DTU I isolates of Trypanosoma cruzi induce upregulation of Galectin-3 in murine myocarditis and fibrosis.

Chagas heart disease is a major public concern since 30% of infected patients develop cardiac alterations. The relationship between Trypanosoma cruzi discrete typing units (DTUs) and the biological properties exhibited by the parasite population has yet to be elucidated. In this study, we analysed the expression of α-smooth muscle actin (α-SMA) and galectin-3 (Gal-3) associated with cardiac extracellular matrix (ECM) remodelling a murine chronic cardiomyopathy induced by Tc I genotypes. We found the induction of myocarditis was associated with the upregulation of Col I, α-SMA, Gal-3, IFN-γ and IL-13, as analysed by q-PCR. In myocardial areas of fibrosis, the intensity of myocarditis and significant ECM remodelling correlated with the presence of Col I-, Gal-3- and α-SMA-positive cells. These results are promising for the further efforts to evaluate the relevance of Gal-3 in Chagas heart disease, since this galectin was proposed as a prognosis marker in heart failure patients.

Q586B2 is a crucial virulence factor during the early stages of Trypanosoma brucei infection that is conserved amongst trypanosomatids.

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment.

Sialic acid removal by trans-sialidase modulates MMP-2 activity during Trypanosoma cruzi infection.

Matrix metalloproteinases (MMPs) not only play a relevant role in homeostatic processes but are also involved in several pathological mechanisms associated with infectious diseases. As their clinical relevance in Chagas disease has recently been highlighted, we studied the modulation of circulating MMPs by Trypanosoma cruzi infection. We found that virulent parasites from Discrete Typing Units (DTU) VI induced higher proMMP-2 and MMP-2 activity in blood, whereas both low (DTU I) and high virulence parasites induced a significant decrease in proMMP-9 plasma activity. Moreover, trans-sialidase, a relevant T. cruzi virulence factor, is involved in MMP-2 activity modulation both in vivo and in vitro. It removes α2,3-linked sialyl residues from cell surface glycoconjugates, which then triggers the PKC/MEK/ERK signaling pathway. Additionally, bacterial sialidases specific for this sialyl residue linkage displayed similar MMP modulation profiles and triggered the same signaling pathways. This novel pathogenic mechanism, dependent on sialic acid removal by the neuraminidase activity of trans-sialidase, can be exploited by different pathogens expressing sialidases with similar specificity. Thus, here we present a new pathogen strategy through the regulation of the MMP network.

Continuous nonradioactive method for screening trypanosomal trans-sialidase activity and its inhibitors.

Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid (SA). Instead of using the corresponding nucleotide sugar as donor of the monosaccharide, the transfer occurs from alpha-2,3-linked SA in the host sialoglycoconjugates to terminal beta-galactopyranosyl units of the parasite mucins. For that purpose, T. cruzi expresses a glycosylphosphatidylinositol-anchored trans-sialidase (TcTS) that is shed into the milieu, being detected in the blood during the acute phase of the infection. The essential role of TcTS in infection and the absence of a similar activity in mammals make this enzyme an attractive target for the development of alternative chemotherapies. However, there is no effective inhibitor toward this enzyme. In vitro, 3'-sialyllactose (SL) as donor and radioactive lactose as acceptor substrate are widely used to measure TcTS activity. The radioactive sialylated product is then isolated by anion exchange chromatography and measured. Here we describe a new nonradioactive assay using SL or fetuin as donor and benzyl beta-d-Fuc-(1-->6)-alpha-d-GlcNAc (1) as acceptor. Disaccharide 1 was easily synthesized by regioselective glycosylation of benzyl alpha-d-GlcNAc with tetra-O-benzoyl-d-fucose followed by debenzoylation. Compound 1 lacks the hydroxyl group at C-6 of the acceptor galactose and therefore is not a substrate for galactose oxidase. Our method relies on the specific quantification of terminal galactose produced by trans-sialylation from the donor to the 6-deoxy-galactose (D-Fuc) unit of 1 by a spectrophotometric galactose oxidase assay. This method may also discriminate sialidase and trans-sialylation activities by running the assay in the absence of acceptor 1.

Anti-inflammatory Role of Galectin-8 During Trypanosoma cruzi Chronic Infection.

Galectins are animal lectins with high affinity for ß-galactosides that drive the immune response through several mechanisms. In particular, the role of galectin-8 (Gal-8) in inflammation remains controversial. To analyze its role in a chronic inflammatory environment, we studied a murine model of Trypanosoma cruzi infection. The parasite induces alterations that lead to the development of chronic cardiomyopathy and/or megaviscera in 30% of infected patients. The strong cardiac inflammation along with fibrosis leads to cardiomyopathy, the most relevant consequence of Chagas disease. By analyzing infected wild-type (iWT) and Gal-8-deficient (iGal-8KO) C57BL/6J mice at the chronic phase (4-5 months post-infection), we observed that the lack of Gal-8 favored a generalized increase in heart, skeletal muscle, and liver inflammation associated with extensive fibrosis, unrelated to tissue parasite loads. Remarkably, increased frequencies of neutrophils and macrophages were observed within cardiac iGal-8KO tissue by flow cytometry. It has been proposed that Gal-8, as well as other galectins, induces the surface expression of the inner molecule phosphatidylserine on activated neutrophils, which serves as an "eat-me" signal for macrophages, favoring viable neutrophil removal and tissue injury protection, a process known as preaparesis. We found that the increased neutrophil rates could be associated with the absence of Gal-8-dependent preaparesis, leading to a diminished neutrophil-clearing capability in macrophages. Thus, our results support that Gal-8 exerts an anti-inflammatory role in chronic T. cruzi infection.

Trans-sialidase inhibition assay detects Trypanosoma cruzi infection in different wild mammal species.

The detection of Trypanosoma cruzi infection in mammals is crucial for understanding the eco-epidemiological role of the different species involved in parasite transmission cycles. Xenodiagnosis (XD) and hemoculture (HC) are routinely used to detect T. cruzi in wild mammals. Serological methods are much more limited because they require the use of specific antibodies to immunoglobulins of each mammalian species susceptible to T. cruzi. In this study we detected T. cruzi infection by trans-sialidase (TS) inhibition assay (TIA). TIA is based on the antibody neutralization of a recombinant TS that avoids the use of anti-immunoglobulins. TS activity is not detected in the co-endemic protozoan parasites Leishmania spp and T. rangeli. In the current study, serum samples from 158 individuals of nine wild mammalian species, previously tested by XD, were evaluated by TIA. They were collected from two endemic areas in northern Argentina. The overall TIA versus XD co-reactivity was 98.7% (156/158). All 18 samples from XD-positive mammals were TIA-positive (co-positivity, 100%) and co-negativity was 98.5% (138/140). Two XD-negative samples from a marsupial (Didelphis albiventris) and an edentate (Dasypus novemcinctus) were detected by TIA. TIA could be used as a novel tool for serological detection of Trypanosoma cruzi in a wide variety of sylvatic reservoir hosts.

Diagnostic peptide discovery: prioritization of pathogen diagnostic markers using multiple features.

The availability of complete pathogen genomes has renewed interest in the development of diagnostics for infectious diseases. Synthetic peptide microarrays provide a rapid, high-throughput platform for immunological testing of potential B-cell epitopes. However, their current capacity prevent the experimental screening of complete "peptidomes". Therefore, computational approaches for prediction and/or prioritization of diagnostically relevant peptides are required. In this work we describe a computational method to assess a defined set of molecular properties for each potential diagnostic target in a reference genome. Properties such as sub-cellular localization or expression level were evaluated for the whole protein. At a higher resolution (short peptides), we assessed a set of local properties, such as repetitive motifs, disorder (structured vs natively unstructured regions), trans-membrane spans, genetic polymorphisms (conserved vs. divergent regions), predicted B-cell epitopes, and sequence similarity against human proteins and other potential cross-reacting species (e.g. other pathogens endemic in overlapping geographical locations). A scoring function based on these different features was developed, and used to rank all peptides from a large eukaryotic pathogen proteome. We applied this method to the identification of candidate diagnostic peptides in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We measured the performance of the method by analyzing the enrichment of validated antigens in the high-scoring top of the ranking. Based on this measure, our integrative method outperformed alternative prioritizations based on individual properties (such as B-cell epitope predictors alone). Using this method we ranked [Formula: see text]10 million 12-mer overlapping peptides derived from the complete T. cruzi proteome. Experimental screening of 190 high-scoring peptides allowed the identification of 37 novel epitopes with diagnostic potential, while none of the low scoring peptides showed significant reactivity. Many of the metrics employed are dependent on standard bioinformatic tools and data, so the method can be easily extended to other pathogen genomes.