Extracellular vesicles of Trypanosoma cruzi tissue-culture cell-derived trypomastigotes: Induction of physiological changes in non-parasitized culture cells

Extracellular vesicles (EVs) are a diverse group of nanoparticles involved in intercellular communication under physiological and pathological conditions. Trypanosoma cruzi, the protozoan that causes Chagas disease, releases EVs that facilitate parasite invasion of the host cell, immunomodulate the host response, and help the parasite to evade this response. However, little is known about how the host cell is altered. In this work, we confirm that EVs of tissue-culture cell-derived trypomastigotes of the Pan4 strain increase cell parasitism. We also demonstrate that EVs affect cell permeability in Vero cells and cardiomyocytes and raise intracellular Ca2+ levels, altering the actin filaments and arresting the cell cycle at the G0/G1 phases. This work seeks to elucidate the way in which EVs influence certain aspects of the cell physiology that favour the establishment of this parasite inside the host cell. The incubation of cells with EVs of trypomastigotes of the Pan4 strain of T. cruzi induce a number of changes in the host cells that include a change in cell permeability and higher intracellular levels of Ca2+ that can alter the dynamics of the actin cytoskeleton and arrest the cell cycle at G0/G1 prior to the DNA synthesis necessary to complete mitosis. These changes aid the invasion of host cells and augment the percentage of cell parasitization.Lissette Retana Moreira was supported by the “Oficina de Asuntos Internacionales y Cooperación Externa (OAICE)” of the University of Costa Rica and the “Ministerio de Ciencia, Tecnología y Telecomunicaciones (MICITT)” and “Consejo Nacional para Investigaciones Científicas y Tecnológicas” (CONICIT)” of the Government of Costa Rica

Extracellular vesicles of trypomastigotes of Trypanosoma cruzi induce changes in ubiquitin‑related processes, cell‑signaling pathways and apoptosis

Beca de Iniciación a la Investigación para estudiantes de Máster” of the University of Granada (Spain)Oficina de Asuntos Internacionales y Cooperación Externa (OAICE) of the Universidad de Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones (MICITT) of the Government of Costa Rica and Fundación Carolina (Spain)TϋCAD2 is funded by the Federal Ministry of Education and Research (BMBF)Baden-Württemberg Ministry of Science as part of the Excellence Strategy of the German Federal and State Governments EXC 2180–390900677ERANet program, Research in prevention of congenital Chagas disease: parasitological, placental and immunological markers (ERANet17/HLH-0142 (Cochaco)Instituto Carlos III, Ministerio de Sanidad, Gobierno de España; Fundación Ramón Areces “Interactoma de las exovesículasMinisterio de Ciencia y Tecnología of the government of Spain funded the project PGC2018- 099424-B-I00Proyect A-BIO-350-UGR18 I+D+i Proyect “Programa Operativo FEDER de Andalucía JJAA” 2014-202

Extracellular vesicles of Trypanosoma cruzi and immune complexes they form with sialylated and non-sialylated IgGs increase small peritoneal macrophage subpopulation and elicit different cytokines profiles

This research was funded by the ERANet program, Research in prevention of congenital Chagas disease: parasitological, placental and immunological markers (ERANet17/HLH-0142 (Cochaco). Instituto Carlos III, Ministerio de Sanidad, Gobierno de Espana; Fundacioin Ramoin Areces "Interactoma de las exovesiculas de T. cruzi y de los inmunocomplejos que forman con las celulas del hospedador: implicaciones en la patologia de la enfermedad de Chagas (2019)". Ministerio de Ciencia y Tecnologia of the Government of Spain funded the project PGC2018-099424-B-I00)". Exovesiculas circulantes como marcadoras de diagnostico, PREcoz de la Enfermedad de CHAGas Fundacion Ramon Areces del XXI Concurso Nacional para la adjudicacion de Ayudas a la Investigacion en Ciencias de la Vida y de la Materia (2022). Ministerio de Ciencia y Tecnologiia of the government of Spain funded the project PGC2018-099424-B-I00.The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2023.1215913/full#supplementary-materialAmerican trypanosomiasis, or Chagas disease, is caused by the protozoan parasite Trypanosoma cruzi and is characterized by the presence of cardiac or gastrointestinal symptoms in a large number of patients during the chronic phase of the disease. Although the origin of the symptoms is not clear, several mechanisms have been described involving factors related to T. cruzi and the host immune response. In this sense, the extracellular vesicles (EVs) secreted by the parasite and the immune complexes (ICs) formed after their recognition by host IgGs (EVs-IgGs) may play an important role in the immune response during infection. The aim of the present work is to elucidate the modulation of the immune response exerted by EVs and the ICs they form by analyzing the variation in the subpopulations of small and large peritoneal macrophages after intraperitoneal inoculation in mice and to evaluate the role of the sialylation of the host IgGs in this immunomodulation. Both macrophage subpopulations were purified and subjected to cytokine expression analysis by RT-qPCR. The results showed an increase in the small peritoneal macrophage subpopulation after intraperitoneal injection of parasite EVs, but a greater increase in this subpopulation was observed when sialylated and non-sialylated ICs were injected, which was similar to inoculation with the trypomastigote stage of the parasite. The cytokine expression results showed the ability of both subpopulations to express inflammatory and non-inflammatory cytokines. These results suggest the role of free EVs in the acute phase of the disease and the possible role of immune complexes in the immune response in the chronic phase of the disease, when the levels of antibodies against the parasite allow the formation of immune complexes. The differential expression of interleukins showed after the inoculation of immune complexes formed with sialylated and non-sialylated IgGs and the interleukins expression induced by EVs, demonstrates that the IgG glycosilation is involved in the type of immune response that dominates in each of the phases of the Chagas disease.ERANet program, Research in prevention of congenital Chagas disease: parasitological, placental and immunological markers PGC2018-099424-B-I00Instituto Carlos III, Ministerio de Sanidad, Gobierno de EspañaFundacioin Ramoin Areces "Interactoma de las exovesiculas de T. cruzi y de los inmunocomplejos que forman con las celulas del hospedador: implicaciones en la patologia de la enfermedad de Chagas (2019)"Spanish Government PGC2018-099424-B-I00Ministerio de Ciencia y Tecnologia of the government of Spain ERANet17/HLH-014

Characterization of Extracellular Vesicles Secreted by a Clinical Isolate of Naegleria fowleri and Identification of Immunogenic Components within Their Protein Cargo

Abstract: Extracellular vesicles (EVs) are small lipid vesicles released by both prokaryotic and eu-karyotic cells, involved in intercellular communication, immunomodulation and pathogenesis. In this study, we performed a characterization of the EVs produced by trophozoites of a clinical isolate of the free-living amoeba Naegleria fowleri (N. fowleri). Size distribution, zeta potential, protein profile and protease activity were analyzed. Under our incubation conditions, EVs of different sizes were observed, with a predominant population ranging from 206 to 227 nm. SDS-PAGE revealed protein bands of 25 to 260 KDa. The presence of antigenic proteins was confirmed by Western blot, which evidenced the strongest recognition by rat polyclonal antibodies raised against N. fowleri in the region close to 80 KDa and included peptidases, as revealed by zymography. Proteins in the selected immunorecognized bands were further identified using nano-ESI-MS/MS. A preliminary proteomic profile of the EVs identified at least 184 proteins as part of the vesicles’ cargo. Protease activity assays, in combination with the use of inhibitors, revealed the predominance of serine proteases. The present characterization uncovers the complexity of EVs produced by N. fowleri, suggesting their potential relevance in the release of virulence factors involved in pathogenicity. Owing to their cargo’s diversity, further research on EVs could reveal new therapeutic targets or biomarkers for developing rapid and accurate diagnostic tools for lethal infections, such as the one caused by this amoeba.Vicerrectoría de Investigación/[803-C1-061]/VI/Costa RicaVicerrectoría de Investigación/[803-C2-600]/VI/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Aplicación de la topología molecular en la predicción de la actividad antiprotozoaria de derivados del benzimidazol

Se ha aplicado la topología molecular a la búsqueda de nuevos derivados del benzimidazol con actividad antiprotozoaria frente a Trichomonas vaginalis y Giardia intestinalis. A partir de los resultados del análisis lineal discriminante, dos modelos basados en cuatro descriptores (en el caso de T. vaginalis) y cinco descriptores (en el caso de G. intestinalis) demostraron ser capaces de predecir correctamente la actividad de cada compuesto analizado. Después de un cribado virtual aplicando ambos modelos, se proponen nuevas estructuras químicas potencialmente activas frente a T. vaginalis y G. intestinalis.Molecular topology has been applied to search for new benzimidazole derivatives with antiprotozoal activity against Trichomonas vaginalis and Giardia intestinalis. From linear discriminant analysis results, two models based on 4 descriptors (in the case of T. vaginalis) and 5 descriptors (in the case of G. intestinalis), demonstrated to be capable to predict correctly the activity of each compound tested. After a virtual screening using both models, new chemical structures potentially active against T. vaginalis and G. intestinalis are proposed.Ciencias Experimentale

Molecular Recognition of Surface Trans-Sialidases in Extracellular Vesicles of the Parasite Trypanosoma cruzi Using Atomic Force Microscopy (AFM)

Trans-sialidases (TS) are important constitutive macromolecules of the secretome present on the surface of Trypanosoma cruzi (T. cruzi) that play a central role as a virulence factor in Chagas disease. These enzymes have been related to infectivity, escape from immune surveillance and pathogenesis exhibited by this protozoan parasite. In this work, atomic force microscopy (AFM)-based single molecule-force spectroscopy is implemented as a suitable technique for the detection and location of functional TS on the surface of extracellular vesicles (EVs) released by tissue-culture cell-derived trypomastigotes (Ex-TcT). For that purpose, AFM cantilevers with functionalized tips bearing the anti-TS monoclonal antibody mAb 39 as a sense biomolecule are engineered using a covalent chemical ligation based on vinyl sulfonate click chemistry; a reliable, simple and efficient methodology for the molecular recognition of TS using the antibody-antigen interaction. Measurements of the breakdown forces between anti-TS mAb 39 antibodies and EVs performed to elucidate adhesion and forces involved in the recognition events demonstrate that EVs isolated from tissue-culture cell-derived trypomastigotes of T. cruzi are enriched in TS. Additionally, a mapping of the TS binding sites with submicrometer-scale resolution is provided. This work represents the first AFM-based molecular recognition study of Ex-TcT using an antibody-tethered AFM probe.ERANet program ERANet17/HLH-0142Spanish Government PGC2018-099424-B-I0

Characterization and functional analysis of the proteins Prohibitin 1 and 2 in Trypanosoma cruzi

Background Chagas disease is the third most important neglected tropical disease. There is no vaccine available, and only two drugs are generally prescribed for the treatment, both of which with a wide range of side effects. Our study of T. cruzi PHBs revealed a pleiotropic function in different stages of the parasite, participating actively in the transformation of the non-infective replicative epimastigote form into metacyclic trypomastigotes and also in the multiplication of intracellular amastigotes. Methodology/principal findings To obtain and confirm our results, we applied several tools and techniques such as electron microscopy, immuno-electron microscopy, bioinformatics analysis and molecular biology. We transfected T. cruzi clones with the PHB genes, in order to overexpress the proteins and performed a CRISPR/Cas9 disruption to obtain partially silenced PHB1 parasites or completely silenced PHB2 parasites. The function of these proteins was also studied in the biology of the parasite, specifically in the transformation rate from non-infective forms to the metacyclic infective forms, and in their capacity of intracellular multiplication. Conclusion/significance This research expands our understanding of the functions of PHBs in the life cycle of the parasite. It also highlights the protective role of prohibitins against ROS and reveals that the absence of PHB2 has a lethal effect on the parasite, a fact that could support the consideration of this protein as a possible target for therapeutic action

Providing an in vitro depiction of microglial cells challenged with immunostimulatory extracellular vesicles of Naegleria fowleri

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a rapid and acute infection of the central nervous system with a fatal outcome in >97% of cases. Due to the infrequent report of cases and diagnostic gaps that hinder the possibility of recovering clinic isolates, studies related to pathogenesis of the disease are scarce. However, the secretion of cytolytic molecules has been proposed as a factor involved in the progression of the infection. Several of these molecules could be included in extracellular vesicles (EVs), making them potential virulence factors and even modulators of the immune response in this infection. In this work, we evaluated the immunomodulatory effect of EVs secreted by two clinic isolates of Naegleria fowleri using in vitro models. For this purpose, characterization analyses between EVs produced by both isolates were first performed, for subsequent gene transcription analyses post incubation of these vesicles with primary cultures from mouse cell microglia and BV-2 cells. Analyses of morphological changes induced in primary culture microglia cells by the vesicles were also included, as well as the determination of the presence of nucleic acids of N. fowleri in the EV fractions. Results revealed increased expression of NOS, proinflammatory cytokines IL-6, TNF-α, and IL-23, and the regulatory cytokine IL-10 in primary cultures of microglia, as well as increased expression of NOS and IL-13 in BV-2 cells. Morphologic changes from homeostatic microglia, with small cellular body and long processes to a more amoeboid morphology were also observed after the incubation of these cells with EVs. Regarding the presence of nucleic acids, specific Naegleria fowleri DNA that could be amplified using both conventional and qPCR was confirmed in the EV fractions. Altogether, these results confirm the immunomodulatory effects of EVs of Naegleria fowleri over microglial cells and suggest a potential role of these vesicles as biomarkers of primary acute meningoencephalitis.Projects C-2600: “Secreción de vesículas extracelulares por Naegleria fowleri y evaluación de su potencial rol inmunomodulador en un modelo in vitro” and C-1061: “Caracterización de antígenos de excreción/secreción y antígenos somáticos en amebas de vida libre mediante el empleo de anticuerpos policlonales producidos en roedores”, supported by Vicerrectoría de Investigación of the University of Costa RicaOficina de Asuntos Internacionales y Cooperación Externa (OAICE) of Universidad de Costa RicaFundación Carolina (Spain)“Contrato de Formación de Profesorado Universitario” of the Ministry of Science, Culture and Sports (Spain

Biophysical and Biochemical Comparison of Extracellular Vesicles Produced by Infective and Non-Infective Stages of Trypanosoma cruzi

Extracellular vesicles (EVs) are small lipid vesicles released by either any prokaryotic or eukaryotic cell, or both, with a biological role in cell-to-cell communication. In this work, we characterize the proteomes and nanomechanical properties of EVs released by tissue-culture cell-derived trypomastigotes (mammalian infective stage; (TCT)) and epimastigotes (insect stage; (E)) of Trypanosoma cruzi, the etiologic agent of Chagas disease. EVs of each stage were isolated by differential centrifugation and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy and atomic force microscopy (AFM). Measurements of zeta-potential were also included. Results show marked differences in the surface molecular cargos of EVs between both stages, with a noteworthy expansion of all groups of trans-sialidase proteins in trypomastigote’s EVs. In contrast, chromosomal locations of trans-sialidases of EVs of epimastigotes were dramatically reduced and restricted to subtelomeric regions, indicating a possible regulatable expression of these proteins between both stages of the parasite. Regarding mechanical properties, EVs of trypomastigotes showed higher adhesion compared to the EVs of epimastigotes. These findings demonstrate the remarkable surface remodeling throughout the life cycle of T. cruzi, which shapes the physicochemical composition of the extracellular vesicles and could have an impact in the ability of these vesicles to participate in cell communication in completely different niches of infection.ERANet programInstituto Carlos IIIMinisterio de Sanidad, Gobierno de EspañaFundación Ramón Areces funded the projects: “Research in prevention of congenital Chagas disease: parasitological, placental and immunological markers” (ERANet17/ HLH-0142 (Cochaco)ERANE-LAC HD 328/2014) and “Interactoma de las exovesículas de T. cruzi y de los inmunocomplejos que forman con las células del hospedador: implicaciones en la patología de la enfermedad de Chagas (2019)”Ministerio de Ciencia y Tecnología of the government of Spain funded the project PGC2018-099424-B-I0