Serological based monitoring of a cohort of patients with chronic Chagas disease treated with benznidazole in a highly endemic area of northern Argentina.

This study aimed to evaluate well-documented diagnostic antigens, named B13, 1F8 and JL7 recombinant proteins, as potential markers of seroconversion in treated chagasic patients. Prospective study, involving 203 patients treated with benznidazole, was conducted from endemic areas of northern Argentina. Follow-up was possible in 107 out of them and blood samples were taken for serology and PCR assays before and 2, 3, 6, 12, 24 and 36 months after treatment initiation. Reactivity against Trypanosoma cruzi lysate and recombinant antigens was measured by ELISA. The rate of decrease of antibody titers showed nonlinear kinetics with an abrupt drop within the first three months after initiation of treatment for all studied antigens, followed by a plateau displaying a low decay until the end of follow-up. At this point, anti-B13, anti-1F8 and anti-JL7 titers were relatively close to the cut-off line, while anti-T. cruzi antibodies still remained positive. At baseline, 60.8% (45/74) of analysed patients tested positive for parasite DNA by PCR and during the follow-up period in 34 out of 45 positive samples (75.5%) could not be detected T. cruzi DNA. Our results suggest that these antigens might be useful as early markers for monitoring antiparasitic treatment in chronic Chagas disease.

Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2.

The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF-2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1ß, TcP2α, TcP2ß and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two-hybrid (Y2H) protein-protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1ß-TcP2α and TcP1ß-TcP2ß. Moreover P2 but not P1 proteins were able to homo-oligomerize. In addition, the region comprising amino acids 210-270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2ß were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF-2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF-2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0.

The heavy chain variable segment gene repertoire in chronic Chagas' heart disease.

Patients chronically infected with Trypanosoma cruzi develop chronic Chagas' heart disease (cChHD). Their Ab response is suspected to be involved in the cardiac pathogenesis. Reactivity of serum Abs from these patients has been extensively studied but little is known about the diversity of the in vivo IgG repertoire. We analyzed 125 variable H chain (VH) genes and compared it to repertoires from healthy individuals, and patients with autoimmune processes and other infections. VH were from plasma cells isolated from heart tissue of three cChHD patients and from a Fab combinatorial library derived from bone marrow of another cChHD patient. The role of the parasite in shaping the Ab repertoire was assessed analyzing VH genes before and after panning against T. cruzi Ag. Among recovered VH genes, a significantly increased representation of VH4 was observed. Plasma cells at the site of cardiac infiltration showed an increased VH1 usage. CDR3 lengths were similar to the ones found in the healthy repertoire and significantly shorter than in other infections. VH derived from anti-T. cruzi Fab and plasma cells showed a higher proportion of hypermutated genes, 46.9% and 43.75%, respectively, vs 30.9% of the cChHD patient repertoire, pointing to the role of parasite Ags in the shaping of the humoral response in Chagas' disease. No histological evidence of germinal center-like structures was observed in heart tissue. In accordance, VH analysis of heart plasmocytes revealed no evidence of clonal B cell expansion, suggesting that they migrated into heart tissue from secondary lymphoid organs.

Human recombinant antibodies against Trypanosoma cruzi ribosomal P2ß protein.

Patients with chronic Chagas' Heart Disease (cChHD) develop an antibody response that is suspected to be involved in the cardiac pathogenesis. The response against Trypanosoma cruzi ribosomal P proteins is of particular interest, as these antibodies can cross-react with host cardiac receptors causing electrophysiological alterations. To better understand the humoral anti-P response we constructed a single-chain variable fragment library derived from a cChHD patient. The variable heavy and light regions were amplified from bone-marrow RNA and subcloned into the vector pComb3X. The phage library was subsequently panned against T. cruzi ribosomal P2ß protein (TcP2ß). We obtained 3 different human recombinant antibodies that specifically reacted with TcP2ß in ELISA and Western blots. Two of them reacted with the C-terminal region of TcP2ß, peptide R13, as the recombinant autoanti-P antibodies from Systemic Lupus Erythematosus (SLE) patients. Interestingly, the third one was specific for TcP2ß but did not recognize R13, confirming the specific nature of the anti-P response in Chagas disease. Neither sequence nor VH usage similarities between Chagas and SLE anti-P autoantibodies were observed. Herein, the first human mAbs against TcP2ß have been obtained and characterized showing that the humoral anti-P response is directed against the parasite and does not include an autoimmune component.

The FIP-1 like polyadenylation factor in trypanosomes and the structural basis for its interaction with CPSF30.

In trypanosomes transcription is polycistronic and individual mRNAs are generated by a trans-splicing/polyadenylation coupled reaction. We identified a divergent trypanosome FIP1-like, a factor required for mRNA 3' end formation from yeasts to human. Here we showed that it is a nuclear protein with a speckled distribution essential for trypanosome viability. A strong interaction was found between TcFIP1-like and TcCPSF30, a component of the polyadenylation complex. We determined the specific amino acids in each protein involved in the interaction. Significant differences were found between the trypanosome interaction surface and its human counterpart. Although CPSF30/FIP1 interaction is known in other organisms, this is the first report mapping the interaction surface at the amino acid level.

Proteomic analysis of the Trypanosoma cruzi ribosomal proteins.

Trypanosoma cruzi is a parasite responsible for Chagas disease. The identification of new targets for chemotherapy is a major challenge for the control of this disease. Several lines of evidences suggest that the translational system in trypanosomatids show important differences compared to other eukaryotes. However, there little is known information about this. We have performed a detailed data mining search for ribosomal protein genes in T. cruzi genome data base combined with mass spectrometry analysis of purified T. cruzi ribosomes. Our results show that T. cruzi ribosomal proteins have approximately 50% sequence identity to yeast ones. Nevertheless, some parasite proteins are longer due to the presence of several N- or C-terminal extensions, which are exclusive of trypanosomatids. In particular, L19 and S21 show C-terminal extensions of 168 and 164 amino acids, respectively. In addition, we detected two 60S subunit proteins that had not been previously detected in the T. cruzi total proteome; namely, L22 and L42.

Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1.

Early in the assembly of eukaryotes the branch-point binding protein (BBP, also called SF1) recognizes the branch point sequence, whereas the heterodimer U2AF, consisting of a 65 and a 35 kDa subunit, contacts the polypyrimidine tract and the AG splice site, respectively. Herein, we identified, cloned and expressed the Trypanosoma cruzi and Trypanosoma brucei U2AF35, U2AF65 and SF1. Trypanosomatid U2AF65 strongly diverged from yeast and human homologues. On the contrary, trypanosomatid SF1 was conserved but lacked the C-terminal sequence present in the mammalian protein. Yeast two hybrid approaches were used to assess their interactions. The interaction between U2AF35 and U2AF65 was very weak or not detectable. However, as in other eukaryotes, the interaction between U2AF65 and SF1 was strong. At the cellular level, these results were confirmed by fractionation and affinity-selection experiments in which SF1 and U2AF65 were affinity-selected with TAP tagged SF1, but not with TAP tagged U2AF35. Silencing one of the three factors affected growth and trans-splicing in the first step of this reaction. Trypanosomes are the first described example of eukaryotic cells in which the interaction of two expressed U2AF factors seemed to be very weak, or not detectable.

The C-terminal end of P proteins mediates ribosome inactivation by trichosanthin but does not affect the pokeweed antiviral protein activity.

Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sdelta. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A(51) residue on 28Sdelta. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T.cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.

Molecular identification of Trypanosoma cruzi I tropism for central nervous system in Chagas reactivation due to AIDS.

Trypanosoma cruzi lineages, microsatellite allelic polymorphism, and mithocondrial gene haplotypes were directly typified from peripheral blood and cerebrospinal fluid specimens of a Bolivian patient with Chagas disease with accompanying AIDS and central nervous system severe involvement. Of note, the patient's blood was infected by a mixture of T. cruzi I and T. cruzi IId/e polyclonal populations while the cerebrospinal fluid showed only a monoclonal T. cruzi I population. Our findings do not corroborate the original assumption of innocuity for T. cruzi I in the southern cone of the Americas and highlight lineage I tropism for central nervous system causing lethal Chagas reactivation.

Gene expression patterns in Euglena gracilis: insights into the cellular response to environmental stress.

To better understand Euglena gracilis gene expression under different stress conditions (Chromium, Streptomycin or darkness), we undertook a survey of the E. gracilis transcriptome by cDNA sequencing and microarray analysis. First, we constructed a non-normalized cDNA library from the E. gracilis UTEX strain and sequenced a total of 1000 cDNAs. Six hundred and ten of these ESTs were similar to either Plantae or Protistae genes (e-value

Mapping of the protein-binding interface between splicing factors SF3b155 and p14 of Trypanosoma cruzi.

SF3b155 and p14 are essential components of spliceosome core that recognize the branch point adenosine, a critical step in splicing in eukaryotes. Trypanosomes are unusual since every transcribed gene is processed by trans-splicing instead of cis-splicing. Thus, the trans-spliceosome emerges as an interesting anti-parasitic drug target since this process is not present in mammalian hosts. Here, we present the orthologues of these proteins in Trypanosoma cruzi that interact strongly with each other. To define similarities and differences with the human pair, we performed a detailed alanine scan analysis that allowed us to identify the regions and the critical amino acids of T. cruzi SF3b155 involved in interaction with p14. We demonstrate that the T. cruzi SF3b155 interface is larger and contains more complex elements than its human counterpart. Additionally, our results provide the first insights into the core of the putative mRNA processing complex of trypanosomes.

Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease.

Congenital transmission of Trypanosoma cruzi may occur in some or all the gestations from a T. cruzi-infected mother. Variable rates of congenital transmission have been reported in different geographical areas where different parasitic strains predominate, suggesting that parasitic genotypes might play a role in the risk of congenital transmission. Moreover, in cases of transmission it is unknown if the whole maternal T. cruzi population or certain clones are preferentially transmitted by the transplacental route. In this study, bloodstream T. cruzi lineages were identified in blood samples from congenitally infected children, transmitting and non-transmitting mothers and unrelated Chagas disease patients, using improved PCR strategies targeted to nuclear genomic markers. T. cruzi IId was the prevalent genotype among 36/38 PCR-positive congenitally infected infants, 5/5 mothers who transmitted congenital Chagas disease, 12/13 mothers who delivered non-infected children and 28/34 unrelated Chagas disease patients, all coming from endemic localities of Argentina and Bolivia. These figures indicate no association between a particular genotype and vertical transmission. Furthermore, minicircle signatures from the maternal and infants' bloodstream trypanosomes were profiled by restriction fragment length polymorphism of the 330-bp PCR-amplified variable regions in seven cases of mothers and congenitally infected infants. Minicircle signatures were nearly identical between each mother and her infant/s and unique to each mother-infant/s case, a feature that was also observed in twin deliveries. Moreover, allelic size polymorphism analysis of microsatellite loci from populations transmitted to twins showed that all clones from the maternal polyclonal population were equally infective to both siblings.

Retinal dysfunction in patients with chronic Chagas' disease is associated to anti-Trypanosoma cruzi antibodies that cross-react with rhodopsin.

To investigate retinal involvement in chronic Chagas' disease, we performed electroretinography and retinal fluorescein angiography studies in chagasic patients. Our results demonstrated a dissociated electrophysiological response characterized by both an abnormal reduction of the electroretinographic b-wave amplitude and a delayed latency, under the dark-adaptated condition. These alterations are compatible with a selective dysfunction of the rods. Antibodies raised against Trypanosoma cruzi that also interact with beta1-adrenergic receptor blocked light stimulation of cGMP-phosphodiesterase in bovine rod membranes. The specificity from the antibody-rhodopsin interaction was confirmed by Western blot analysis and antigenic competition experiments. Our results suggest an immunomediated rhodopsin blockade. T. cruzi infection probably induces an autoimmune response against rhodopsin in the chronic phase of Chagas' disease through a molecular mimicry mechanism similar to that described previously on cardiac human beta1-adrenergic and M2-cholinergic receptors, all related to the same subfamily of G-protein-coupled receptors.

The VIPER elements of trypanosomes constitute a novel group of tyrosine recombinase-enconding retrotransposons.

VIPER was initially characterized as a 2326bp LTR-like retroelement associated to SIRE, a short interspersed repetitive element specific of Trypanosoma cruzi. It carried a single ORF that coded for a putative reverse transcriptase-RNAse H protein, suggesting that it could be a truncated copy of a longer retroelement. Herein we report the identification and characterization of a complete 4480bp long VIPER in the T. cruzi genome. The complete VIPER harbored three non-overlapped domains encoding for a GAG-like, a tyrosine recombinase and a reverse transcriptase-RNAse H proteins. VIPER elements were also found in the genomes of Trypanosoma brucei and Trypanosoma vivax, but not in Leishmania sp. On the basis of its reverse transcriptase phylogeny, VIPER was classified as an LTR retroelement. However, VIPER was structurally related to the tyrosine recombinase encoding retroelements, DIRS and Ngaro. Phylogenetic analysis showed that VIPER's tyrosine recombinase grouped with the transposases RCI1 of Escherichia coli and Ye24 and Ye72 of Haemophilus influenzae within a major branch of prokaryotic recombinases. Taken together, VIPER's structure, the nature of its tyrosine recombinase, the unique features of its reverse transcriptase catalytic consensus motif and the fact that it was found in Trypanosomes, an early branching eukaryote, suggest that VIPER may be the closest relative of the founder element of the tyrosine recombinase encoding retrotransposons known up to date. Our analysis revealed that tyrosine recombinase-encoding retroelements were originated as early in evolution as non-LTR retroelements and suggests that VIPER, Ngaro and DIRS elements may constitute a third group of retrotransposons, distinct from both LTR and non-LTR retroelements.

Evolution of non-LTR retrotransposons in the trypanosomatid genomes: Leishmania major has lost the active elements.

The ingi and L1Tc non-LTR retrotransposons--which constitute the ingi clade--are abundant in the genome of the trypanosomatid species Trypanosoma brucei and Trypanosoma cruzi, respectively. The corresponding retroelements, however, are not present in the genome of a closely related trypanosomatid, Leishmania major. To study the evolution of non-LTR retrotransposons in trypanosomatids, we have analyzed all ingi/L1Tc elements and highly degenerate ingi/L1Tc-related sequences identified in the recently completed T. brucei, T. cruzi and L. major genomes. The coding sequences of 242 degenerate ingi/L1Tc-related elements (DIREs) in all three genomes were reconstituted by removing the numerous frame shifts. Three independent phylogenetic analyses conducted on the conserved domains encoded by these elements show that all DIREs, including the 52 L. major DIREs, form a monophyletic group belonging to the ingi clade. This indicates that the trypanosomatid ancestor contained active mobile elements that have been retained in the Trypanosoma species, but were lost from L. major genome, where only remnants (DIRE) are detectable. All 242 DIREs analyzed group together according to their species origin with the exception of 11 T. cruzi DIREs which are close to the T. brucei ingi/DIRE families. Considering the absence of known horizontal transfer between the African T. brucei and the South-American T. cruzi, this suggests that this group of elements evolved at a lower rate when compared to the other trypanosomatid elements. Interestingly, the only nucleotide sequence conserved between ingi and L1Tc (the first 79 residues) is also present at the 5'-extremity of all the full length DIREs and suggests a possible role for this conserved motif, as well as for DIREs.

Contribution of yeast artificial chromosome-based physical maps to the final assembly of the Trypanosoma cruzi genome.

This chapter describes the methodology used both in performing the electrophoretic karyotype of the protozoan parasite Trypanosoma cruzi and mapping the genetic markers of the chromosomal bands, the construction of chromosome-specific YAC contigs, and their use to assign a chromosomal location to whole genome shotgun sequences.

Protein-protein interaction map of the Trypanosoma cruzi ribosomal P protein complex.

The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. Four out of five ribosomal P proteins of Trypanosoma cruzi, TcP0, TcP1alpha, TcP2alpha, and TcP2beta had been previously characterized. Data mining of the T. cruzi genome data base allowed the identification of the fifth member of this protein group, a novel P1 protein, named P1beta. To gain insight into the assembly of the stalk, a yeast two-hybrid based protein interaction map was generated. A parasite specific profile of interactions amongst the ribosomal P proteins of T. cruzi was evident. The TcP0 protein was able to interact with all both P1 and both P2 proteins. Moreover, the interactions between P2beta with P1alpha as well as with P2alpha were detected, as well as the ability of TcP2beta to homodimerize. A quantitative evaluation of the interactions established that the strongest interacting pair was TcP0-TcP1beta.

Analysis of the highly efficient pre-mRNA processing region HX1 of Trypanosoma cruzi.

Gene expression in trypanosomes is controlled mainly by post-transcriptional processes. This study was designed to analyse HX1, one of the TcP2beta upstream intergenic regions. It is an efficient pre-mRNA processing region that has been widely and successfully used in Trypanosoma cruzi transfection vectors. Herein we compared its performance with other regions within the same locus, and we identified the sequence elements responsible for the HX1 efficiency in trans-splicing and protein synthesis. Our mutational analysis showed the flexibility of the branch point site selection for HX1 trans-splicing process. We demonstrated also that its 12 nt 5'UTR sequence contributes to both trans-splicing and translation efficiency. The natural insertion of the repetitive element short interspersed repetitive element (SIRE) in one of the HX1 polypyrimidine tracts decreases the translated protein level by 40%. In this report, we demonstrated that this reduction is a consequence of a decrease of five-fold in the level of processed mRNA balanced by an increased efficiency of translation due to the inclusion of a 38 nt SIRE specific sequence in the 5'UTR of the mRNA.

Molecular diagnosis and typing of Trypanosoma cruzi populations and lineages in cerebral Chagas disease in a patient with AIDS.

Trypanosoma cruzi DNA was amplified from an intracranial biopsy and peripheral blood of an HIV patient with encephalitis; this episode was indicative of AIDS and congenital Chagas disease. The analysis of a micro-satellite locus revealed a multiclonal parasite population at the brain lesion with a more complex minicircle signature than that profiled in blood using restriction fragment length polymorphism (RFLP)-PCR and low stringency single primer (LSSP) PCR. Interestingly, different sublineages of T. cruzi II were detected in blood and brain by means of spliced-leader and 24salpha ribosomal-DNA amplifications. Quantitative-competitive PCR monitored the decrease of parasitic load during treatment and secondary prophylaxis with benznidazole. The synergy between parasiticidal plus anti-retroviral treatments probably allowed the patient a longer survival than usually achieved in similar episodes. This is the first case report demonstrating a differential distribution of natural parasite populations and sublineages in Chagas disease reactivation, showing the proliferation of cerebral variants not detectable in peripheral blood.

Preliminary structural studies of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi, a part of the P0/P1/P2 complex.

The Trypanosoma cruzi ribosomal P0 protein (TcP0) is part of the ribosomal stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. The TcP0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by Trypanosoma cruzi infection. The structural properties of TcP0 have been explored by circular dichroism, tryptophan fluorescence and limited proteolysis experiments. These studies were complemented by secondary structure consensus prediction analysis. The results suggest that the tertiary structure of TcP0 could be described as a compact, stable, trypsin-resistant, 200 residues long N-terminal domain belonging to the alpha/beta class and a more flexible, degradable, helical, 123 residues long C-terminal domain which could be involved in the formation of an unusual hydrophobic zipper with the ribosomal P1/P2 proteins to form the P0/P1/P2 complex.