Paracrine Signaling Mediated by the Cytosolic Tryparedoxin Peroxidase of Trypanosoma cruzi.

Peroxiredoxins are abundant and ubiquitous proteins that participate in different cellular functions, such as oxidant detoxification, protein folding, and intracellular signaling. Under different cellular conditions, peroxiredoxins can be secreted by different parasites, promoting the induction of immune responses in hosts. In this work, we demonstrated that the cytosolic tryparedoxin peroxidase of Trypanosoma cruzi (cTXNPx) is secreted by epimastigotes and trypomastigotes associated with extracellular vesicles and also as a vesicle-free protein. By confocal microscopy, we show that cTXNPx can enter host cells by an active mechanism both through vesicles and as a recombinant protein. Transcriptomic analysis revealed that cTXNPx induces endoplasmic reticulum stress and interleukin-8 expression in epithelial cells. This analysis also suggested alterations in cholesterol metabolism in cTXNPx-treated cells, which was confirmed by immunofluorescence showing the accumulation of LDL and the induction of LDL receptors in both epithelial cells and macrophages. BrdU incorporation assays and qPCR showed that cTXNPx has a mitogenic, proliferative, and proinflammatory effect on these cells in a dose-dependent manner. Importantly, we also demonstrated that cTXNPx acts as a paracrine virulence factor, increasing the susceptibility to infection in cTXNPx-pretreated epithelial cells by approximately 40%. Although the results presented in this work are from in vitro studies and likely underestimate the complexity of parasite-host interactions, our work suggests a relevant role for this protein in establishing infection.

Overoxidation and Oligomerization of Trypanosoma cruzi Cytosolic and Mitochondrial Peroxiredoxins.

Peroxiredoxins (Prxs) have been shown to be important enzymes for trypanosomatids, counteracting oxidative stress and promoting cell infection and intracellular survival. In this work, we investigate the in vitro sensitivity to overoxidation and the overoxidation dynamics of Trypanosoma cruzi Prxs in parasites in culture and in the infection context. We showed that recombinant m-TXNPx, in contrast to what was observed for c-TXNPx, exists as low molecular mass forms in the overoxidized state. We observed that T. cruzi Prxs were overoxidized in epimastigotes treated with oxidants, and a significant proportion of the overoxidized forms were still present at least 24 h after treatment suggesting that these forms are not actively reversed. In in vitro infection experiments, we observed that Prxs are overoxidized in amastigotes residing in infected macrophages, demonstrating that inactivation of at least part of the Prxs by overoxidation occurs in a physiological context. We have shown that m-TXNPx has a redox-state-dependent chaperone activity. This function may be related to the increased thermotolerance observed in m-TXNPx-overexpressing parasites. This study suggests that despite the similarity between protozoan and mammalian Prxs, T. cruzi Prxs have different oligomerization dynamics and sensitivities to overoxidation, which may have implications for their function in the parasite life cycle and infection process.

Genome-wide chromatin interaction map for Trypanosoma cruzi.

Trypanosomes are eukaryotic, unicellular parasites, such as Trypanosoma brucei, which causes sleeping sickness, and Trypanosoma cruzi, which causes Chagas disease. Genomes of these parasites comprise core regions and species-specific disruptive regions that encode multigene families of surface glycoproteins. Few transcriptional regulators have been identified in these parasites, and the role of spatial organization of the genome in gene expression is unclear. Here we mapped genome-wide chromatin interactions in T. cruzi using chromosome conformation capture (Hi-C), and we show that the core and disruptive regions form three-dimensional chromatin compartments named C and D. These chromatin compartments differ in levels of DNA methylation, nucleosome positioning and chromatin interactions, affecting genome expression dynamics. Our data reveal that the trypanosome genome is organized into chromatin-folding domains and transcription is affected by the local chromatin structure. We propose a model in which epigenetic mechanisms affect gene expression in trypanosomes.

New insights into phenotype and genotype relationships in Neospora caninum.

The successful isolation of four new Neospora caninum strains from different regions and with different backgrounds (obtained from an abortion storm or congenitally infected and asymptomatic calves) allowed us previously to characterize natural isolates, finding differences in phenotype and microsatellites. Given the variability observed, we wondered in this work whether these differences had consequences in virulence, invasion and vertical transmission using cell cultures and murine neosporosis models. In addition, we performed the genomic analysis and SNP comparative studies of the NcURU isolates. The results obtained in this work allowed us to establish that NcURU isolates are of low virulence and have unique phenotypic characteristics. Likewise, sequencing their genomes has allowed us to delve into the genetic singularities underlying these phenotypes, as well as the common mutated genes. This work opens a new perspective for diagnostic purposes and formulating possible vaccines based on attenuated strains.

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.

Comparative microRNA profiling of Trypanosoma cruzi infected human cells.

Introduction: Trypanosoma cruzi, the causative agent of Chagas disease, can infect almost any nucleated cell in the mammalian host. Although previous studies have described the transcriptomic changes that occur in host cells during parasite infection, the understanding of the role of post-transcriptional regulation in this process is limited. MicroRNAs, a class of short non-coding RNAs, are key players in regulating gene expression at the post-transcriptional level, and their involvement in the host-T. cruzi interplay is a growing area of research. However, to our knowledge, there are no comparative studies on the microRNA changes that occur in different cell types in response to T. cruzi infection. Methods and results: Here we investigated microRNA changes in epithelial cells, cardiomyocytes and macrophages infected with T. cruzi for 24 hours, using small RNA sequencing followed by careful bioinformatics analysis. We show that, although microRNAs are highly cell type-specific, a signature of three microRNAs -miR-146a, miR-708 and miR-1246, emerges as consistently responsive to T. cruzi infection across representative human cell types. T. cruzi lacks canonical microRNA-induced silencing mechanisms and we confirm that it does not produce any small RNA that mimics known host microRNAs. We found that macrophages show a broad response to parasite infection, while microRNA changes in epithelial and cardiomyocytes are modest. Complementary data indicated that cardiomyocyte response may be greater at early time points of infection. Conclusions: Our findings emphasize the significance of considering microRNA changes at the cellular level and complement previous studies conducted at higher organizational levels, such as heart samples. While miR-146a has been previously implicated in T. cruzi infection, similarly to its involvement in many other immunological responses, miR-1246 and miR-708 are demonstrated here for the first time. Given their expression in multiple cell types, we anticipate our work as a starting point for future investigations into their role in the post-transcriptional regulation of T. cruzi infected cells and their potential as biomarkers for Chagas disease.

Biochemical characterization of GAF domain of free-R-methionine sulfoxide reductase from Trypanosoma cruzi.

Trypanosoma cruzi is the causal agent of Chagas Disease and is a unicellular parasite that infects a wide variety of mammalian hosts. The parasite exhibits auxotrophy by L-Met; consequently, it must be acquired from the extracellular environment of the host, either mammalian or invertebrate. Methionine (Met) oxidation produces a racemic mixture (R and S forms) of methionine sulfoxide (MetSO). Reduction of L-MetSO (free or protein-bound) to L-Met is catalyzed by methionine sulfoxide reductases (MSRs). Bioinformatics analyses identified the coding sequence for a free-R-MSR (fRMSR) enzyme in the genome of T. cruzi Dm28c. Structurally, this enzyme is a modular protein with a putative N-terminal GAF domain linked to a C-terminal TIP41 motif. We performed detailed biochemical and kinetic characterization of the GAF domain of fRMSR in combination with mutant versions of specific cysteine residues, namely, Cys12, Cys98, Cys108, and Cys132. The isolated recombinant GAF domain and full-length fRMSR exhibited specific catalytic activity for the reduction of free L-Met(R)SO (non-protein bound), using tryparedoxins as reducing partners. We demonstrated that this process involves two Cys residues, Cys98 and Cys132. Cys132 is the essential catalytic residue on which a sulfenic acid intermediate is formed. Cys98 is the resolutive Cys, which forms a disulfide bond with Cys132 as a catalytic step. Overall, our results provide new insights into redox metabolism in T. cruzi, contributing to previous knowledge of L-Met metabolism in this parasite.

Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango.

Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe-4S]2+ prosthetic group which yields an inactive [3Fe-4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe-S clusters. A high yield of pure ACO2 (≥99%, 22 ± 2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102 M-1 s-1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 °C for [4Fe-4S]2+ and [3Fe-4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe-S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe-4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe-4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation.

Trypanosoma cruzi Isolates Naturally Adapted to Congenital Transmission Display a Unique Strategy of Transplacental Passage.

Chagas disease is mainly transmitted by vertical transmission (VT) in nonendemic areas and in endemic areas where vector control programs have been successful. For the present study, we isolated natural Trypanosoma cruzi strains vertically transmitted through three generations and proceeded to study their molecular mechanism of VT using mice. No parasitemia was detected in immunocompetent mice, but the parasites were able to induce an immune response and colonize different organs. VT experiments revealed that infection with different strains did not affect mating, pregnancy, or resorption, but despite low parasitemia, VT strains reached the placenta and resulted in higher vertical transmission rates than strains of either moderate or high virulence. While the virulent strain modulated more than 2,500 placental genes, VT strains modulated 150, and only 29 genes are shared between them. VT strains downregulated genes associated with cell division and replication and upregulated immunomodulatory genes, leading to anti-inflammatory responses and tolerance. The virulent strain stimulated a strong proinflammatory immune response, and this molecular footprint correlated with histopathological analyses. We describe a unique placental response regarding the passage of T. cruzi VT isolates across the maternal-fetal interphase, challenging the current knowledge derived mainly from studies of laboratory-adapted or highly virulent strains. IMPORTANCE The main findings of this study are that we determined that there are Trypanosoma cruzi strains adapted to transplacental transmission and completely different from the commonly used laboratory reference strains. This implies a specific strategy for the vertical transmission of Chagas disease. It is impressive that the strains specialized for vertical transmission modify the gene expression of the placenta in a totally different way than the reference strains. In addition, we describe isolates of T. cruzi that cannot be transmitted transplacentally. Taken together, these results open up new insights into the molecular mechanisms of this insect vector-independent transmission form.

New Insights into the Role of the Trypanosoma cruzi Aldo-Keto Reductase TcAKR.

Chagas disease is a zoonotic infectious disease caused by the protozoan parasite Trypanosoma cruzi. It is distributed worldwide, affecting around 7 million people; there is no effective treatment, and it constitutes a leading cause of disability and premature death in the Americas. Only two drugs are currently approved for the treatment, Benznidazole and Nifurtimox, and both have to be activated by reducing the nitro-group. The T. cruzi aldo-keto reductase (TcAKR) has been related to the metabolism of benznidazole. TcAKR has been extensively studied, being most efforts focused on characterizing its implication in trypanocidal drug metabolism; however, little is known regarding its biological role. Here, we found that TcAKR is confined, throughout the entire life cycle, into the parasite mitochondria providing new insights into its biological function. In particular, in epimastigotes, TcAKR is associated with the kinetoplast, which suggests additional roles of the protein. The upregulation of TcAKR, which does not affect TcOYE expression, was correlated with an increase in PGF2α, suggesting that this enzyme is related to PGF2α synthesis in T. cruzi. Structural analysis showed that TcAKR contains a catalytic tetrad conserved in the AKR superfamily. Finally, we found that TcAKR is also involved in Nfx metabolization.

Insights into the Cell Division of Neospora caninum.

Neospora caninum is an apicomplexan protozoan parasite responsible for causing neosporosis in a range of animal species. It results in substantial economic losses in the livestock industry and poses significant health risks to companion and wild animals. Central to its survival and pathogenicity is the process of cell division, which remains poorly understood in this parasite. In this study, we explored the cell division of Neospora caninum using a combination of modern and classic imaging tools, emphasizing its pivotal role in perpetuating the parasite's life cycle and contributing to its ability to persist within host organisms. We described the intricacies of endodyogeny in Neospora caninum, detailing the dynamics of the cell assembly and the nuclear division by ultrastructure expansion microscopy and regular confocal microscopy. Furthermore, we explored the centrosome dynamics, the centrioles and the apicoplast through the advancement of the cell cycle. Our analysis described with unprecedented detail, the endodyogeny in this parasite. By advancing our understanding of these molecular mechanisms, we aimed to inspire innovative strategies for disease management and control, with the ultimate goal of mitigating the devastating impact of neosporosis on animal health and welfare.

Trypanosoma cruzi-Derived Molecules Induce Anti-Tumour Protection by Favouring Both Innate and Adaptive Immune Responses.

Lung cancer remains the leading cause of cancer mortality worldwide. Thus, the development of strategies against this type of cancer is of high value. Parasite infections can correlate with lower cancer incidence in humans and their use as vaccines has been recently explored in preclinical models. In this study, we investigated whether immunisations with a Trypanosoma cruzi lysate from epimastigotes protect from lung tumour growth in mice. We also explore the role of parasite glycans in the induction of the protective immune response. A pre-clinical murine cancer model using the lung tumour cell line LL/2 was used to evaluate the anti-tumour potential, both in preventive and therapeutic settings, of a T. cruzi epimastigote-derived protein lysate. Immunisation with the parasite lysate prevents tumour growth and induces both humoral and cellular anti-tumour immune responses to LL-2 cancer cells. The induced immunity and tumour protection were associated with the activation of natural killer (NK) cells, the production of interferon-γ (IFN-γ) and tumour cell cytotoxicity. We also show that mannose residues in the T. cruzi lysate induce Toll-like receptor (TLR) signalling. The evaluated T. cruzi lysate possesses anti-tumour properties likely by activating innate and adaptive immunity in a process where carbohydrates seem to be essential.

A holstein heifer infected with Neospora caninum NcUru3 congenitally transmits this strain to a viable offspring although infection does not protect her from aborting by a different N. caninum genotype in the subsequent gestation.

Neospora caninum is a leading cause of bovine abortion worldwide. Although the genetic diversity of this apicomplexan parasite has long been recognized, there is little information on whether infection with different genotypes results in different clinical outcomes or whether infection by a given genotype impairs protective immunity against abortion induced by different genotypes. Here, we provide evidence supporting that natural subclinical infection with isolate NcUru3 of N. caninum in a pregnant heifer did not provide protection against abortion caused by a different N. caninum genotype in the subsequent gestation. A Holstein heifer delivered a healthy calf congenitally infected with N. caninum. Specific anti-N. caninum IgG was detected by indirect ELISA in sera obtained from the dam at calving and the calf before ingestion of colostrum, indicating in utero exposure to the parasite in the latter. A N. caninum strain named NcUru3 was isolated and characterized by multilocus microsatellite typing from the brain of this neonate euthanized at 9 days of age. Sixty days after calving, the cow got pregnant, although she aborted spontaneously at ~6 months of gestation. Pathologic examination of the aborted fetus and placenta revealed typical lesions of neosporosis, including encephalitis, myocarditis, hepatitis, myositis, and placentitis. Neospora caninum DNA was amplified from the fetal brain, heart, kidney, and placenta, and multilocus microsatellite typing revealed a genotype that differed from isolate NcUru3 at the level of microsatellite marker 6A (MS6A). Serum obtained from the dam at the time of abortion had IgG that cross-recognized isolate NcUru3, as demonstrated by immunoblotting, indicating that the humoral immune response did not prevent the other genotype from infecting the fetus and inducing fetoplacental lesions and abortion. This is the first description of one same dam transmitting two N. caninum genotypes to her offspring in subsequent gestations.

Genomic and proteomic analysis of Tausonia pullulans reveals a key role for a GH15 glucoamylase in starch hydrolysis.

Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. KEY POINTS: • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch.

Clinical and epidemiological features of tuberculosis isolated from critically ill patients.

Human tuberculosis is still a major world health concern. In Uruguay, contrary to the world trend, an increase in cases has been observed since 2006. Although the incidence of MDR-resistant strains is low and no cases of XDR-TB were registered, an increase in the number of patients with severe tuberculosis requiring critical care admission was observed. As a first aim, we performed the analysis of the genetic structure of strains isolated from patients with severe tuberculosis admitted to an intensive care unit. We compared these results with those corresponding to the general population observing a statistically significant increase in the Haarlem genotypes among ICU patients (53.3% vs 34.7%; p<0.05). In addition, we investigated the association of clinical outcomes with the genotype observing a major incidence of hepatic dysfunctions among patients infected with the Haarlem strain (p<0.05). The cohort presented is one of the largest studied series of critically ill patients with tuberculosis.

Indigenous Ancestry and Admixture in the Uruguayan Population.

The Amerindian group known as the Charrúas inhabited Uruguay at the timing of European colonial contact. Even though they were extinguished as an ethnic group as a result of a genocide, Charrúan heritage is part of the Uruguayan identity both culturally and genetically. While mitochondrial DNA studies have shown evidence of Amerindian ancestry in living Uruguayans, here we undertake whole-genome sequencing of 10 Uruguayan individuals with self-declared Charruan heritage. We detect chromosomal segments of Amerindian ancestry supporting the presence of indigenous genetic ancestry in living descendants. Specific haplotypes were found to be enriched in "Charrúas" and rare in the rest of the Amerindian groups studied. Some of these we interpret as the result of positive selection, as we identified selection signatures and they were located mostly within genes related to the infectivity of specific viruses. Historical records describe contacts of the Charrúas with other Amerindians, such as Guaraní, and patterns of genomic similarity observed here concur with genomic similarity between these groups. Less expected, we found a high genomic similarity of the Charrúas to Diaguita from Argentinian and Chile, which could be explained by geographically proximity. Finally, by fitting admixture models of Amerindian and European ancestry for the Uruguayan population, we were able to estimate the timing of the first pulse of admixture between European and Uruguayan indigenous peoples in approximately 1658 and the second migration pulse in 1683. Both dates roughly concurring with the Franciscan missions in 1662 and the foundation of the city of Colonia in 1680 by the Spanish.

GH Overexpression Alters Spermatic Cells MicroRNAome Profile in Transgenic Zebrafish.

Overexpression of growth hormone (GH) in gh-transgenic zebrafish of a highly studied lineage F0104 has earlier been reported to cause increased muscle growth. In addition to this, GH affects a broad range of cellular processes in transgenic fish, such as morphology, physiology, and behavior. Reports show changes such as decreased sperm quality and reduced reproductive performance in transgenic males. It is hypothesized that microRNAs are directly involved in the regulation of fertility potential during spermatogenesis. The primary aim of our study was to verify whether gh overexpression disturbs the sperm miRNA profile and influences the sperm quality in transgenic zebrafish. We report a significant increase in body weight of gh-transgenic males along with associated reduced sperm motility and other kinetic parameters in comparison to the non-transgenic group. MicroRNA transcriptome sequencing of gh-transgenic zebrafish sperms revealed expressions of 186 miRNAs, among which six miRNA were up-regulated (miR-146b, miR-200a-5p, miR-146a, miR-726, miR-184, and miR-738) and sixteen were down-regulated (miR-19d-3p, miR-126a-5p, miR-126b-5p, miR-22a-5p, miR-16c-5p, miR-20a-5p, miR-126b-3p, miR-107a-3p, miR-93, miR-2189, miR-202-5p, miR-221-3p, miR-125a, miR-125b-5p, miR-126a-3p, and miR-30c-5p) in comparison to non-transgenic zebrafish. Some of the dysregulated miRNAs were previously reported to be related to abnormalities in sperm quality and reduced reproduction ability in other species. In this study, an average of 134 differentially expressed miRNAs-targeted genes were predicted using the in silico approach. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that the genes of affected pathways were primarily related to spermatogenesis, sperm motility, and cell apoptosis. Our results suggested that excess GH caused a detrimental effect on sperm microRNAome, consequently reducing the sperm quality and reproductive potential of zebrafish males.

Preclinical Studies in Anti-Trypanosomatidae Drug Development.

The trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania are the causative agents of human African trypanosomiasis, Chagas Disease and Leishmaniasis, respectively. These infections primarily affect poor, rural communities in the developing world, and are responsible for trapping sufferers and their families in a disease/poverty cycle. The development of new chemotherapies is a priority given that existing drug treatments are problematic. In our search for novel anti-trypanosomatid agents, we assess the growth-inhibitory properties of >450 compounds from in-house and/or "Pathogen Box" (PBox) libraries against L. infantum, L. amazonensis, L.braziliensis, T. cruzi and T. brucei and evaluate the toxicities of the most promising agents towards murine macrophages. Screens using the in-house series identified 17 structures with activity against and selective toward Leishmania: Compounds displayed 50% inhibitory concentrations between 0.09 and 25 µM and had selectivity index values >10. For the PBox library, ~20% of chemicals exhibited anti-parasitic properties including five structures whose activity against L. infantum had not been reported before. These five compounds displayed no toxicity towards murine macrophages over the range tested with three being active in an in vivo murine model of the cutaneous disease, with 100% survival of infected animals. Additionally, the oral combination of three of them in the in vivo Chagas disease murine model demonstrated full control of the parasitemia. Interestingly, phenotyping revealed that the reference strain responds differently to the five PBox-derived chemicals relative to parasites isolated from a dog. Together, our data identified one drug candidate that displays activity against Leishmania and other Trypanosomatidae in vitro and in vivo, while exhibiting low toxicity to cultured mammalian cells and low in vivo acute toxicity.

Maxicircle architecture and evolutionary insights into Trypanosoma cruzi complex.

We sequenced maxicircles from T. cruzi strains representative of the species evolutionary diversity by using long-read sequencing, which allowed us to uncollapse their repetitive regions, finding that their real lengths range from 35 to 50 kb. T. cruzi maxicircles have a common architecture composed of four regions: coding region (CR), AT-rich region, short (SR) and long repeats (LR). Distribution of genes, both in order and in strand orientation are conserved, being the main differences the presence of deletions affecting genes coding for NADH dehydrogenase subunits, reinforcing biochemical findings that indicate that complex I is not functional in T. cruzi. Moreover, the presence of complete minicircles into maxicircles of some strains lead us to think about the origin of minicircles. Finally, a careful phylogenetic analysis was conducted using coding regions of maxicircles from up to 29 strains, and 1108 single copy nuclear genes from all of the DTUs, clearly establishing that taxonomically T. cruzi is a complex of species composed by group 1 that contains clades A (TcI), B (TcIII) and D (TcIV), and group 2 (1 and 2 do not coincide with groups I and II described decades ago) containing clade C (TcII), being all hybrid strains of the BC type. Three variants of maxicircles exist in T. cruzi: a, b and c, in correspondence with clades A, B, and C from mitochondrial phylogenies. While A and C carry maxicircles a and c respectively, both clades B and D carry b maxicircle variant; hybrid strains also carry the b- variant. We then propose a new nomenclature that is self-descriptive and makes use of both the phylogenetic relationships and the maxicircle variants present in T. cruzi.