Small molecule mediators of host-T. cruzi-environment interactions in Chagas disease.

Small molecules (less than 1,500 Da) include major biological signals that mediate host-pathogen-microbiome communication. They also include key intermediates of metabolism and critical cellular building blocks. Pathogens present with unique nutritional needs that restrict pathogen colonization or promote tissue damage. In parallel, parts of host metabolism are responsive to immune signaling and regulated by immune cascades. These interactions can trigger both adaptive and maladaptive metabolic changes in the host, with microbiome-derived signals also contributing to disease progression. In turn, targeting pathogen metabolic needs or maladaptive host metabolic changes is an important strategy to develop new treatments for infectious diseases. Trypanosoma cruzi is a single-celled eukaryotic pathogen and the causative agent of Chagas disease, a neglected tropical disease associated with cardiac and intestinal dysfunction. Here, we discuss the role of small molecules during T. cruzi infection in its vector and in the mammalian host. We integrate these findings to build a theoretical interpretation of how maladaptive metabolic changes drive Chagas disease and extrapolate on how these findings can guide drug development.

Unveiling the Peptidase Network Orchestrating Hemoglobin Catabolism in Rhodnius prolixus.

Chagas disease is transmitted to humans by obligatory hematophagous insects of Triatominae subfamily, which feeds on various hosts to acquire their nutritional sustenance derived from blood proteins. Hemoglobin (Hb) digestion is a pivotal metabolic feature of triatomines, representing a key juncture in their competence toward Trypanosoma cruzi; however, it remains poorly understood. To explore the Hb digestion pathway in Rhodnius prolixus, a major Chagas disease vector, we employed an array of approaches for activity profiling of various midgut-associated peptidases using specific substrates and inhibitors. Dissecting the individual contribution of each peptidase family in Hb digestion has unveiled a predominant role played by aspartic proteases and cathepsin B-like peptidases. Determination of peptidase-specific cleavage sites of these key hemoglobinases, in conjunction with mass spectrometry-based identification of in vivo Hb-derived fragments, has revealed the intricate network of peptidases involved in the Hb digestion pathway. This network is initiated by aspartic proteases and subsequently sustained by cysteine proteases belonging to the C1 family. The process is continued simultaneously by amino and carboxypeptidases. The comprehensive profiling of midgut-associated aspartic proteases by quantitative proteomics has enabled the accurate revision of gene annotations within the A1 family of the R. prolixus genome. Significantly, this study also serves to illuminate a potentially important role of the anterior midgut in blood digestion. The expanded repertoire of midgut-associated proteases presented in this study holds promise for the identification of novel targets aimed at controlling the transmission of Chagas disease.

A spatially resolved elemental nanodomain organization within acidocalcisomes in Trypanosoma cruzi.

How are ions distributed in the three-dimensional (3D) volume confined in a nanoscale compartment? Regulation of ionic flow in the intracellular milieu has been explained by different theoretical models and experimentally demonstrated for several compartments with microscale dimensions. Most of these models predict a homogeneous distribution of ions seconds or milliseconds after an initial diffusion step formed at the ion translocation site, leaving open questions when it comes to ion/element distribution in spaces/compartments with nanoscale dimensions. Due to the influence of compartment size on the regulation of ionic flow, theoretical variations of classical models have been proposed, suggesting heterogeneous distributions of ions/elements within nanoscale compartments. Nonetheless, such assumptions have not been fully proven for the 3D volume of an organelle. In this work, we used a combination of cutting-edge electron microscopy techniques to map the 3D distribution of diffusible elements within the whole volume of acidocalcisomes in trypanosomes. Cryofixed cells were analyzed by scanning transmission electron microscopy tomography combined with elemental mapping using a high-performance setup of X-ray detectors. Results showed the existence of elemental nanodomains within the acidocalcisomes, where cationic elements display a self-excluding pattern. These were validated by Pearson correlation analysis and in silico molecular dynamic simulations. Formation of element domains within the 3D space of an organelle is demonstrated. Distribution patterns that support the electrodiffusion theory proposed for nanophysiology models have been found. The experimental pipeline shown here can be applied to a variety of models where ion mobilization plays a crucial role in physiological processes.

Fatty acid elongases 1-3 have distinct roles in mitochondrial function, growth, and lipid homeostasis in Trypanosoma cruzi.

Trypanosomatids are a diverse group of uniflagellate protozoan parasites that include globally relevant pathogens such as Trypanosoma cruzi, the causative agent of Chagas disease. Trypanosomes lack the fatty acid synthase system typically used for de novo fatty acid (FA) synthesis in other eukaryotes. Instead, these microbes have evolved a modular FA elongase (ELO) system comprised of individual ELO enzymes (ELO1-4) that can operate processively to generate long chain- and very long chain-FAs. The importance of ELO's for maintaining lipid homeostasis in trypanosomatids is currently unclear, given their ability to take up and utilize exogenous FAs for lipid synthesis. To assess ELO function in T. cruzi, we generated individual KO lines, Δelo1, Δelo2, and Δelo3, in which the genes encoding ELO1-3 were functionally disrupted in the parasite insect stage (epimastigote). Using unbiased lipidomic and metabolomic analyses, in combination with metabolic tracing and biochemical approaches, we demonstrate that ELO2 and ELO3 are required for global lipid homeostasis, whereas ELO1 is dispensable for this function. Instead, ELO1 activity is needed to sustain mitochondrial activity and normal growth in T. cruzi epimastigotes. The cross-talk between microsomal ELO1 and the mitochondrion is a novel finding that, we propose, merits further examination of the trypanosomatid ELO pathway as critical for central metabolism.

RNA-Seq reveals that overexpression of TcUBP1 switches the gene expression pattern toward that of the infective form of Trypanosoma cruzi.

Trypanosomes regulate gene expression mainly by using posttranscriptional mechanisms. Key factors responsible for carrying out this regulation are RNA-binding proteins, affecting subcellular localization, translation, and/or transcript stability. Trypanosoma cruzi U-rich RNA-binding protein 1 (TcUBP1) is a small protein that modulates the expression of several surface glycoproteins of the trypomastigote infective stage of the parasite. Its mRNA targets are known, but the impact of its overexpression at the transcriptome level in the insect-dwelling epimastigote cells has not yet been investigated. Thus, in the present study, by using a tetracycline-inducible system, we generated a population of TcUBP1-overexpressing parasites and analyzed its effect by RNA-Seq methodology. This allowed us to identify 793 up- and 371 downregulated genes with respect to the wildtype control sample. Among the upregulated genes, it was possible to identify members coding for the TcS superfamily, MASP, MUCI/II, and protein kinases, whereas among the downregulated transcripts, we found mainly genes coding for ribosomal, mitochondrial, and synthetic pathway proteins. RNA-Seq comparison with two previously published datasets revealed that the expression profile of this TcUBP1-overexpressing replicative epimastigote form resembles the transition to the infective metacyclic trypomastigote stage. We identified novel cis-regulatory elements in the 3'-untranslated region of the affected transcripts and confirmed that UBP1m, a signature TcUBP1 binding element previously characterized in our laboratory, is enriched in the list of stabilized genes. We can conclude that the overall effect of TcUBP1 overexpression on the epimastigote transcriptome is mainly the stabilization of mRNAs coding for proteins that are important for parasite infection.

Nonstandard RNA/RNA interactions likely enhance folding and stability of segmented ribosomes.

The ribosome is the molecular factory that catalyzes all coded protein synthesis in extant organisms. Eukaryotic ribosomes are typically assembled out of four rRNAs; namely, 5S, 5.8S, 18S, and 28S. However, the 28S rRNA of some trypanosomatid organisms has been found to be segmented into six independent rRNAs of different sizes. The two largest segments have multiple sites where they jointly form stems comprised of standard base pairs that can hold them together. However, such regions of interaction are not observed among the four smaller RNAs. Early reports suggested that trypanosomatid segmented ribosome assembly was essentially achieved thanks to their association with rProteins. However, examination of cryo-EM ribosomal structures from Trypanosoma brucei, Leishmania donovani, and Trypanosoma cruzi reveals several long-range nonstandard RNA/RNA interactions. Most of these interactions are clusters of individual hydrogen bonds and so are not readily predictable. However, taken as a whole, they represent significant stabilizing energy that likely facilitates rRNA assembly and the overall stability of the segmented ribosomes. In the context of origin of life studies, the current results provide a better understanding of the true nature of RNA sequence space and what might be possible without an RNA replicase.

Trypanosoma cruzi strain and starvation-driven mitochondrial RNA editing and transcriptome variability.

Trypanosoma cruzi is a unicellular protistan parasitic species that is comprised of strains and isolates exhibiting high levels of genetic and metabolic variability. In the insect vector, it is known to be highly responsive to starvation, a signal for progression to a life stage in which it can infect mammalian cells. Most mRNAs encoded in its mitochondrion require the targeted insertion and deletion of uridines to become translatable transcripts. This study defined differences in uridine-insertion/deletion RNA editing among three strains and established the mechanism whereby abundances of edited (and, thus, translatable) mitochondrial gene products increase during starvation. Our approach utilized our custom T-Aligner toolkit to describe transcriptome-wide editing events and reconstruct editing products from high-throughput sequencing data. We found that the relative abundance of mitochondrial transcripts and the proportion of mRNAs that are edited varies greatly between analyzed strains, a characteristic that could potentially impact metabolic capacity. Starvation typically led to an increase in overall editing activity rather than affecting a specific step in the process. We also determined that transcripts CR3, CR4, and ND3 produce multiple open reading frames that, if translated, would generate different proteins. Finally, we quantitated the inherent flexibility of editing in T. cruzi and found it to be higher relative to that in a related trypanosomatid lineage. Over time, new editing domains or patterns could prove advantageous to the organism and become more widespread within individual transcriptomes or among strains.

H2B.V demarcates divergent strand-switch regions, some tDNA loci, and genome compartments in Trypanosoma cruzi and affects parasite differentiation and host cell invasion.

Histone variants play a crucial role in chromatin structure organization and gene expression. Trypanosomatids have an unusual H2B variant (H2B.V) that is known to dimerize with the variant H2A.Z generating unstable nucleosomes. Previously, we found that H2B.V protein is enriched in tissue-derived trypomastigote (TCT) life forms, a nonreplicative stage of Trypanosoma cruzi, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among parasite life forms. Here, we performed the first genome-wide profiling of histone localization in T. cruzi using epimastigotes and TCT life forms, and we found that H2B.V was preferentially located at the edges of divergent transcriptional strand switch regions, which encompass putative transcriptional start regions; at some tDNA loci; and between the conserved and disrupted genome compartments, mainly at trans-sialidase, mucin and MASP genes. Remarkably, the chromatin of TCT forms was depleted of H2B.V-enriched peaks in comparison to epimastigote forms. Interactome assays indicated that H2B.V associated specifically with H2A.Z, bromodomain factor 2, nucleolar proteins and a histone chaperone, among others. Parasites expressing reduced H2B.V levels were associated with higher rates of parasite differentiation and mammalian cell infectivity. Taken together, H2B.V demarcates critical genomic regions and associates with regulatory chromatin proteins, suggesting a scenario wherein local chromatin structures associated with parasite differentiation and invasion are regulated during the parasite life cycle.

AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models.

The AlphaFold Protein Structure Database (AlphaFold DB, https://alphafold.ebi.ac.uk) is an openly accessible, extensive database of high-accuracy protein-structure predictions. Powered by AlphaFold v2.0 of DeepMind, it has enabled an unprecedented expansion of the structural coverage of the known protein-sequence space. AlphaFold DB provides programmatic access to and interactive visualization of predicted atomic coordinates, per-residue and pairwise model-confidence estimates and predicted aligned errors. The initial release of AlphaFold DB contains over 360,000 predicted structures across 21 model-organism proteomes, which will soon be expanded to cover most of the (over 100 million) representative sequences from the UniRef90 data set.

Influence of CK2 protein kinase activity on the interaction between Trypanosoma cruzi and its vertebrate and invertebrate hosts.

Chagas disease, endemic from Latin America, is caused by Trypanosoma cruzi and is transmitted by triatomine feces. This parasite undergoes complex morphological changes through its life cycle, promoted by significant changes in signal transduction pathways. The activity of protein kinase CK2 has been described in trypanosomatids. Using a specific peptide and radioactive ATP, we identified CK2 activity on the cellular surface and the cytoplasmic content in Trypanosoma cruzi, apart from the secreted form. Dephosphorylated casein promoted an increase of 48% in the secreted CK2 activity. Total extract of peritoneal macrophages from BALB/c and inactivated human serum promoted an increase of 67% and 36%, respectively, in this activity. The protein secreted by parasites was purified by HPLC and had shown compatibility with the catalytic subunit of mammalian CK2. Incubation of the parasites with CK2 inhibitors, added to the culture medium, prevented their growth. The opposite was observed when CK2 activators were used. Results of interaction between Trypanosoma cruzi and the gut of the vector have revealed that, in the presence of CK2 inhibitors, there is a reduction in the association rate. A similar inhibition profile was seen in the Trypanosoma cruzi-macrophages interaction, confirming the importance of this enzyme in the life cycle of this protozoan.

Kinetic and Structural Characterization of Trypanosoma cruzi Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferases and Repurposing of Transition-State Analogue Inhibitors.

Over 70 million people are currently at risk of developing Chagas Disease (CD) infection, with more than 8 million people already infected worldwide. Current treatments are limited and innovative therapies are required. Trypanosoma cruzi, the etiological agent of CD, is a purine auxotroph that relies on phosphoribosyltransferases to salvage purine bases from their hosts for the formation of purine nucleoside monophosphates. Hypoxanthine-guanine-xanthine phosphoribosyltransferases (HGXPRTs) catalyze the salvage of 6-oxopurines and are promising targets for the treatment of CD. HGXPRTs catalyze the formation of inosine, guanosine, and xanthosine monophosphates from 5-phospho-d-ribose 1-pyrophosphate and the nucleobases hypoxanthine, guanine, and xanthine, respectively. T. cruzi possesses four HG(X)PRT isoforms. We previously reported the kinetic characterization and inhibition of two isoforms, TcHGPRTs, demonstrating their catalytic equivalence. Here, we characterize the two remaining isoforms, revealing nearly identical HGXPRT activities in vitro and identifying for the first time T. cruzi enzymes with XPRT activity, clarifying their previous annotation. TcHGXPRT follows an ordered kinetic mechanism with a postchemistry event as the rate-limiting step(s) of catalysis. Its crystallographic structures reveal implications for catalysis and substrate specificity. A set of transition-state analogue inhibitors (TSAIs) initially developed to target the malarial orthologue were re-evaluated, with the most potent compound binding to TcHGXPRT with nanomolar affinity, validating the repurposing of TSAIs to expedite the discovery of lead compounds against orthologous enzymes. We identified mechanistic and structural features that can be exploited in the optimization of inhibitors effective against TcHGPRT and TcHGXPRT concomitantly, which is an important feature when targeting essential enzymes with overlapping activities.

Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate.

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

The high identity of the Trypanosoma cruzi Group-I of trans-sialidases points them as promising vaccine immunogens.

Trans-sialidase (TS) superfamily of proteins comprises eight subgroups, being the proteins of Group-I (TS-GI) promising immunogens in vaccine approaches against Trypanosoma cruzi. Strikingly, TS-GI antigenic variability among parasite lineages and their influence on vaccine development has not been previously analyzed. Here, a search in GenBank detects 49 TS-GI indexed sequences, whereas the main infecting human different parasite discrete typing units (DTU) are represented. In silico comparison among these sequences indicate that they share an identity above 92%. Moreover, the antigenic regions (T-cell and B-cell epitopes) are conserved in most sequences or present amino acid substitutions that scarcely may alter the antigenicity. Additionally, since the generic term TS is usually used to refer to different immunogens of this broad family, a further in silico analysis of the TS-GI-derived fragments tested in preclinical vaccines was done to determine the coverage and identity among them, showing that overall amino acid identity of vaccine immunogens is high, but the segment coverage varies widely. Accordingly, strong H-2K, H-2I, and B-cell epitopes are dissimilarly represented among vaccine TS-derived fragments depending on the extension of the TG-GI sequence used. Moreover, bioinformatic analysis detected a set of 150 T-cell strong epitopes among the DTU-indexed sequences that strongly bind human HLA-I supertypes. In all currently reported experimental vaccines based on TS-GI fragments, mapping these 150 epitopes showed that they are moderately represented. However, despite vaccine epitopes do not present all the substitutions observed in the DTUs, these regions of the proteins are equally recognized by the same HLAs.  Interestingly, the predictions regarding global and South American population coverage estimated in these 150 epitopes are similar to the estimations in experimental vaccines when the complete sequence of TS-GI is used as an antigen. In silico prediction also shows that a number of these MHC-I restricted T-cell strong epitopes could be also cross-recognized by HLA-I supertypes and H-2Kb or H-2Kd backgrounds, indicating that these mice may be used to improve and facilitate the development of new TS-based vaccines and suggesting an immunogenic and protective potential in humans. Further molecular docking analyses were performed to strengthen these results. Taken together, different strategies that would cover more or eventually fully of these T-cell and also B-cell epitopes to reach a high level of coverage are considered.

Blocking activation of CD4-CD8- T cells modulates their cytotoxic potential and decreases the expression of inflammatory and chemotactic receptors.

CD4-CD8- (double negative - DN) T cells represent a small fraction of circulating T lymphocytes but are a major source of pro-inflammatory cytokines in patients with infectious diseases, including chronic Chagas cardiomyopathy (CCC), one of the deadliest cardiopathies known. Chagas disease is caused by an infection with the protozoan parasite Trypanosoma cruzi and can lead to either an asymptomatic form or a high-mortality cardiac disease. While circulating DN T cells represent a major inflammatory cytokine-expressing cell population in Chagas disease, their potential to be recruited to the heart and to perform cytotoxicity has not been determined. Our previous studies showed that blocking DN T cell activation decreases the expression of IFN-gamma, a cytokine involved in the severity of CCC. Here, studying a well-characterized cohort of Chagas patients with CCC or the asymptomatic form of Chagas disease (indeterminate form, IND), we evaluated the expression of cytotoxic molecules, cytokine and chemokine receptors in γδ+ and αß+ DN T cells by multiparameter flow cytometry, and investigated whether blocking the activation of DN T cells influences the expression of these molecules. We observed that DN T cells from CCC display a higher expression of granzyme A, perforin, inflammatory molecules, and inflammatory chemokine receptors than cells from IND. Messenger RNA coding for these molecules is also upregulated in the heart of CCC patients. Importantly, blocking the activation of DN T cells from CCC modulates their cytotoxic potential and the expression of inflammatory and of chemokine receptors, suggesting that targeting DN T cell activation may be a valid strategy to reduce recruitment to the heart, inflammation, cytotoxicity and, thereby diminish CCC progression and severity.

Trypanosoma cruzi Fibrillarins: Two paralogous proteins with non-identical signals for nuclear transport.

Trypanosoma cruzi is a parasitic protozoa causative of Chagas disease. As part of our interest in studying the basic biology of this microorganism, this work reports our observations related to the characterization of motifs and structural domains present in two fibrillarin isoforms (TcFib1 and TcFib2) that were found to be necessary for the nuclear targeting of these nucleolar proteins. Previous characterization of these proteins indicated that they share 68.67% of identical amino acids and are both expressed as nucleolar proteins in T. cruzi epimastigotes. Using an approach based on the transfection of recombinant genes encoding fluorescent fibrillarin-EGFP fusion proteins, this study found evidence for the presence of 4 motifs or protein domains that help target these proteins to the nucleus: The GAR domain and carboxyl terminus in both TcFibs, as well as two lysines and a computationally predicted cNLS in TcFib1. As a distinctive feature, the GAR domain of TcFib2 proved to be essential for the nuclear localization of this protein paralog. Such a difference between TcFib1 and Tcfib2 nuclear localization signals can be explained as the presence of two partially related nuclear import pathways for the two fibrillarin homologues in this organism.

Fatty acid oxidation participates in resistance to nutrient-depleted environments in the insect stages of Trypanosoma cruzi.

Trypanosoma cruzi, the parasite causing Chagas disease, is a digenetic flagellated protist that infects mammals (including humans) and reduviid insect vectors. Therefore, T. cruzi must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard, it is well known that T. cruzi has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established has been the capability of T. cruzi to use glucose and amino acids to support the differentiation process occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities of using externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production. Additionally, we show that electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid (TCA) cycle, which can be used to feed anabolic pathways such as the de novo biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization into the mitochondrion diminishes the survival to severe starvation, and impairs metacyclogenesis.

Structural differences between hypoxanthine phosphoribosyltransferase family members highlight opportunities for antiparasitic drug design in neglected diseases.

Approaches to identify novel druggable targets for treating neglected diseases include computational studies that predict possible interactions of drugs and their molecular targets. Hypoxanthine phosphoribosyltransferase (HPRT) plays a central role in the purine salvage pathway. This enzyme is essential for the survival of the protozoan parasite T. cruzi, the causal agent of Chagas disease, and other parasites related to neglected diseases. Here we found dissimilar functional behaviours between TcHPRT and the human homologue, HsHPRT, in the presence of substrate analogues that can lie in differences in their oligomeric assemblies and structural features. To shed light on this issue, we carried out a comparative structural analysis between both enzymes. Our results show that HsHPRT is considerably more resistant to controlled proteolysis than TcHPRT. Moreover, we observed a variation in the length of two key loops depending on the structural arrangement of each protein (groups D1T1 and D1T1'). Such variations might be involved in inter-subunit communication or influencing the oligomeric state. Besides, to understand the molecular basis that govern D1T1 and D1T1' folding groups, we explored the distribution of charges on the interaction surfaces of TcHPRT and HsHPRT, respectively. To know whether the rigidity degree bears effect on the active site, we studied the flexibility of both proteins. The analysis performed here illuminates the underlying reasons and significance behind each protein's preference for one or the other quaternary arrangement that can be exploited for therapeutic approaches.

Gene editing of putative cAMP and Ca2+ -regulated proteins using an efficient cloning-free CRISPR/Cas9 system in Trypanosoma cruzi.

Trypanosoma cruzi, the agent of Chagas disease, must adapt to a diversity of environmental conditions that it faces during its life cycle. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Cyclic AMP (cAMP) and Calcium (Ca2+ ) signaling pathways regulate critical cellular processes in this parasite, such as differentiation, osmoregulation, host cell invasion and cell bioenergetics. Although the use of CRISPR/Cas9 technology prompted reverse genetics approaches for functional analysis in T. cruzi, it is still necessary to expand the toolbox for genome editing in this parasite, as for example to perform multigene analysis. Here we used an efficient T7RNAP/Cas9 strategy to tag and delete three genes predicted to be involved in cAMP and Ca2+ signaling pathways: a putative Ca2+ /calmodulin-dependent protein kinase (CAMK), Flagellar Member 6 (FLAM6) and Cyclic nucleotide-binding domain/C2 domain-containing protein (CC2CP). We endogenously tagged these three genes and determined the subcellular localization of the tagged proteins. Furthermore, the strategy used to knockout these genes allows us to presume that TcCC2CP is an essential gene in T. cruzi epimastigotes. Our results will open new venues for future research on the role of these proteins in T. cruzi.

Allylpolyalkoxybenzene Inhibitors of Galactonolactone Oxidase from Trypanosoma cruzi.

Inhibition of biosynthetic pathways of compounds essential for Trypanosoma cruzi is considered as one of the possible action mechanisms of drugs against Chagas disease. Here, we investigated the inhibition of galactonolactone oxidase from T. cruzi (TcGAL), which catalyzes the final step in the synthesis of vitamin C, an antioxidant that T. cruzi is unable to assimilate from outside and must synthesize itself, and identified allylbenzenes from plant sources as a new class of TcGAL inhibitors. Natural APABs (apiol, dillapiol, etc.) inhibited TcGAL with IC50 = 20-130 µM. The non-competitive mechanism of TcGAL inhibition by apiol was established. Conjugation of APABs with triphenylphosphonium, which ensures selective delivery of biologically active substances to the mitochondria, increased the efficiency and/or the maximum percentage of TcGAL inhibition compared to nonmodified APABs.

Differential expression of meiosis and homologous recombination-related genes in the life cycle of Trypanosoma cruzi.

Trypanosoma cruzi has a complex life cycle consisting of four morphological and distinct biological stages. Although some authors suggest that T. cruzi primarily follows clonal reproduction, recent genomic and transcriptomic studies indicate an unorthodox capacity for recombination. We aimed to estimate the differential gene expression of 10 meiosis/homologous recombination-related genes during the T. cruzi life cycle, including epimastigotes, under two different types of stress (oxidative stress and pH changes). We performed RT-qPCR tests using novel-designed primers to estimate the differential gene expression (∆Ct and ∆∆Ct) of nine genes (SPO11, HAP2, RAD50, MRN complex, BRCA2, DMC1, MND1, and RPA1) and RAD51, which was previously reported. Our results show basal expression of all genes during the life cycle, indicating their hypothetical role in several cellular processes but with specific signatures of differential gene expression during the life cycle (HAP2, RPA, RAD50, BRCA2, MND1, and DMC1) and oxidative stress (RPA, MRE11, NBS1, BRCA2, MND1, and RAD51). Additionally, we found that the MRN complex has an independent level of expression in T. cruzi, with profiles of MRE11 and NBS1 upregulated in some stages. Recent studies on other trypanosomatids have highlighted the influence of HAP2 and RPA in recombination and hybridization. If T. cruzi uses the same repertoire of genes, our findings could suggest that metacyclogenesis may be the putative step that the parasite uses to undergo recombination. Likewise, our study reveals the differential profiles of genes expressed in response to oxidative and pH stress. Further studies are necessary to confirm our findings and understand the recombination mechanism in T. cruzi.