An α-Gal antigenic surrogate as a biomarker of treatment evaluation in Trypanosoma cruzi-infected children. A retrospective cohort study.

BACKGROUND: Proper evaluation of therapeutic responses in Chagas disease is hampered by the prolonged persistence of antibodies to Trypanosoma cruzi measured by conventional serological tests and by the lack of sensitivity of parasitological tests. Previous studies indicated that tGPI-mucins, an α-Gal (α-d-Galp(1→3)-ß-d-Galp(1→4)-d-GlcNAc)-rich fraction obtained from T. cruzi trypomastigotes surface coat, elicit a strong and protective antibody response in infected individuals, which disappears soon after successful treatment. The cost and technical difficulties associated with tGPI-mucins preparation, however, preclude its routine implementation in clinical settings. METHODS/PRINCIPLE FINDINGS: We herein developed a neoglycoprotein consisting of a BSA scaffold decorated with several units of a synthetic α-Gal antigenic surrogate (α-d-Galp(1→3)-ß-d-Galp(1→4)-ß-d-Glcp). Serological responses to this reagent, termed NGP-Tri, were monitored by means of an in-house enzyme-linked immunosorbent assay (α-Gal-ELISA) in a cohort of 82 T. cruzi-infected and Benznidazole- or Nifurtimox-treated children (3 days to 16 years-old). This cohort was split into 3 groups based on the age of patients at the time of treatment initiation: Group 1 comprised 24 babies (3 days to 5 months-old; median = 26 days-old), Group 2 comprised 31 children (7 months to 3 years-old; median = 1.0-year-old) and Group 3 comprised 26 patients (3 to 16 years-old; median = 8.4 years-old). A second, control cohort (Group 4) included 39 non-infected infants (3 days to 5 months-old; median = 31 days-old) born to T. cruzi-infected mothers. Despite its suboptimal seroprevalence (58.4%), α-Gal-ELISA yielded shorter median time values of negativization (23 months [IC 95% 7 to 36 months] vs 60 months [IC 95% 15 to 83 months]; p = 0.0016) and higher rate of patient negative seroconversion (89.2% vs 43.2%, p < 0.005) as compared to conventional serological methods. The same effect was verified for every Group, when analyzed separately. Most remarkably, 14 out of 24 (58.3%) patients from Group 3 achieved negative seroconversion for α-Gal-ELISA while none of them were able to negativize for conventional serology. Detailed analysis of patients showing unconventional serological responses suggested that, in addition to providing a novel tool to shorten follow-up periods after chemotherapy, the α-Gal-ELISA may assist in other diagnostic needs in pediatric Chagas disease. CONCLUSIONS/SIGNIFICANCE: The tools evaluated here provide the cornerstone for the development of an efficacious, reliable, and straightforward post-therapeutic marker for pediatric Chagas disease.

Deep serological profiling of the Trypanosoma cruzi TSSA antigen reveals different epitopes and modes of recognition by Chagas disease patients.

BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, displays a highly structured population, with multiple strains that can be grouped into 6-7 evolutionary lineages showing variable eco-epidemiological traits and likely also distinct disease-associated features. Previous works have shown that antibody responses to 'isoforms' of the polymorphic parasite antigen TSSA enable robust and sensitive identification of the infecting strain with near lineage-level resolution. To optimize the serotyping performance of this molecule, we herein used a combination of immunosignaturing approaches based on peptide microarrays and serum samples from Chagas disease patients to establish a deep linear B-cell epitope profiling of TSSA. METHODS/PRINCIPLE FINDINGS: Our assays revealed variations in the seroprevalence of TSSA isoforms among Chagas disease populations from different settings, hence strongly supporting the differential distribution of parasite lineages in domestic cycles across the Americas. Alanine scanning mutagenesis and the use of peptides of different lengths allowed us to identify key residues involved in antibody pairing and the presence of three discrete B-cell linear epitopes in TSSAII, the isoform with highest seroprevalence in human infections. Comprehensive screening of parasite genomic repositories led to the discovery of 9 novel T. cruzi TSSA variants and one TSSA sequence from the phylogenetically related bat parasite T. cruzi marinkellei. Further residue permutation analyses enabled the identification of diagnostically relevant or non-relevant substitutions among TSSA natural polymorphisms. Interestingly, T. cruzi marinkellei TSSA displayed specific serorecognition by one chronic Chagas disease patient from Colombia, which warrant further investigations on the diagnostic impact of such atypical TSSA. CONCLUSIONS/SIGNIFICANCE: Overall, our findings shed new light into TSSA evolution, epitope landscape and modes of recognition by Chagas disease patients; and have practical implications for the design and/or evaluation of T. cruzi serotyping strategies.

Characterization of ADAT2/3 molecules in Trypanosoma cruzi and regulation of mucin gene expression by tRNA editing.

Adenosine-to-inosine conversion at position 34 (A34-to-I) of certain tRNAs is essential for expanding their decoding capacity. This reaction is catalyzed by the adenosine deaminase acting on tRNA (ADAT) complex, which in Eukarya is formed by two subunits: ADAT2 and ADAT3. We herein identified and thoroughly characterized the ADAT molecules from the protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas Disease. TcADAT2 and TcADAT3 spontaneously form a catalytically active complex, as shown by expression in engineered bacteria and/or by the increased ex vivo tRNA A-to-I deamination activity of T. cruzi epimastigotes overexpressing TcADAT subunits. Importantly, enhanced TcADAT2/3 activity in transgenic parasites caused a shift in their in vivo tRNAThrAGU signature, which correlated with significant changes in the expression of the Thr-rich TcSMUG proteins. To our knowledge, this is the first evidence indicating that T. cruzi tRNA editing can be modulated in vivo, in turn post-transcriptionally changing the expression of specific genes. Our findings suggest tRNA editing/availability as a forcible step in controlling gene expression and driving codon adaptation in T. cruzi. Moreover, we unveil certain differences between parasite and mammalian host tRNA editing and processing, such as cytosine-to-uridine conversion at position 32 of tRNAThrAGU in T. cruzi, that may be exploited for the identification of novel druggable targets of intervention.

Serological Approaches for Trypanosoma cruzi Strain Typing.

Trypanosoma cruzi, the protozoan agent of Chagas' disease, displays a complex population structure made up of multiple strains showing a diverse ecoepidemiological distribution. Parasite genetic variability may be associated with disease outcome, hence stressing the need to develop methods for T. cruzi typing in vivo. Serological typing methods that exploit the presence of host antibodies raised against polymorphic parasite antigens emerge as an appealing approach to address this issue. These techniques are robust, simple, cost-effective, and are not curtailed by methodological/biological limitations intrinsic to available genotyping methods. Here, we critically assess the progress towards T. cruzi serotyping and discuss the opportunity provided by high-throughput immunomics to improve this field.

Trypanosoma cruzi surface mucins are involved in the attachment to the Triatoma infestans rectal ampoule.

BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, is a protozoan parasite transmitted to humans by blood-sucking triatomine vectors. However, and despite its utmost biological and epidemiological relevance, T. cruzi development inside the digestive tract of the insect remains a poorly understood process. METHODS/PRINCIPLE FINDINGS: Here we showed that Gp35/50 kDa mucins, the major surface glycoproteins from T. cruzi insect-dwelling forms, are involved in parasite attachment to the internal cuticle of the triatomine rectal ampoule, a critical step leading to its differentiation into mammal-infective forms. Experimental evidence supporting this conclusion could be summarized as follows: i) native and recombinant Gp35/50 kDa mucins directly interacted with hindgut tissues from Triatoma infestans, as assessed by indirect immunofluorescence assays; ii) transgenic epimastigotes over-expressing Gp35/50 kDa mucins on their surface coat exhibited improved attachment rates (~2-3 fold) to such tissues as compared to appropriate transgenic controls and/or wild-type counterparts; and iii) certain chemically synthesized compounds derived from Gp35/50 kDa mucins were able to specifically interfere with epimastigote attachment to the inner lining of T. infestans rectal ampoules in ex vivo binding assays, most likely by competing with or directly blocking insect receptor(s). A solvent-exposed peptide (smugS peptide) from the Gp35/50 kDa mucins protein scaffolds and a branched, Galf-containing trisaccharide (Galfß1-4[Galpß1-6]GlcNAcα) from their O-linked glycans were identified as main adhesion determinants for these molecules. Interestingly, exogenous addition of a synthetic Galfß1-4[Galpß1-6]GlcNAcα derivative or of oligosaccharides containing this structure impaired the attachment of Dm28c but not of CL Brener epimastigotes to triatomine hindgut tissues; which correlates with the presence of Galf residues on the Gp35/50 kDa mucins' O-glycans on the former but not the latter parasite clone. CONCLUSION/SIGNIFICANCE: These results provide novel insights into the mechanisms underlying T. cruzi-triatomine interplay, and indicate that inter-strain variations in the O-glycosylation of Gp35/50 kDa mucins may lead to differences in parasite differentiation and hence, in parasite transmissibility to the mammalian host. Most importantly, our findings point to Gp35/50 kDa mucins and/or the Galf biosynthetic pathway, which is absent in mammals and insects, as appealing targets for the development of T. cruzi transmission-blocking strategies.

Synthesis and characterization of α-d-Galp-(1 → 3)-ß-d-Galp epitope-containing neoglycoconjugates for chagas disease serodiagnosis.

The immunodominant epitope α-d-Galp-(1 → 3)-ß-d-Galp-(1 → 4)-d-GlcNAc, expressed in the mucins of the infective trypomastigote stage of Trypanosoma cruzi has been proposed for multiple clinical applications, from serodiagnosis of protozoan caused diseases to xenotransplantation or cancer vaccinology. It was previously shown that the analogue trisaccharide, with glucose in the reducing end instead of GlcNAc, was as efficient as the natural trisaccharide for recognition of chagasic antibodies. Here we describe the synthesis of α-d-Galp-(1 → 3)-ß-d-Galp-(1 → 4)-d-Glcp functionalized as the 6-aminohexyl glycoside and its conjugation to BSA using the squarate method. The conjugate of 6-aminohexyl α-d-Galp-(1 → 3)-ß-d-Galp was also prepared. Both neoglycoconjugates were recognized by serum samples of Trypanosoma cruzi-infected individuals and thus, are promising tools for the improvement of Chagas disease diagnostic applications.

Homologous Expression of Glycosylphosphatidylinositol-Anchored Glycoproteins in Trypanosoma cruzi.

The surface coat of Trypanosoma cruzi is covered with glycosylphosphatidylinositol (GPI)-anchored glycoproteins (GAGPs) that contribute to parasite protection and to the establishment of a persistent infection in both the insect vector and the mammalian host. Multiple GAGPs that vary by amino acid sequence and/or posttranslational modifications are co-expressed on the parasite surface coat, hence curtailing structural/functional analyses on these molecules. Studies in our lab have indicated that GAGP-tagged variants expressed by transfected parasites undergo analogous posttranslational processing than endogenous ones and therefore constitute suitable tools to overcome these limitations. In this chapter, we detail the entire methodological pipeline for the efficient homologous expression of GAGPs in T. cruzi: from a simple strategy for the simultaneously cloning and tagging of the gene of interest to the biochemical validation of the parasite-expressed product.

Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins.

BACKGROUND: TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3-5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen. METHODS/PRINCIPLE FINDINGS: Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis. CONCLUSION/SIGNIFICANCE: This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications.

New human isolates of Trypanosoma cruzi confirm the predominance of hybrid lineages in domestic transmission cycle of the Argentinean Chaco.

Trypanosoma cruzi, the etiological agent of Chagas disease, was initially classified into 6 Discrete Typing Units (DTUs). The hybrid DTUs TcV and TcVI are the most frequent in domestic transmission cycles throughout the Southern Cone countries of South America. Here, we genotyped parasite isolates from human residents in Pampa del Indio municipality, Chaco, to further characterize the structure of T. cruzi populations, and to assess the degree of overlapping between the domestic and sylvatic transmission cycles. Artificial xenodiagnostic tests were performed to blood samples from 125 T. cruzi-seropositive people (age range, 3-70 years) who represented 14.3% of all seropositive residents identified. Parasites were obtained from feces of T. cruzi-infected Triatoma infestans examined 30 or 60 days after blood-feeding, and grown in vitro. The cultured parasites were genotyped by means of two PCR-based protocols. DTUs were determined from 39 (31%) patients residing in 28 dwellings. The only DTUs identified were TcV (92%) and TcVI (8-36%). Households with more than one parasite isolate consistently displayed the same DTU. Further sequencing of a fragment of the TcMK gene from selected samples argue against the occurrence of mixed TcV-TcVI infections in the study population. Sequencing data revealed an unexpected degree of genetic variability within TcV including two apparently robust subgroups of isolates. Our results for human residents confirm the predominance of hybrid lineages (TcV and to a much lesser extent TcVI) and the absence of sylvatic genotypes (TcI and TcIII) in (peri)domestic transmission cycles in the Argentinean Chaco area. 245 words.

The Trypomastigote Small Surface Antigen from Trypanosoma cruzi Improves Treatment Evaluation and Diagnosis in Pediatric Chagas Disease.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi Assessment of parasitological cure upon treatment with available drugs relies on achieving consistent negative results in conventional parasitological and serological tests, which may take years to assess. Here, we evaluated the use of a recombinant T. cruzi antigen termed trypomastigote small surface antigen (TSSA) as an early serological marker of drug efficacy in T. cruzi-infected children. A cohort of 78 pediatric patients born to T. cruzi-infected mothers was included in this study. Only 39 of the children were infected with T. cruzi, and they were immediately treated with trypanocidal drugs. Serological responses against TSSA were evaluated in infected and noninfected populations during the follow-up period using an in-house enzyme-linked immunosorbent assay (ELISA) and compared to conventional serological methods. Anti-TSSA antibody titers decreased significantly faster than anti-whole parasite antibodies detected by conventional serology both in T. cruzi-infected patients undergoing effective treatment and in those not infected. The differential kinetics allowed a significant reduction in the required follow-up periods to evaluate therapeutic responses or to rule out maternal-fetal transmission. Finally, we present the case of a congenitally infected patient with an atypical course in whom TSSA provided an early marker for T. cruzi infection. In conclusion, we showed that TSSA was efficacious both for rapid assessment of treatment efficiency and for early negative diagnosis in infants at risk of congenital T. cruzi infection. Based upon these findings we propose the inclusion of TSSA for refining the posttherapeutic cure criterion and other diagnostic needs in pediatric Chagas disease.

The Trypomastigote Small Surface Antigen (TSSA) regulates Trypanosoma cruzi infectivity and differentiation.

BACKGROUND: TSSA (Trypomastigote Small Surface Antigen) is an antigenic, adhesion molecule displayed on the surface of Trypanosoma cruzi trypomastigotes. TSSA displays substantial sequence identity to members of the TcMUC gene family, which code for the trypomastigote mucins (tGPI-mucins). In addition, TSSA bears sequence polymorphisms among parasite strains; and two TSSA variants expressed as recombinant molecules (termed TSSA-CL and TSSA-Sy) were shown to exhibit contrasting features in their host cell binding and signaling properties. METHODS/PRINCIPLE FINDINGS: Here we used a variety of approaches to get insights into TSSA structure/function. We show that at variance with tGPI-mucins, which rely on their extensive O-glycoslylation to achieve their protective function, TSSA seems to be displayed on the trypomastigote coat as a hypo-glycosylated molecule. This has a functional correlate, as further deletion mapping experiments and cell binding assays indicated that exposition of at least two peptidic motifs is critical for the engagement of the 'adhesive' TSSA variant (TSSA-CL) with host cell surface receptor(s) prior to trypomastigote internalization. These motifs are not conserved in the 'non-adhesive' TSSA-Sy variant. We next developed transgenic lines over-expressing either TSSA variant in different parasite backgrounds. In strict accordance to recombinant protein binding data, trypomastigotes over-expressing TSSA-CL displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. These phenotypes could be specifically counteracted by exogenous addition of peptides spanning the TSSA-CL adhesion motifs. In addition, and irrespective of the TSSA variant, over-expression of this molecule leads to an enhanced trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation. CONCLUSION/SIGNIFICANCE: In this study we provided novel evidence indicating that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi trypomastigotes but also on the phenotypic variability displayed by parasite strains.

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.

Mapping antigenic motifs in the trypomastigote small surface antigen from Trypanosoma cruzi.

The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease.

Cytokine production but lack of proliferation in peripheral blood mononuclear cells from chronic Chagas' disease cardiomyopathy patients in response to T. cruzi ribosomal P proteins.

BACKGROUND: Trypanosoma cruzi ribosomal P proteins, P2ß and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with ß1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2ß, the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2ß or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells. CONCLUSIONS/SIGNIFICANCE: Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection.