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Summary Large-scale mapping of antigens and epitopes is pivotal for developing immunotherapies but challenging, especially for eukaryotic pathogens, owing to their large genomes. Here, we developed an integrated platform for genome phage display (gPhage) to show that unbiased libraries of the eukaryotic parasite Trypanosoma cruzi enable the identification of thousands of antigens recognized by serum samples from patients with Chagas disease. Because most of these antigens are hypothetical proteins, gPhage provides evidence of their expression during infection. We built and validated a comprehensive map of Chagas disease antibody response to show how linear and putative conformation epitopes, many rich in repetitive elements, allow the parasite to evade a buildup of neutralizing antibodies directed against protein domains that mediate infection pathogenesis. Thus, the gPhage platform is a reproducible and effective tool for rapid simultaneous identification of epitopes and antigens, not only in Chagas disease but perhaps also in globally emerging/reemerging acute pathogens., Graphical abstract, Highlights • Genomic shotgun phage display (gPhage) of eukaryotes is feasible and promising. • gPhage allows rapid antigen ID and epitope mapping, including 3D structures. • Conformation epitopes can be identified and validated by using the gPhage platform. • Most Chagas disease antigens are hypothetical proteins rich in repetitive elements., Parasitology; Sequence analysis; Systems biology

Trypanosoma cruzi is a flagellate protozoa being the etiological agent of Chagas disease, a neglected tropical disease, which still poses a public health problem worldwide. The intricate molecular changes during T. cruzi-host interaction have been explored using different largescale omics techniques. However, protein stability is largely unknown. Thermal proteome profiling (TPP) methodology has the potential to characterize proteome-wide stability highlighting key proteins during T. cruzi infection and life stage transition from the invertebrate to the mammalian host. In the present work, T. cruzi epimastigotes and trypomastigotes cell lysates were subjected to TPP workflow and analyzed by quantitative large-scale mass spectrometry-based proteomics to fit a melting profile for each protein. A total of 2884 proteins were identified and associated to 1741 melting curves being 1370 in trypomastigotes (TmAVG 53.53 °C) and 1279 in epimastigotes (TmAVG 50.89 °C). A total of 453 proteins were identified with statistically different melting profiles between the two life stages. Proteins associated to pathogenesis and intracellular transport had regulated melting temperatures. Membrane and glycosylated proteins had a higher average Tm in trypomastigotes compared to epimastigotes. This study represents the first large-scale comparison of parasite protein stability between life stages. Significance Trypanosoma cruzi, a unicellular flagellate parasite, is the etiological agent of Chagas disease, endemic in South America and affecting more that 7 million people worldwide. There is an intense research to identify novel chemotherapeutic and diagnostic targets of Chagas disease. Proteomic approaches have helped in elucidating the quantitative proteome and PTMs changes of T. cruzi during life cycle transition and upon different biotic and abiotic stimuli. However, a comprehensive knowledge of the protein-protein interaction and protein conformation is still missing. In order to fill this gap, this manuscript elucidates the T. cruzi Y strain proteome-wide thermal stability map in the epimastigote and trypomastigote life stages. Comparison between life stages showed a higher average melting temperature stability for trypomastigotes than epimastigotes indicating a host temperature adaptation. Both presented a selective thermal stability shift for cellular compartments, molecular functions and biological processes based on the T. cruzi life stage. Membrane and glycosylated proteins presented a higher thermal stability in trypomastigotes when compared to the epimastigotes.

Trypanosoma cruzi trypomastigotes adhere to extracellular matrix (ECM) to invade mammalian host cells regulating intracellular signaling pathways. Herein, resin-assisted enrichment of thiols combined with mass spectrometry were employed to map site-specific S-nitrosylated (SNO) proteins from T. cruzi trypomastigotes incubated (MTy) or not (Ty) with ECM. We confirmed the reduction of S-nitrosylation upon incubation with ECM, associated with a rewiring of the subcellular distribution and intracellular signaling pathways. Forty, 248 and 85 SNO-peptides were identified only in MTy, Ty or in both conditions, respectively. SNO proteins were enriched in ribosome, transport, carbohydrate and lipid metabolisms. Nitrosylation of histones H2B and H3 on Cys64 and Cys126, respectively, is described. Protein-protein interaction networks revealed ribosomal proteins, proteins involved in carbon and fatty acid metabolism to be among the enriched protein complexes. Kinases, phosphatases and enzymes involved in the metabolism of carbohydrates, lipids and amino acids were identified as nitrosylated and phosphorylated, suggesting a post-translational modifications crosstalk. In silico mapping of nitric oxide synthase (NOS) genes, previously uncharacterized, matched to four putative T. cruzi proteins expressing C-terminal NOS domain. Our results provide the first site-specific characterization of S-nitrosylated proteins in T. cruzi and their modulation upon ECM incubation before infection of the mammalian hosts. SIGNIFICANCE: Protein S-nitrosylation represents a major molecular mechanism for signal transduction by nitric oxide. We present for the first time a proteomic profile of S-nitrosylated proteins from infective forms of T. cruzi, showing a decrease in SNO proteins after incubation of the parasite with the extracellular matrix, a necessary step for the parasite invasion of the host mammalian cells. We also show for the first time nitrosylation of H2B (Cys64) and H3 (Cys126) histones, sites not conserved in higher eukaryotic cells, and suggest that some specific histone isoforms are sensitive to NO signaling. S-nitrosylation in H2B and H3 histones are more abundant in MTy. Moreover, proteins involved in translation, glycolytic pathway and fatty acid metabolism are enriched in the present dataset. Comparison of the SNO proteome and the phosphoproteome, obtained previously under the same experimental conditions, show that most of the proteins sharing both modifications are involved in metabolic pathways, transport and ribosome function. The data suggest that both PTMs are involved in reprogramming the metabolism of T. cruzi in response to environmental changes. Although NO synthesis was detected in T. cruzi, the identification of NOS remains elusive. Analysis in silico showed two genes similar in domains to NADPH-dependent cytochrome-P450 reductase and two putative oxidoreductases, but no oxygenase domain of NOS was mapped in the T. cruzi genome. It is tempting to speculate that NO synthase-like from T. cruzi and its early NO-mediated pathways triggered in response to host interaction constitute potential diagnostic and therapeutic targets.

Summary This protocol describes the genomic phage (gPhage) display platform, a large-scale antigen and epitope mapping technique. We constructed a gPhage display peptide library of a eukaryotic organism, Trypanosoma cruzi (causative agent of Chagas disease), to map the antibody response landscape against the parasite. Here, we used an organism with a relatively large but intronless genome, although future applications could include other prevalent or (re)emerging infectious organisms carrying genomes with a limited number of introns. For complete details on the use and execution of this protocol, please refer to Teixeira et al. (2021)., Graphical abstract, Highlights • A streamlined protocol to build genome shotgun phage display libraries • Scalable to different organisms, eukaryotes or prokaryotes • Optimizes key steps for high throughput identification of antibody antigens/epitopes • gPhage is also a platform to study conformational and difficult-to-validate antigens, This protocol describes the genomic phage (gPhage) display platform, a large-scale antigen and epitope mapping technique. We constructed a gPhage display peptide library of a eukaryotic organism, Trypanosoma cruzi (causative agent of Chagas disease), to map the antibody response landscape against the parasite. Here, we used an organism with a relatively large but intronless genome, although future applications could include other prevalent or (re)emerging infectious organisms carrying genomes with a limited number of introns.

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions., Author summary T. cruzi, the etiological agent of Chagas’ disease, interacts with ECM in order to invade cells and establish the infection in the mammalian host. Studying this interaction, our group has recently described changes in the nitration profile of histones upon incubation of the parasite with ECM. Herein we used a proteomic approach to identify nitrated DNA binding proteins in T. cruzi. We also used ChIP-Seq to investigate whether the contact of the parasite with host ECM is capable of promoting changes in the DNA binding pattern of nitrated proteins. We observed significant alterations in the nitration levels and DNA binding profile of T. cruzi histones upon parasite incubation with ECM.

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 Sao Paulo, on this subject.

The OX513A strain ofLa souche OX513A d'La cepa OX513A deتمثل سلالة OX513A منOX513A 类Вид комаров Aedes aegypti под названием OX513A, разработанный британской компанией Oxitec, экспрессирует ограничивающий популяцию своего носителя трансген, который препятствует развитию личинки во взрослую особь. В апреле 2014 года Бразильская национальная техническая комиссия по биобезопасности завершила оценку рисков OX513A и пришла к выводу, что этот вид не представляет новых биологических угроз для человека или окружающей среды и может быть выпущен в Бразилии. Тогда Бразилия стала первой страной, в которой был разрешен неограниченный выпуск генетически измененных комаров. В ходе оценки комиссия опубликовала исчерпывающий перечень предполагаемых рисков и провела их систематический анализ. Такие риски включали потенциальную способность комаров неполовозрелой стадии, несущих трансген, дожить до репродуктивного возраста в случае неудавшейся экспрессии трансгена или его подавления, вызванного воздействием достаточного количества тетрациклина на комаров во внешней среде. Среди других предполагаемых рисков — потенциальная аллергенность или токсичность белков, образующихся в результате экспрессии гена, возможность дрейфа генов или повышенного распространения болезнетворных организмов для человека и заселение свободных мест размножения другими видами переносчиков. На фоне эпидемии вируса Зика лица, ответственные за разработку стратегических решений, регулирующие органы, а также общественность увеличили внимание к роли Ae. Aegypti в качестве переносчиков и выразили большую обеспокоенность по поводу выпуска мужских особей вида OX513A. По этой причине авторы данной статьи провели повторную оценку потенциальных рисков. Была подтверждена безопасность и высокое потенциальное значение выпуска генетически измененных комаров для борьбы с распространяемыми Ae. aegypti заболеваниями, такими как чикунгунья, денге и болезнь, вызываемая вирусом Зика.

Trypanosoma cruzi, the etiological agent of Chagas’ disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host., Author summary Adhesion of Trypanosoma cruzi to distinct elements of ECM involving different surface proteins from the infective stage of the parasite has been described. Despite the relevance of ECM for T. cruzi infection, the signaling pathways triggered in trypomastigotes upon interactions with ECM are less well understood. In previous work we demonstrated the dephosphorylation of proteins, such as α-tubulin, paraflagellar rod proteins and ERK 1/2 in trypomastigotes incubated with either laminin or fibronectin. Further, we described changes in the S-nitrosylation and nitration pattern of proteins from trypomastigote incubated with ECM. To expand our knowledge on ECM triggered parasite signaling we applied quantitative proteomic and phosphoproteomic studies to trypomastigotes incubated with ECM (MTy) compared to controls (Ty). Our results indicate relevant changes in total protein and phosphoprotein profiles in MTy. The kinases implicated in the modifications were suggested by bioinformatic analyses, as well as the number of modifications and the frequency of amino acids per peptide that have been modified. Proteins involved in metabolic processes, including enzymes from the glycolytic pathway, phosphatases and kinases were the most representative groups among the proteins modified by phosphorylation. Quantification of metabolites in MTy and Ty also indicated that glucose metabolism is impaired in trypomastigotes incubated with ECM. The significant inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy associated with phosphorylation levels, strongly suggests that trypomastigotes reprogram their metabolism in response to interaction with the extracellular matrix, an obligatory step prior to host cell invasion.

Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving insect and mammalian hosts and distinct developmental stages. During T. cruzi developmental stages, glycoproteins play important role in the host-parasite interaction, such as cellular recognition, host cell invasion and adhesion, and immune evasion. In this study, comprehensive glycoprofiling analysis was performed in the epimastigote and trypomastigote stages of T. cruzi using two glycopeptide enrichment strategies, lectin-based and hydrophilic interaction liquid chromatography, followed by high resolution LC-MS/MS. Following deglycosylation, a total of 1306 N-glycosylation sites in NxS/T/C motifs were identified from 690 T. cruzi glycoproteins. Among them, 170 and 334 glycoproteins were exclusively identified in epimastigotes and trypomastigotes, respectively. Besides, global site-specific characterization of the N- and O-linked glycan heterogeneity in the two life stages of T. cruzi was achieved by intact glycopeptide analysis, revealing 144/466 unique N-linked and 10/97 unique O-linked intact glycopeptides in epimastigotes/trypomastigotes, respectively. Conclusively, this study documents the significant T. cruzi stage-specific expression of glycoproteins that can help to better understand the T. cruzi phenotype and response caused by the interaction with different hosts during its complex life cycle. Biological significance Chagas disease caused by the protozoan Trypanosoma cruzi is a neglected disease which affects millions of people especially in Latin America. The absence of efficient drugs and vaccines against Chagas disease stimulates the search for novel targets. Glycoproteins are very attractive therapeutic candidate targets since they mediate key processes in the host-parasite interaction, such as cellular recognition, host cell invasion and adhesion, and immune evasion. This study aimed to provide an in depth characterization of the N-linked and O-linked glycoproteome of two T. cruzi life stages: epimastigotes and trypomastigotes. Mass spectrometry-based proteomics showed interesting stage-specific glycoproteome signatures that are valuable to better understand the importance of protein glycosylation in epimastigotes and trypomastigotes and to expand the repertoire of potential therapeutic targets against Chagas disease.

Different types of shed vesicles as, for example, exosomes, plasma-membrane-derived vesicles or microparticles, are the focus of intense research in view of their potential role in cell–cell communication and under the perspective that they might be good tools for immunotherapy, vaccination or diagnostic purposes. This review discusses ways employed by pathogenic trypanosomatids to interact with the host by shedding vesicles that contain molecules important for the establishment of infection, as opposed to previous beliefs considering them as a waste of cellular metabolism. Trypanosomatids are compared with Apicomplexa, which circulate parasite antigens bound to vesicles shed by host cells. The knowledge of the origin and chemical composition of these different vesicles might lead to the understanding of the mechanisms that determine their biological function.

A análise de risco de OGM ou transgênicos segue protocolos internacionais através das quais se pergunta sobre o possível dano (efeito adverso), a forma de aparecimento do dano (riscos), os riscos significativos que mereçam análise detalhada e de que forma o risco identificado será observado e medido (avaliação). Assim como no Brasil, análises de risco efetuadas em outros países nos últimos quinze anos nunca evidenciaram, nos produtos liberados, efeitos adversos à saúde humana e animal ou ao ambiente. O excesso de regulamentação favorece as grandes empresas que têm recursos e tempo para atender a todas as demandas regulatórias. No entanto, pequenas empresas ou estatais como a Embrapa não têm orçamento para cumprir com os exageros do excesso de regulamentação. Por isso, o Brasil, tendo em vista a experiência dos outros países, além de sua própria experiência, deve introduzir mais racionalidade no debate e começar a desregular o que está regulado em demasia. As plantas transgênicas, além de inócuas, trazem vantagens econômicas e ao meio ambiente. Comprovadamente, reduzem o uso de água por exigir menos inseticidas e economizam o uso de óleo diesel reduzindo a emissão de CO2 na atmosfera.

SUMMARYTrypanosoma cruziis a protozoan parasite that infects vertebrates, causing in humans a pathological condition known as Chagas’ disease. The infection of host cells byT. cruziinvolves a vast collection of molecules, including a family of 85 kDa GPI-anchored glycoproteins belonging to the gp85/trans-sialidase superfamily, which contains a conserved cell-binding sequence (VTVXNVFLYNR) known as FLY, for short. Herein, it is shown that BALB/c mice administered with a single dose (1μg/animal, intraperitoneally) of FLY-synthetic peptide are more susceptible to infection byT. cruzi, with increased systemic parasitaemia (2-fold) and mortality. Higher tissue parasitism was observed in bladder (7·6-fold), heart (3-fold) and small intestine (3·6-fold). Moreover, an intense inflammatory response and increment of CD4+T cells (1·7-fold) were detected in the heart of FLY-primed and infected animals, with a 5-fold relative increase of CD4+CD25+FoxP3+T (Treg) cells. Mice treated with anti-CD25 antibodies prior to infection, showed a decrease in parasitaemia in the FLY model employed. In conclusion, the results suggest that FLY facilitatesin vivoinfection byT. cruziand concurs with other factors to improve parasite survival to such an extent that might influence the progression of pathology in Chagas’ disease.

Trypanosoma cruzi epimastigotes adhere in vivo to the luminal surface of their triatomid vector digestive tract by molecular mechanisms, as yet, unknown. Here, we show that the administration of 0.5 μM epimastigote major surface glycoinositolphospholipids (GIPLs) to the infected bloodmeal inhibits up to 90% parasite infection in Rhodnius prolixus. The parasite behavior was investigated in vitro using fragments of the insect midgut. The addition of GIPLs in concentration as low as 50–100 nM impaired 95% the attachment of epimastigotes. Previous treatment of GIPLs with trifluoroacetic acid to remove the terminal β-galactofuranosyl residues reversed 50% the epimastigote in vitro attachment. The binding sites of purified GIPLs on the luminal surface of the posterior midgut were exposed by immunofluorescence microscopy. These observations indicate that GIPLs are one of the components involved in the adhesion of T. cruzi to the luminal insect midgut surface and possibly one of the determinants of parasite infection in the insect vector.

Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.

Both invasion of the host cell by T. cruzi and its establishment into the mammalian host are critical steps. In this review, the adhesion step and the intracellular survival in non-professional phagocytes are particularly focused on, with special emphasis on the role of Gp85/trans-sialidase (Gp85/TS) superfamily. Excellent reviews have been published lately, some covering other aspects of T. cruzi - host interaction and will be cited instead of the original articles due to limited number of listed references. IUBMB Life, 59: 274-279, 2007

Trypanosoma cruzi is the etiologic agent of Chagas disease. Two main distinct forms are present in the mammalian host: trypomastigote, an infective form, and the amastigote, a typical replicative form. Succeeding the host cell invasion, trypomastigotes differentiate to amastigotes, a process known as amastigogenesis. Amastigogenesis is characterized by parasite body remodeling, with drastic reduction of flagellum, and changes in protein profile and energetic metabolism. Our aim is to explore the role of nitric oxide as a signaling molecule during the amastigogenesis process, which must be strictly regulated. We report herein that acid pH (6.0) is essential for T. cruzi amastigogenesis. Also, during amastigogenesis there is a progressive solubilization of the paraflagellar protein, a flagellum marker. Moreover, the process is dependent on (•)NO concentration, since it is suppressed by 1mM SNAP, a (•)NO donor, and favored by 10mM L-NAME, a NOS inhibitor. Accordingly, S-nitrosylation of selective proteins occurs in amastigogenesis. Additionally, amastigogenesis is affected by IBMX (PDE inhibitor) treatment, suggesting the importance of cyclic nucleotides signaling. Furthermore, tubulin stability is also affected by the (•)NO availability. Along amastigogenesis, flagellum disassembling is accompanied by changes in a-tubulin tyrosylation and polyglutamylation levels. Taken together, these results suggest (•)NO participation in trypomastigote differentiation to amastigotes in T. cruzi.

Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans-sialidase (TS) superfamily and other α-galactosyl (α-Gal)-containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL-14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α-galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL-14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF-α and IL-6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL-10 in the chronically infected mice. A high frequency of IL-10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF-α and IL-10. Finally, dendritic cells produced TNF-α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase.Keywords: Trypanosoma cruzi; extracellular vesicles; innate and chronic immunity; TLR2; α-galactosyl(Published: 26 November 2015)Citation: Journal of Extracellular Vesicles 2015, 4: 28734 - http://dx.doi.org/10.3402/jev.v4.28734

Trypanosoma cruzi trypomastigotes invade a great variety of mammalian cells, with several molecules being implicated in this complex event. Herein, the sequence GGIALAG present in prokineticin-2 receptor (PKR2), selected by phage display technology, is described as a new T. cruzi receptor for the Tc85 group of glycoproteins belonging to the gp85/TS superfamily and involved in cellular invasion of mammalian hosts. This finding is confirmed by the inhibitory activity of MCF10-A (human mammary) cell invasion by T. cruzi either by anti-PKR2 antibodies (77 %) or GGIALAG-synthetic peptide (42 %). Furthermore, interference RNA (iRNA) inhibition of PKR2 expression in MCF10-A cells reduces T. cruzi invasion by 50 %. The binding site of Tc85 to PKR2 was localized at the C-terminal end of the molecule, upstream of the conserved FLY sequence, previously implicated in parasite cell invasion. PKR2, a receptor formed by seven membrane-spanning α-helical segments, is mainly present in the central nervous system, peripheral organs, and mature blood cells. Due to its wide distribution, PKR2 could be a suitable receptor for T. cruzi natural infection, contributing to the parasite dissemination throughout the mammalian organism. These findings augment the number and diversity of possible in vivo receptors for T. cruzi and reassure the multiplicity of Tc85 binding sites to mammalian hosts.

This present work reports on development of an amperometric immunosensor for the diagnosis of Chagas’ disease using a specific glycoprotein of the trypomastigote surface, which belongs to the Tc85-11 protein family of Trypanosoma cruzi (T. cruzi). An atomically flat gold surface on a silicon substrate and gold screen-printed electrodes were functionalized with cystamine and later activated with glutaraldehyde (GA), which was used to form covalent bonds with the purified recombinant antigen (Tc85-11). The antigen reacts with the antibody from the serum, and the affinity reaction was monitored directly using atomic force microscopy or amperometry through a secondary antibody tagged to peroxidase (HRP). Surface imaging allowed to us to differentiate the modification steps and antigen–antibody interaction allowed to distinguish the affinity reactions. In the amperometric immunosensor, peroxidase catalyses the L2 formation in the presence of hydrogen peroxide and potassium iodide, and the reduction current intensity was measured at a given potential with screen-printed electrodes. The immunosensor was applied to sera of chagasic patients and patients having different systemic diseases.

Trypanosoma cruzi proteins from epimastigote membranes, herein referred as antigens, have been used for the construction of an amperometric immunosensor for serological diagnosis of Chagas’ disease. The proteins used had a molecular mass ranging from 30 to 100 kDa. The gold electrode was treated with cysteamine and glutaraldehyde prior to antigen immobilization. Antibodies present in the serum of patients with Chagas’ disease were captured by the immobilized antigens and the affinity interaction was monitored by chronoamperometry at a potential of −400 mV (versus Ag pseudo-reference electrode) using peroxidase-labeled IgG conjugate and hydrogen peroxide, iodide substrate. The incubation time to allow maximum antigen–antibody and antibody–peroxidase-labeled IgG interactions was 20 min with a reactivity threshold at −0.104 μA.

Chagas' disease is a zoonosis caused by the parasite Trypanosoma cruzi, a haematic protozoan, transmitted by insects from the Reduviidae family. This constitutes a relevant health and socio-economic problem in the Americas, with 11 - 18 million people infected, and approximately 100 million people at risk. The therapeutic possibilities rely into two drugs, nifurtimox and benznidazole, that were discovered more than thirty years ago, and are mainly successful during the acute phase of the disease. In the majority of the cases the disease is diagnosed in the chronic phase, when the therapy is inefficient and the probability of cure is low. In addition, these drugs are highly toxic, with systemic side effects on patients. Trypanosoma cruzi has a metabolism largely based on the consumption of amino acids, mainly proline, aspartate and glutamate, which constitute the main carbon and energy sources in the insect stage of the parasite life cycle. These amino acids also participate in the differentiation process of the replicative non-infective form (epimastigote) to the non-replicative infective form (trypomastigote). In particular, the participation of proline in the intracellular differentiation cycle, which occurs in the mammalian host, was recently demonstrated. In addition, an arginine kinase has been described in T. cruzi and T. brucei, which converts free arginine to phosphoarginine, a phosphagen with a role as an energy reservoir. Arginine kinase seems to be an essential component of energy management during stress conditions. Taken together, these data indicate that amino acid metabolism may provide multiple as yet unexplored targets for therapeutic drugs.

Trypanosoma cruzi expresses a set of glycoproteins encoded by the gp85/trans-sialidase gene superfamily. In this report a structure model is proposed for a cloned member of the superfamily, the Tc85-11 protein. The structure consists of an N-terminus beta-propeller and a C-terminus beta-sandwich interconnected by an alpha-helix. The recombinant protein, corresponding to the N-domain (Tc85-N), binds to laminin in a selective manner. Six synthetic 20-mer peptides from the N-domain adhere onto the surface of LLC-MK(2) cells and two of these peptides specifically inhibit the Tc85-N/laminin interaction, indicating that they are the laminin-binding sites of the molecule. Thus, Tc85-11 and other related members of the family appear to be good candidates to play an important role in T. cruzi infection via a laminin mediated host-parasite interaction.

The "amino acid/auxin permeases" is probably the most represented family of transporters in the Trypanosoma cruzi genome. Using a high-throughput searching routine and preliminary data from the T. cruzi genome project, more than 15,000 sequences were iteratively assembled into contigs, and 60 open reading frames corresponding to different putative amino acid transporters, clustered in 12 groups, were detected and characterized in silico. T. cruzi genomic organization of such sequences showed that these putative amino acid transporter genes are in an unusually large number and arranged in repeat clusters comprising about 0.2% of the genome. These data suggest that the family has evolved following tandem duplication events and constitutes a novel family of variable proteins in protozoan organisms. The mRNA expression of the predicted genes was demonstrated in infective and non-infective parasite forms. Orthologous sequences were also identified in other unicellular parasites such as Leishmania spp., Plasmodium spp., and Trypanosoma brucei.

Summary Using as the host cell, a proline-requiring mutant of Chinese hamster ovary cell (CHO-K1), it was possible to arrest the differentiation of amastigote forms of Trypanosoma cruzi at the intermediate intracellular epimastigote-like stage. Complete differentiation to the trypomastigote stage was obtained by addition of L -proline to the medium. This effect was more pro- nounced using the T. cruzi CL-14 clone that differen- tiates fully at 33 ∞ ∞ ∞ C (permissive temperature) and poorly at 37 ∞ C (restrictive temperature). A synchro- nous differentiation of T. cruzi inside the host-cell is then possible by temperature switching in the pres- ence of proline. It was found that differentiation of intracellular epimastigotes and trypomastigote burst- ing were proline concentration dependent. The intra- cellular concentration of proline was measured as well as the transport capacity of proline by each stage of the parasite. Amastigotes have the highest concen- tration of free proline (8.09 ± 1.46 mM) when com- pared to trypomastigotes (3.81 ± 1.55) or intracellular epimastigote-like forms (0.45 ± 0.06 mM). In spite of having the lowest content of intracellular free proline, intracellular epimastigotes maintained the highest levels of L -proline transport compared to trypomas- tigotes and intracellular amastigotes, providing evi- dence for a high turnover for the L -proline pool in that parasite stage. This is the first report to establish a relationship between proline concentration and intra- cellular differentiation of Trypanosoma cruzi in the mammalian host.