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.

The Trypanosoma cruzi Antigen and Epitope Atlas: antibody specificities in Chagas disease patients across the Americas.

During an infection the immune system produces pathogen-specific antibodies. These antibody repertoires become specific to the history of infections and represent a rich source of diagnostic markers. However, the specificities of these antibodies are mostly unknown. Here, using high-density peptide arrays we examined the human antibody repertoires of Chagas disease patients. Chagas disease is a neglected disease caused by Trypanosoma cruzi, a protozoan parasite that evades immune mediated elimination and mounts long-lasting chronic infections. We describe a proteome-wide search for antigens, characterised their linear epitopes, and show their reactivity on 71 individuals from diverse human populations. Using single-residue mutagenesis we revealed the core functional residues for 232 of these epitopes. Finally, we show the diagnostic performance of identified antigens on challenging samples. These datasets enable the study of the Chagas antibody repertoire at an unprecedented depth and granularity, while also providing a rich source of serological biomarkers.

Targeting trypanosomes: how chemogenomics and artificial intelligence can guide drug discovery.

Trypanosomatids are protozoan parasites that cause human and animal neglected diseases. Despite global efforts, effective treatments are still much needed. Phenotypic screens have provided several chemical leads for drug discovery, but the mechanism of action for many of these chemicals is currently unknown. Recently, chemogenomic screens assessing the susceptibility or resistance of parasites carrying genome-wide modifications started to define the mechanism of action of drugs at large scale. In this review, we discuss how genomics is being used for drug discovery in trypanosomatids, how integration of chemical and genomics data from these and other organisms has guided prioritisations of candidate therapeutic targets and additional chemical starting points, and how these data can fuel the expansion of drug discovery pipelines into the era of artificial intelligence.

Biological features of TcM: A new Trypanosoma cruzi isolate from Argentina classified into TcV lineage.

Trypanosoma cruzi, the etiologic agent of Chagas disease (CD) presents a wide genetic and phenotypic diversity that is classified into seven lineages or discrete typing units (DTU: TcI to TcVI and Tcbat). Although isolates and strains that belong to a particular group can share some attributes, such as geographic distribution, others like growth rate, cell tropism, and response to treatment can be highly variable. In addition, studies that test new trypanocidal drugs are frequently conducted on T. cruzi strains maintained for a long time in axenic culture, resulting in changes in parasite virulence and other important features. This work aimed to isolate and characterize a new T. cruzi strain from a chronic Chagas disease patient. The behavior of this isolate was studied by using standard in vitro assays and in vivo mice infection tests and compared with the T. cruzi Y strain (TcY), broadly used in research laboratories worldwide. Data showed that TcM behaves as a slow-growing strain in vitro that develops chronic infections in mice and displays high tropism to muscular tissues, in accordance with its clinical performance. In contrast, the Y strain behaved as an acute strain that can infect different types of cells and tissues. Interestingly, TcM, which belongs to DTU TcV, is more susceptible to benznidazole than TcY, a TcII strain considered moderately resistant to this drug. These differential properties contribute to the characterization of a TcV strain, one of the main lineages in the southern countries of South America, and open the possibility to introduce changes that improve the management of Chagas patients in the future.

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.

APRANK: Computational Prioritization of Antigenic Proteins and Peptides From Complete Pathogen Proteomes.

Availability of highly parallelized immunoassays has renewed interest in the discovery of serology biomarkers for infectious diseases. Protein and peptide microarrays now provide a rapid, high-throughput platform for immunological testing and validation of potential antigens and B-cell epitopes. However, there is still a need for tools to prioritize and select relevant probes when designing these arrays. In this work we describe a computational method called APRANK (Antigenic Protein and Peptide Ranker) which integrates multiple molecular features to prioritize potentially antigenic proteins and peptides in a given pathogen proteome. These features include subcellular localization, presence of repetitive motifs, natively disordered regions, secondary structure, transmembrane spans and predicted interaction with the immune system. We trained and tested this method with a number of bacteria and protozoa causing human diseases: Borrelia burgdorferi (Lyme disease), Brucella melitensis (Brucellosis), Coxiella burnetii (Q fever), Escherichia coli (Gastroenteritis), Francisella tularensis (Tularemia), Leishmania braziliensis (Leishmaniasis), Leptospira interrogans (Leptospirosis), Mycobacterium leprae (Leprae), Mycobacterium tuberculosis (Tuberculosis), Plasmodium falciparum (Malaria), Porphyromonas gingivalis (Periodontal disease), Staphylococcus aureus (Bacteremia), Streptococcus pyogenes (Group A Streptococcal infections), Toxoplasma gondii (Toxoplasmosis) and Trypanosoma cruzi (Chagas Disease). We have evaluated this integrative method using non-parametric ROC-curves and made an unbiased validation using Onchocerca volvulus as an independent data set. We found that APRANK is successful in predicting antigenicity for all pathogen species tested, facilitating the production of antigen-enriched protein subsets. We make APRANK available to facilitate the identification of novel diagnostic antigens in infectious diseases.

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.

Screening and Identification of Metacaspase Inhibitors: Evaluation of Inhibition Mechanism and Trypanocidal Activity.

A common strategy to identify new antiparasitic agents is the targeting of proteases, due to their essential contributions to parasite growth and development. Metacaspases (MCAs) are cysteine proteases present in fungi, protozoa, and plants. These enzymes, which are associated with crucial cellular events in trypanosomes, are absent in the human host, thus arising as attractive drug targets. To find new MCA inhibitors with trypanocidal activity, we adapted a continuous fluorescence enzymatic assay to a medium-throughput format and carried out screening of different compound collections, followed by the construction of dose-response curves for the most promising hits. We used MCA5 from Trypanosoma brucei (TbMCA5) as a model for the identification of inhibitors from the GlaxoSmithKline HAT and CHAGAS chemical boxes. We also assessed a third collection of nine compounds from the Maybridge database that had been identified by virtual screening as potential inhibitors of the cysteine peptidase falcipain-2 (clan CA) from Plasmodium falciparum Compound HTS01959 (from the Maybridge collection) was the most potent inhibitor, with a 50% inhibitory concentration (IC50) of 14.39 µM; it also inhibited other MCAs from T. brucei and Trypanosoma cruzi (TbMCA2, 4.14 µM; TbMCA3, 5.04 µM; TcMCA5, 151 µM). HTS01959 behaved as a reversible, slow-binding, and noncompetitive inhibitor of TbMCA2, with a mechanism of action that included redox components. Importantly, HTS01959 displayed trypanocidal activity against bloodstream forms of T. brucei and trypomastigote forms of T. cruzi, without cytotoxic effects on Vero cells. Thus, HTS01959 is a promising starting point to develop more specific and potent chemical structures to target MCAs.

TDR Targets 6: driving drug discovery for human pathogens through intensive chemogenomic data integration.

The volume of biological, chemical and functional data deposited in the public domain is growing rapidly, thanks to next generation sequencing and highly-automated screening technologies. These datasets represent invaluable resources for drug discovery, particularly for less studied neglected disease pathogens. To leverage these datasets, smart and intensive data integration is required to guide computational inferences across diverse organisms. The TDR Targets chemogenomics resource integrates genomic data from human pathogens and model organisms along with information on bioactive compounds and their annotated activities. This report highlights the latest updates on the available data and functionality in TDR Targets 6. Based on chemogenomic network models providing links between inhibitors and targets, the database now incorporates network-driven target prioritizations, and novel visualizations of network subgraphs displaying chemical- and target-similarity neighborhoods along with associated target-compound bioactivity links. Available data can be browsed and queried through a new user interface, that allow users to perform prioritizations of protein targets and chemical inhibitors. As such, TDR Targets now facilitates the investigation of drug repurposing against pathogen targets, which can potentially help in identifying candidate targets for bioactive compounds with previously unknown targets. TDR Targets is available at https://tdrtargets.org.

Potent and selective inhibitors for M32 metallocarboxypeptidases identified from high-throughput screening of anti-kinetoplastid chemical boxes.

Enzymes of the M32 family are Zn-dependent metallocarboxypeptidases (MCPs) widely distributed among prokaryotic organisms and just a few eukaryotes including Trypanosoma brucei and Trypanosoma cruzi, the causative agents of sleeping sickness and Chagas disease, respectively. These enzymes are absent in humans and several functions have been proposed for trypanosomatid M32 MCPs. However, no synthetic inhibitors have been reported so far for these enzymes. Here, we present the identification of a set of inhibitors for TcMCP-1 and TbMCP-1 (two trypanosomatid M32 enzymes sharing 71% protein sequence identity) from the GlaxoSmithKline HAT and CHAGAS chemical boxes; two collections grouping 404 compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. For this purpose, we adapted continuous fluorescent enzymatic assays to a medium-throughput format and carried out the screening of both collections, followed by the construction of dose-response curves for the most promising hits. As a result, 30 micromolar-range inhibitors were discovered for one or both enzymes. The best hit, TCMDC-143620, showed sub-micromolar affinity for TcMCP-1, inhibited TbMCP-1 in the low micromolar range and was inactive against angiotensin I-converting enzyme (ACE), a potential mammalian off-target structurally related to M32 MCPs. This is the first inhibitor reported for this family of MCPs and considering its potency and specificity, TCMDC-143620 seems to be a promissory starting point to develop more specific and potent chemical tools targeting M32 MCPs from trypanosomatid parasites.

FastqCleaner: an interactive Bioconductor application for quality-control, filtering and trimming of FASTQ files.

BACKGROUND: Exploration and processing of FASTQ files are the first steps in state-of-the-art data analysis workflows of Next Generation Sequencing (NGS) platforms. The large amount of data generated by these technologies has put a challenge in terms of rapid analysis and visualization of sequencing information. Recent integration of the R data analysis platform with web visual frameworks has stimulated the development of user-friendly, powerful, and dynamic NGS data analysis applications. RESULTS: This paper presents FastqCleaner, a Bioconductor visual application for both quality-control (QC) and pre-processing of FASTQ files. The interface shows diagnostic information for the input and output data and allows to select a series of filtering and trimming operations in an interactive framework. FastqCleaner combines the technology of Bioconductor for NGS data analysis with the data visualization advantages of a web environment. CONCLUSIONS: FastqCleaner is an user-friendly, offline-capable tool that enables access to advanced Bioconductor infrastructure. The novel concept of a Bioconductor interactive application that can be used without the need for programming skills, makes FastqCleaner a valuable resource for NGS data analysis.

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.

Author Correction: Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Next-generation ELISA diagnostic assay for Chagas Disease based on the combination of short peptidic epitopes.

Chagas Disease, caused by the protozoan Trypanosoma cruzi, is a major health and economic problem in Latin America for which no vaccine or appropriate drugs for large-scale public health interventions are yet available. Accurate diagnosis is essential for the early identification and follow up of vector-borne cases and to prevent transmission of the disease by way of blood transfusions and organ transplantation. Diagnosis is routinely performed using serological methods, some of which require the production of parasite lysates, parasite antigenic fractions or purified recombinant antigens. Although available serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. Short peptides spanning linear B-cell epitopes have proven ideal serodiagnostic reagents in a wide range of diseases. Recently, we have conducted a large-scale screening of T. cruzi linear B-cell epitopes using high-density peptide chips, leading to the identification of several hundred novel sequence signatures associated to chronic Chagas Disease. Here, we performed a serological assessment of 27 selected epitopes and of their use in a novel multipeptide-based diagnostic method. A combination of 7 of these peptides were finally evaluated in ELISA format against a panel of 199 sera samples (Chagas-positive and negative, including sera from Leishmaniasis-positive subjects). The multipeptide formulation displayed a high diagnostic performance, with a sensitivity of 96.3% and a specificity of 99.15%. Therefore, the use of synthetic peptides as diagnostic tools are an attractive alternative in Chagas' disease diagnosis.

High-resolution profiling of linear B-cell epitopes from mucin-associated surface proteins (MASPs) of Trypanosoma cruzi during human infections.

BACKGROUND: The Trypanosoma cruzi genome bears a huge family of genes and pseudogenes coding for Mucin-Associated Surface Proteins (MASPs). MASP molecules display a 'mosaic' structure, with highly conserved flanking regions and a strikingly variable central and mature domain made up of different combinations of a large repertoire of short sequence motifs. MASP molecules are highly expressed in mammal-dwelling stages of T. cruzi and may be involved in parasite-host interactions and/or in diverting the immune response. METHODS/PRINCIPLE FINDINGS: High-density microarrays composed of fully overlapped 15mer peptides spanning the entire sequences of 232 non-redundant MASPs (~25% of the total MASP content) were screened with chronic Chagasic sera. This strategy led to the identification of 86 antigenic motifs, each one likely representing a single linear B-cell epitope, which were mapped to 69 different MASPs. These motifs could be further grouped into 31 clusters of structurally- and likely antigenically-related sequences, and fully characterized. In contrast to previous reports, we show that MASP antigenic motifs are restricted to the central and mature region of MASP polypeptides, consistent with their intracellular processing. The antigenicity of these motifs displayed significant positive correlation with their genome dosage and their relative position within the MASP polypeptide. In addition, we verified the biased genetic co-occurrence of certain antigenic motifs within MASP polypeptides, compatible with proposed intra-family recombination events underlying the evolution of their coding genes. Sequences spanning 7 MASP antigenic motifs were further evaluated using distinct synthesis/display approaches and a large panel of serum samples. Overall, the serological recognition of MASP antigenic motifs exhibited a remarkable non normal distribution among the T. cruzi seropositive population, thus reducing their applicability in conventional serodiagnosis. As previously observed in in vitro and animal infection models, immune signatures supported the concurrent expression of several MASPs during human infection. CONCLUSIONS/SIGNIFICANCE: In spite of their conspicuous expression and potential roles in parasite biology, this study constitutes the first unbiased, high-resolution profiling of linear B-cell epitopes from T. cruzi MASPs during human infection.

Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes.

American Trypanosomiasis or Chagas disease is a prevalent, neglected and serious debilitating illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The current chemotherapy is limited only to nifurtimox and benznidazole, two drugs that have poor efficacy in the chronic phase and are rather toxic. In this scenario, more efficacious and safer drugs, preferentially acting through a different mechanism of action and directed against novel targets, are particularly welcome. Cruzipain, the main papain-like cysteine peptidase of T. cruzi, is an important virulence factor and a chemotherapeutic target with excellent pre-clinical validation evidence. Here, we present the identification of new Cruzipain inhibitory scaffolds within the GlaxoSmithKline HAT (Human African Trypanosomiasis) and Chagas chemical boxes, two collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. We have adapted a continuous enzymatic assay to a medium-throughput format and carried out a primary screening of both collections, followed by construction and analysis of dose-response curves of the most promising hits. Using the identified compounds as a starting point a substructure directed search against CHEMBL Database revealed plausible common scaffolds while docking experiments predicted binding poses and specific interactions between Cruzipain and the novel inhibitors.

A Multilayer Network Approach for Guiding Drug Repositioning in Neglected Diseases.

Drug development for neglected diseases has been historically hampered due to lack of market incentives. The advent of public domain resources containing chemical information from high throughput screenings is changing the landscape of drug discovery for these diseases. In this work we took advantage of data from extensively studied organisms like human, mouse, E. coli and yeast, among others, to develop a novel integrative network model to prioritize and identify candidate drug targets in neglected pathogen proteomes, and bioactive drug-like molecules. We modeled genomic (proteins) and chemical (bioactive compounds) data as a multilayer weighted network graph that takes advantage of bioactivity data across 221 species, chemical similarities between 1.7 105 compounds and several functional relations among 1.67 105 proteins. These relations comprised orthology, sharing of protein domains, and shared participation in defined biochemical pathways. We showcase the application of this network graph to the problem of prioritization of new candidate targets, based on the information available in the graph for known compound-target associations. We validated this strategy by performing a cross validation procedure for known mouse and Trypanosoma cruzi targets and showed that our approach outperforms classic alignment-based approaches. Moreover, our model provides additional flexibility as two different network definitions could be considered, finding in both cases qualitatively different but sensible candidate targets. We also showcase the application of the network to suggest targets for orphan compounds that are active against Plasmodium falciparum in high-throughput screens. In this case our approach provided a reduced prioritization list of target proteins for the query molecules and showed the ability to propose new testable hypotheses for each compound. Moreover, we found that some predictions highlighted by our network model were supported by independent experimental validations as found post-facto in the literature.

Neglected Tropical Diseases in the Post-Genomic Era.

Neglected tropical diseases (NTDs) are a group of viral, bacterial, and eukaryotic parasitic diseases that are especially endemic in low-income populations, with a large health and economic impact on both the developing and developed world. The structure and dynamics of the genomes of the organisms causing these diseases, as well as the modes of expression, exchange, and transmission of their genetic information, often deviate from those found in classical, model organism-centric textbooks. We assess the role of basic and applied genetic research in our understanding of key aspects of their biology and evolution, and discuss the impact of novel high-throughput approaches spawned by the post-genomic era on the development of next-generation drugs, vaccines, molecular epidemiology, and/or diagnostic tools for these important pathogens.

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.