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Syzygium aromaticum has a diversity of biological activities due to the chemical compounds found in its plant products such as total phenolic compounds and flavonoids. The present work describes the chemical analysis and antimicrobial, antioxidant, and antitrypanosomal activity of the essential oil of S. aromaticum. Eugenol (53.23%) as the major compound was verified by gas chromatography-mass spectrometry. S. aromaticum essential oil was more effective against S. aureus (MIC 50 μg/mL) than eugenol (MIC 250 μg/mL). Eugenol presented higher antioxidant activity than S. aromaticum essential oil, with an EC50 of 12.66 and 78.98 µg/mL, respectively. S. aromaticum essential oil and eugenol exhibited Trypanosoma cruzi inhibitory activity, with IC50 of 28.68 ± 1.073 and 31.97 ± 1.061 μg/mL against epimastigotes and IC50 of 64.51 ± 1.658 and 45.73 ± 1.252 μg/mL against intracellular amastigotes, respectively. Both compounds presented low cytotoxicity, with S. aromaticum essential oil displaying 15.5-fold greater selectivity for the parasite than the cells. Nitrite levels in T. cruzi-stimulated cells were reduced by essential oil (47.01%; p = 0.002) and eugenol (48.05%; p = 0.003) treatment. The trypanocidal activity of S. aromaticum essential oil showed that it is reasonable to use it in future research in the search for new therapeutic alternatives for trypanosomiasis.

Background Chagas disease, caused by the protozoan parasite Trypanosoma cruzi , is a neglected tropical illness affecting an estimated 6–7 million people worldwide. The currently approved drugs have significant limitations, but antimicrobial peptides (AMPs) have emerged as promising therapeutic alternatives. Members of the cruzioseptin family, a group of AMPs derived from the frog Cruziohyla calcarifer , have demonstrated anti- T. cruzi activity, but their mode of action remains poorly understood. Herein, T. cruzi epimastigotes were used to identify active cruzioseptins and investigate their mechanism of action through untargeted metabolomics and molecular dynamics simulations. Methods Synthetic versions of three previously unstudied cruzioseptins (CZS-5, CZS-7, and CZS-11) were evaluated for their effects on T. cruzi X-1081 epimastigotes via microplate assays with resazurin-based viability measurements. CZS-1, a peptide with known anti- T. cruzi activity, was also included. Selectivity was assessed via hemolysis assays on human erythrocytes. To evaluate membrane damage, DNA leakage assays and scanning electron microscopy (SEM) were performed on epimastigotes treated with CZS-5. In addition, the interaction of cruzioseptins with the epimastigote membrane was modeled using molecular dynamics simulations. To explore additional mechanisms of action, a multiplatform metabolomic analysis (HILIC-LC-QTOF-MS and GC-QTOF-MS) was conducted to identify altered metabolites in epimastigotes treated with CZS-5. Results Among the tested cruzioseptins, CZS-5 exhibited the highest potency (IC 50  = 4.7 ± 1.0 µM) and selectivity (SI = 50.3). This peptide induced DNA leakage from epimastigotes and caused surface alterations, suggesting membrane damage. Molecular dynamics simulations indicated that CZS-5 may exert its effects through the formation of toroidal pores in the parasite membrane. Untargeted metabolomic analysis revealed 118 altered metabolites in CZS-5-treated epimastigotes, with significant enrichment of glycerophospholipids (40.7%), supporting the involvement of membrane disruption. In addition, metabolic pathways were affected, suggesting complementary mechanisms of action, including oxidative stress and disruptions in energy metabolism. Conclusions CZS-5 was identified as a potent cruzioseptin with multiple potential mechanisms of action in the epimastigotes stage of T. cruzi . Further validation is needed in clinically relevant parasite stages to assess its potential as a therapeutic agent. Graphical Abstract

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.

The causative agent of Chagas disease undergoes drastic morphological and biochemical modifications as it passes between hosts and transitions from extracellular to intracellular stages. The osmotic and mechanical aspects of these cellular transformations are not understood. Here we identify and characterize a novel mechanosensitive channel in T rypanosoma cruzi (TcMscS) belonging to the superfamily of small-conductance mechanosensitive channels (MscS). TcMscS is activated by membrane tension and forms a large pore permeable to anions, cations, and small osmolytes. The channel changes its location from the contractile vacuole complex in epimastigotes to the plasma membrane as the parasites develop into intracellular amastigotes. TcMscS knockout parasites show significant fitness defects, including increased cell volume, calcium dysregulation, impaired differentiation, and a dramatic decrease in infectivity. Our work provides mechanistic insights into components supporting pathogen adaptation inside the host, thus opening the exploration of mechanosensation as a prerequisite for protozoan infectivity.

Background Trypanosoma cruzi uses several strategies to survive in different hosts. A key step in the life-cycle of this parasite is metacyclogenesis, which involves various morphological, biochemical, and genetic changes that induce the differentiation of non-pathogenic epimastigotes into pathogenic metacyclic trypomastigotes. During metacyclogenesis, T. cruzi displays distinct morphologies and ultrastructural features, which have not been fully characterized. Results We performed a temporal description of metacyclogenesis using different microscopy techniques that resulted in the identification of three intermediate forms of T. cruzi : intermediates I, II and III. Such classification was based on morphological and ultrastructural aspects as the location of the kinetoplast in relation to the nucleus, kinetoplast shape and kDNA topology. Furthermore, we suggested that metacyclic trypomastigotes derived from intermediate forms that had already detached from the substrate. We also found that changes in the kinetoplast morphology and kDNA arrangement occurred only after the repositioning of this structure toward the posterior region of the cell body. These changes occurred during the later stages of differentiation. In contrast, changes in the nucleus shape began as soon as metacyclogenesis was initiated, while changes in nuclear ultrastructure, such as the loss of the nucleolus, were only observed during later stages of differentiation. Finally, we found that kDNA networks of distinct T. cruzi forms present different patterns of DNA topology. Conclusions Our study of T. cruzi metacyclogenesis revealed important aspects of the morphology and ultrastructure of this intriguing cell differentiation process. This research expands our understanding of this parasite’s fascinating life-cycle. It also highlights the study of T. cruzi as an important and exciting model system for investigating diverse aspects of cellular, molecular, and evolutionary biology.

Various methods, including blood culture, are employed to isolate Trypanosoma cruzi . However, there is currently no standardized protocol for parasite culture, and the effectiveness of available techniques varies. In this study, we developed a standardized closed blood culture system (CBCS) designed to support the survival of trypomastigotes and their differentiation into epimastigotes, from sample collection through to primary laboratory isolation. Blood samples were artificially infected with varying concentrations of T. cruzi trypomastigotes to assess the performance of the CBCS. The CBCS enabled successful isolation and exponential growth of the parasite, demonstrating performance comparable to that of conventional culture methods. All assays included a reference control culture, which served as a benchmark for comparison. No contamination events were observed, and it was possible to isolate and expand the parasite population from an initial sample containing as few as ten trypomastigotes. The standardized CBCS protocol demonstrated good precision, as confirmed by repeatability and reproducibility tests, which showed acceptable variability among replicates.

Trypanosoma cruzi is a protist parasite that is the causative agent of Chagas disease, a neglected tropical disease endemic to the Americas. T . cruzi cells are highly polarized and undergo morphological changes as they cycle within their insect and mammalian hosts. Work on related trypanosomatids has described cell division mechanisms in several life-cycle stages and identified a set of essential morphogenic proteins that serve as markers for key events during trypanosomatid division. Here, we use Cas9-based tagging of morphogenic genes, live-cell imaging, and expansion microscopy to study the cell division mechanism of the insect-resident epimastigote form of T . cruzi , which represents an understudied trypanosomatid morphotype. We find that T . cruzi epimastigote cell division is highly asymmetric, producing one daughter cell that is significantly smaller than the other. Daughter cell division rates differ by 4.9 h, which may be a consequence of this size disparity. Many of the morphogenic proteins identified in T . brucei have altered localization patterns in T . cruzi epimastigotes, which may reflect fundamental differences in the cell division mechanism of this life cycle stage, which widens and shortens the cell body to accommodate the duplicated organelles and cleavage furrow rather than elongating the cell body along the long axis of the cell, as is the case in life-cycle stages that have been studied in T . brucei . This work provides a foundation for further investigations of T . cruzi cell division and shows that subtle differences in trypanosomatid cell morphology can alter how these parasites divide.

Cruzipains are the main papain-like cysteine proteases of Trypanosoma cruzi , the protozoan parasite that causes Chagas disease. Encoded by a multigenic family, previous studies have estimated the presence of dozens of copies spread over multiple chromosomes in different parasite strains. Here, we describe the complete gene repertoire of cruzipain in three parasite strains, their genomic organization, and expression pattern throughout the parasite life cycle. Furthermore, we have analyzed primary sequence variations among distinct family members as well as structural differences between the main groups of cruzipains. Based on phylogenetic inferences and residue positions crucial for enzyme function and specificity, we propose the classification of cruzipains into two families (I and II), whose genes are distributed in two or three separate clusters in the parasite genome, according with the strain. Family I comprises nearly identical copies to the previously characterized cruzipain 1/cruzain, whereas Family II encompasses three structurally distinct sub-types, named cruzipain 2, cruzipain 3, and cruzipain 4. RNA-seq data derived from the CL Brener strain indicates that Family I genes are mainly expressed by epimastigotes, whereas trypomastigotes mainly express Family II genes. Significant differences in the active sites among the enzyme sub-types were also identified, which may play a role in their substrate selectivity and impact their inhibition by small molecules.