Murine colon organoids as a novel model to study Trypanosoma cruzi infection and interactions with the intestinal epithelium

Chagas disease (CD) is a life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 50,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne), but congenital and oral transmission have also been reported. The acute phase of CD presents mild symptoms but may develop into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In this immune-privileged reservoir, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models that approximate to the biological and structural complexity of this tissue. Therefore, to understand the role played by the intestinal tissue during transmission and chronic infection, physiological models resembling the organ complexity are needed. Here we addressed this issue by establishing and characterizing adult stem cell-derived colonoid infection models that are clinically relevant for CD. 3D and 2D systems of murine intestinal organoids infected with T. cruzi Dm28c (a highly virulent strain associated with oral outbreaks) were analyzed at different time points by confocal microscopy. T. cruzi was able to invade and replicate in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between organoids and cell lines (primate and intestinal human cell lines). So far, this represents the first evidence of the potential that these cellular systems offer for the study of host-pathogen interactions and the discovery of effective anti-chagasic drugs.Agencia Nacional de Investigación e InnovaciónPasteur NetworkFOCEM (MERCOSUR Structural Convergence Fund

Modeling host-parasite interaction in chagas disease with murine intestinal organoids

Chagas disease (CD) is a potentially life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 10,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne) and, to a minor extent, by blood transfusion, organ transplantation, laboratory accidents, congenitally and orally (food-borne). The acute phase of CD presents mild symptoms. If left untreated, it develops into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In these immune-privileged reservoirs, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models resembling the biological and structural complexity of this organ. Therefore, to understand the pathophysiological role played by the intestinal tissue during transmission and chronic infection, we evaluated the progression of T. cruzi infection of murine colon organoids. In order to model CD, 3D and 2D systems of murine intestinal organoids were infected with T. cruzi Dm28c, a strain that has been associated with high virulence and oral outbreaks. At different time points, the presence and load of parasites in the organoids, as well as the host cell morphology were evaluated by confocal microscopy, and compared to those obtained with a classical infection model (Vero cells). We show that the parasite invades and replicates in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between the primary and the tumoral (Vero) cells. So far, this represents the first evidence of the potential of these nearly physiological cellular systems to study host-pathogen interaction for CD and/or for the future evaluation of anti-chagasic drugs.Agencia Nacional de Investigación e Innovación (ANII)FOCEM (MERCOSUR Structural Convergence Fund

Nox2-derived superoxide radical is crucial to control acute Trypanosoma cruzi infection

Carolina Prolo: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay -- Damián Estrada: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay -- Lucía Piacenza: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay -- Diego Benítez: Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Uruguay -- Marcelo A. Comini: Laboratory Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Uruguay -- Rafael Radi: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay -- María Noel Álvarez: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Departamento de Educación Médica, Facultad de Medicina, Universidad de la República, Montevideo, UruguayTrypanosoma cruzi is a flagellated protozoan that undergoes a complex life cycle between hematophagous insects and mammals. In humans, this parasite causes Chagas disease, which in thirty percent of those infected, would result in serious chronic pathologies and even death. Macrophages participate in the first stages of infection, mounting a cytotoxic response which promotes massive oxidative damage to the parasite. On the other hand, T. cruzi is equipped with a robust antioxidant system to repeal the oxidative attack from macrophages. This work was conceived to explicitly assess the role of mammalian cell-derived superoxide radical in a murine model of acute infection by T. cruzi. Macrophages derived from Nox2-deficient (gp91phox-/-) mice produced marginal amounts of superoxide radical and were more susceptible to parasite infection than those derived from wild type (wt) animals. Also, the lack of superoxide radical led to an impairment of parasite differentiation inside gp91phox-/- macrophages. Biochemical or genetic reconstitution of intraphagosomal superoxide radical formation in gp91phox-/- macrophages reverted the lack of control of infection. Along the same line, gp91phox-/- infected mice died shortly after infection. In spite of the higher lethality, parasitemia did not differ between gp91phox-/- and wt animals, recapitulating an observation that has led to conflicting interpretations about the importance of the mammalian oxidative response against T. cruzi. Importantly, gp91phox-/- mice presented higher and disseminated tissue parasitism, as evaluated by both qPCR- and bioimaging-based methodologies. Thus, this work supports that Nox2-derived superoxide radical plays a crucial role to control T. cruzi infection in the early phase of a murine model of Chagas disease

The lineage-specific, intrinsically disordered N-terminal extension of monothiol glutaredoxin 1 from trypanosomes contains a regulatory region

Glutaredoxins (Grx) are small proteins conserved throughout all the kingdoms of life that are engaged in a wide variety of biological processes and share a common thioredoxin-fold. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (FeS) metabolism. They contain a single thiol group in their active site and use low molecular mass thiols such as glutathione as ligand for binding FeS-clusters. In this study, we investigated molecular aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei brucei, a mitochondrial class II Grx that fulfills an indispensable role in vivo. Mitochondrial 1CGrx1 from trypanosomes differs from orthologues in several features including the presence of a parasite-specific N-terminal extension (NTE) whose role has yet to be elucidated. Previously we have solved the structure of a truncated form of 1CGrx1 containing only the conserved glutaredoxin domain but lacking the NTE. Our aim here is to investigate the effect of the NTE on the conformation of the protein. We therefore solved the NMR structure of the full-length protein, which reveals subtle but significant differences with the structure of the NTE-less form. By means of different experimental approaches, the NTE proved to be intrinsically disordered and not involved in the non-redox dependent protein dimerization, as previously suggested. Interestingly, the portion comprising residues 65–76 of the NTE modulates the conformational dynamics of the glutathione-binding pocket, which may play a role in iron-sulfur cluster assembly and delivery. Furthermore, we disclosed that the class II-strictly conserved loop that precedes the active site is critical for stabilizing the protein structure. So far, this represents the first communication of a Grx containing an intrinsically disordered region that defines a new protein subgroup within class II Grx

Potencial de los organoides intestinales murinos para el estudio de la enfermedad de Chagas

Chagas disease (CD) is a potentially life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 10,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne) and, to a minor extent, by blood transfusion, organ transplantation, laboratory accidents, congenitally and orally (food- borne). The acute phase of CD presents mild symptoms. If left untreated, it develops into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In these immune-privileged reservoirs, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models resembling the biological and structural complexity of this organ. Therefore, to understand the pathophysiological role played by the intestinal tissue during transmission and chronic infection, we evaluated the progression of T. cruzi infection of murine colon organoids. In order to model CD, 3D and 2D systems of murine intestinal organoids were infected with T. cruzi Dm28c, a strain that has been associated with high virulence and oral outbreaks. At different time points, the presence and load of parasites in the organoids, as well as the host cell morphology were evaluated by confocal microscopy, and compared to those obtained with a classical infection model (Vero cells). We show that the parasite invades and replicates in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between the primary and the tumoral (Vero) cells. So far, this represents the first evidence of the potential of these nearly physiological cellular systems to study host-pathogen interaction for CD and/or for the future evaluation of anti-chagasic drugs.Agencia Nacional de Investigación e Innovación (ANII

An essential thioredoxin-type protein of Trypanosoma brucei acts as redox-regulated mitochondrial chaperone

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes

Ensemble learning application to discover new trypanothione synthetase inhibitors

Trypanosomatid-caused diseases are among the neglected infectious diseases with the highest disease burden, affecting about 27 million people worldwide and, in particular, socio-economically vulnerable populations. Trypanothione synthetase (TryS) is considered one of the most attractive drug targets within the thiol-polyamine metabolism of typanosomatids, being unique, essential and druggable. Here, we have compiled a dataset of 401 T. brucei TryS inhibitors that includes compounds with inhibitory data reported in the literature, but also in-house acquired data. QSAR classifiers were derived and validated from such dataset, using publicly available and open-source software, thus assuring the portability of the obtained models. The performance and robustness of the resulting models were substantially improved through ensemble learning. The performance of the individual models and the model ensembles was further assessed through retrospective virtual screening campaigns. At last, as an application example, the chosen model-ensemble has been applied in a prospective virtual screening campaign on DrugBank 5.1.6 compound library. All the in-house scripts used in this study are available on request, whereas the dataset has been included as supplementary material.Facultad de Ciencias ExactasLaboratorio de Investigación y Desarrollo de Bioactivo

Mechanistic and biological characterisation of novel N5-substituted paullones targeting the biosynthesis of trypanothione in Leishmania

Trypanothione synthetase (TryS) produces N1,N8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N5-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC50 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species

Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system.

Background: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. Methods: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target_s orthologues, focusing mainly on post-translational modifications. Findings: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. Interpretation: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection

Drug repurposing screening validated by experimental assays identifies two clinical drugs targeting SARS-CoV-2 main protease

The COVID-19 pandemic prompted several drug repositioning initiatives with the aim to rapidly deliver pharmacological candidates able to reduce SARSCoV- 2 dissemination and mortality. A major issue shared by many of the in silico studies addressing the discovery of compounds or drugs targeting SARS-CoV- 2 molecules is that they lacked experimental validation of the results. Here we present a computer-aided drug-repositioning campaign against the indispensable SARS-CoV-2 main protease (MPro or 3CLPro) that involved the development of ligand-based ensemble models and the experimental testing of a small subset of the identified hits. The search method explored random subspaces of molecular descriptors to obtain linear classifiers. The best models were then combined by selective ensemble learning to improve their predictive power. Both the individual models and the ensembles were validated by retrospective screening, and later used to screen the DrugBank, Drug Repurposing Hub and Sweetlead libraries for potential inhibitors of MPro. From the 4 in silico hits assayed, atpenin and tinostamustine inhibited MPro (IC50 1 μM and 4 μM, respectively) but not the papain-like protease of SARSCoV- 2 (drugs tested at 25 μM). Preliminary kinetic characterization suggests that tinostamustine and atpenin inhibit MPro by an irreversible and acompetitive mechanisms, respectively. Both drugs failed to inhibit the proliferation of SARSCoV- 2 in VERO cells. The virtual screening method reported here may be a powerful tool to further extent the identification of novel MPro inhibitors. Furthermore, the confirmed MPro hits may be subjected to optimization or retrospective search strategies to improve their molecular target and anti-viral potency.Laboratorio de Investigación y Desarrollo de Bioactivo