated recognize this glycoconjugate and that an important part of such recognition can be attributed to galf residues. Furthermore, no correlation among reactivity to LPPG, strain zymodeme and clinical forms of the disease was found.

Cell surface antigens of Trypanosoma cruzi: possible correlation with the interiorization process in mammalian cells.

Differences were observed in the pattern of the cell surface proteins of evolutive stages of Trypanosoma cruzi after radioiodination of the cells and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity chromatography revealed that surface glycoproteins also vary in the epimastigote and trypomastigote forms of the parasite. The cell surface antigens of the two forms were identified after radioiodination or biosynthetic labeling with [35S]methionine and incubation with different antisera. Both epimastigotes and trypomastigotes share two main antigens, possibly glycoproteins, of apparent molecular weight 95 000 and 80 000, respectively, which are recognized by all antisera tested. On the other hand, the trypomastigote form possesses somewhat more cell surface antigens recognized both by rabbit anti-trypomastigote and by human Chagasic sera. Sequential immunoprecipitations with heterologous and homologous antisera established that an 85 000 and some higher molecular weight antigens are specific to the trypomastigote form. Preincubation of the trypomastigotes with sera from Chagasic patients elicits a 60% inhibition of the parasite interiorization into mammalian cells in culture when compared with normal human serum or anti-epimastigote serum. This result suggests that the antigens specific to the trypomastigote form are involved in the interiorization process.

Direct sialic acid transfer from a protein donor to glycolipids of trypomastigote forms of Trypanosoma cruzi.

Labeled sialoglycolipids were purified from tissue culture-derived trypomastigotes incubated with [3H]fetuin. Thin layer chromatography of [3H]sialoglycolipids showed three components with the same migration as gangliosides extracted from parasites incubated with [3H]palmitic acid. Neuraminidase treatment or mild acid hydrolysis confirmed the presence of [3H]sialyl residues in sialoglycolipids synthesized after [3H]fetuin incubation. Labeling was not observed when parasites were incubated with free [3H]sialic acid (C7 derivative), suggesting that sialyl residues are directly transferred in vivo to gangliosides, by an enzymatic reaction possibly catalysed by a sialyl transferase (transglycosylase). Sonicated extracts of trypomastigotes incubated with [3H]fetuin catalysed the labeling of endogenous glycoconjugates as well as of bovine brain gangliosides. The transglycosylase activity was found associated with the particulate fraction and could be solubilized with Triton X-100. The specific activity of the sialic acid transglycosylase in epimastigotes is 17% of that found in trypomastigotes. Addition of an excess free sialic acid did not inhibit the reaction, suggesting that transfer does not occur via a pool of free sialic acid.

Correlation of tunicamycin-sensitive surface glycoproteins from Trypanosoma cruzi with parasite interiorization into mammalian cells.

Trypomastigote forms of Trypanosoma cruzi lose infectivity to cultured mammalian cells when exposed to tunicamycin. Upon reincubation into fresh medium, parasites recover their full penetration capacity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides showed that tunicamycin-treated parasites present several components with altered electrophoretic mobility when compared with controls. Immunoprecipitation with rabbit hyperimmune and human chagasic sera indicated that the surface antigens of approximate molecular masses of 175-180, 120-125, 90-95 and 85 kDa are not encountered in tunicamycin-treated trypomastigotes. By affinity chromatography on wheat germ agglutinin-Sepharose, it was observed that the trypomastigote-specific 85 kDa glycoprotein (Tc-85) is affected by the drug. The other affected components are glycoproteins with affinity for concanavalin A. The results suggest that tunicamycin-sensitive surface glycoproteins from T. cruzi are involved in the parasite interiorization into mammalian cells.

The evolution of two Trypanosoma cruzi subgroups inferred from rRNA genes can be correlated with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host specificity.

The agent of Chagas disease, Trypanosoma cruzi, is divided into two highly divergent genetic subgroups, lineages 1 and 2, which include all typed strains isolated from humans, insect vectors, and sylvatic mammals. The evolutionary origin of these two T. cruzi lineages and the clinical importance of their identification, have been the subject of intense debate. Here, using molecular phylogenetic analysis, we found that the distance between the two T. cruzi lineages is equivalent to the distance between genera Leishmania and Endotrypanum. Also, we confirmed that T. rangeli is more closely related to T. cruzi than to T. brucei using the rDNA sequence from a human strain of T. rangeli. Phylogenetic trees based on small subunit rDNA sequences further suggest that the two T. cruzi lineages diverged between 88 and 37 million years (Myr) ago. We hypothesize that lineage 2 is indigenous to South America while lineage 1 has been introduced to South America recently, along with North American placental mammals, after the connection of the Americas in the Pliocene (5 Myr ago) or with caviomorph rodents and primates in the Oligocene (38 Myr ago). This would explain the preferential association of T. cruzi lineage 2 with marsupials and of lineage 1 with human disease. These two T. cruzi lineages are likely to be distinct species, or at least subspecies, because of their different ecological and epidemiological traits and estimated long period of independent evolution.

Sequence of the 24S alpha ribosomal RNA gene and characterization of a corresponding pseudogene from Trypanosoma cruzi.

Two genomic clones which cover a 30-kb region containing the ribosomal RNA cistron from Trypanosoma cruzi have been isolated. The location of the 18S, 24S alpha and 24S beta RNA species within the cistron was determined. The complete sequences of the genes corresponding to the 24S alpha RNA and to a small RNA (S1), as well as two internal transcribed spacers were obtained by sequencing a cDNA and a genomic fragment. A locus containing sequences related to the 24S alpha RNA has been determined. Sequence data and structural characterization of this locus strongly suggest that this region contains a 24S alpha RNA pseudogene.

DNA markers define two major phylogenetic lineages of Trypanosoma cruzi.

Parasitic protozoa within the taxon Trypanosoma cruzi are considered to be derived from multiple clonal lineages, and show broad genetic diversity as a result of propagation with little or no genetic exchange. We have analyzed a wide sample of T. cruzi isolates from vertebrate and invertebrate hosts by PCR amplification of a ribosomal RNA gene sequence, a mini-exon gene sequence and random amplified polymorphic DNA (RAPD). Amplification of the distinct rDNA and mini-exon gene sequences indicated a dimorphism within both of the tandemly-repeated genes: 125 or 110 bp products for rDNA and 300 or 350 bp products for the mini-exon. Within individual isolates, one of three associations was observed: the 125 bp rDNA product with the 300 bp mini-exon product (defined as group 1), the 110 bp rDNA product with the 350 bp mini-exon product (defined as group 2) and the presence of both rDNA amplification products with the mini-exon group 1 product (group 1/2). The RAPD analysis showed variability between individual isolates, however, tree analysis clearly indicated the presence of two major branches. Interestingly, the rDNA/mini-exon group 2 isolates correlated precisely with one branch of the RAPD-derived tree; group 1 and group 1/2 isolates correlated with the other branch. Our studies show a clear division of T. cruzi into two major lineages presenting a high phylogenetic divergence. Hypotheses are discussed to explain the origin of the two lineages as well as isolates that are hybrid for group 1 and 2 rDNA markers.

Molecular epidemiology of American trypanosomiasis in Brazil based on dimorphisms of rRNA and mini-exon gene sequences.

American trypanosomiasis is transmitted in nature via a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, or via a domestic cycle where the parasite comes into contact with humans through domiciliated triatomines. The pool of T. cruzi isolates consists of sub-populations presenting a broad genetic diversity. In contrast to the heterogeneity suggested by isoenzyme analysis, PCR amplification of sequences from the 24S alpha rRNA gene and from the non-transcribed spacer of the mini-exon gene indicated dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 157 T. cruzi isolates obtained from humans, triatomines and sylvatic mammalian reservoirs from 12 Brazilian states were analysed by the 24S alpha RNA and mini-exon typing approaches. The stocks were classified into the two proposed lineages and according to the domestic or sylvatic cycle of the parasite. Data presented provide evidence for a strong association of T. cruzi lineage 1 with the domestic cycle, while in the sylvatic cycle both lineages circulate equally. Molecular typing of human parasite isolates from three well-characterised endemic regions of Chagas disease (Minas Gerais, Paraiba and Piaui) and from Amazonas State, where T. cruzi is enzootic, suggests that in some endemic areas in Brazil there is a preferential linkage between both cycles mediated by lineage-1 stocks.

Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences.

Traditional molecular and biochemical methods, such as schizodeme analysis, karyotyping, DNA fingerprinting, and enzyme electrophoretic profiles, have shown a large variability among Trypanosoma cruzi isolates. In contrast to those results, polymerase chain reaction (PCR) amplification of sequences from the 24S alpha ribosomal RNA gene and from the mini-exon gene nontranscribed spacer indicated a dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 86 T. cruzi field stocks (68 isolated from humans with defined presentations of Chagas' disease and 18 from triatomines) derived from four Brazilian geographic areas were typed by the PCR assay based on the DNA sequences of the mini-exon and 24S alpha rRNA genes. These stocks were ordered into the two major T. cruzi lineages. Lineage 1 was associated mainly with human isolates and lineage 2 with the sylvatic cycle of the parasite.

Trypanosoma cruzi: different surface antigens of trypomastigotes are targets of lytic antibodies.

Polyclonal antisera were obtained in rabbits following immunization with disrupted epimastigote or trypomastigote forms; 8-methoxypsoralen-inactivated trypomastigotes; and surface trypomastigote antigens shed into the medium. High antibody levels were induced by all preparations as observed by indirect immunofluorescence and ELISA. However, antibodies promoting complement-mediated lysis of bloodstream forms were only detected in animals immunized with inactivated living trypomastigotes and shed surface antigens. Immunoprecipitation of radioiodinated parasites showed that sera with lytic antibodies bound strongly to a wide range of membrane polypeptides from 72 to 160 kDa. Immunoadsorption of antibodies from a serum with high lytic activity on specific classes of trypomastigote polypeptides indicated that independent antigens are targets of lytic antibodies and that common epitopes may exist in different trypomastigote components.