Participação de vesículas extracelulares na dinâmica de infecção de Trypanosoma cruzi em células hospedeiras

Orientador: Prof. Dr. Marcel Ivan RamirezTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa : Curitiba, 29/06/2023Inclui referênciasResumo: O protozoário Trypanosoma cruzi é o agente etiológico da Doença de Chagas, que inicialmente se restringia às Américas, porém tem se disseminado pelo mundo todo, atingindo milhões de indivíduos. Durante a infecção no hospedeiro, as diferentes formas evolutivas do parasito enfrentam distintos cenários: enquanto as formas metacíclicas são responsáveis pelo primeiro contato com o hospedeiro e devem sobrepassar o ataque do sistema imunológico inato do mamífero, as formas tripomastigotas sanguíneas (ou, experimentalmente, as formas tripomastigotas derivadas de cultivo) são responsáveis pela persistência da infecção e lidam com uma resposta imune adquirida. Para o sucesso da infecção, T. cruzi apresenta diferentes mecanismos para evadir ao sistema imune e invadir células do hospedeiro, expressando diferentes moléculas em sua superfície e liberando vesículas extracelulares (VEs). As VEs são pequenas nanopartículas compostas por uma bicamada lipídica, de tamanho, conteúdo e composição de membrana heterogêneos e dinâmicos. Atualmente, as VEs compreendem microvesículas e exossomos, conforme seu tamanho e biogênese. Nosso grupo mostrou que as VEs derivadas da interação entre T. cruzi e células hospedeiras tem capacidade de inibir o sistema complemento e aumentam a invasão de formas metacíclicas de T. cruzi às células de mamíferos. Visto que as diferentes formas infectivas do parasito enfrentam diferentes cenários durante a infecção e que as VEs representam um importante componente da comunicação celular entre patógenos com o hospedeiro, o presente projeto objetivou caracterizar VEs derivadas da interação de formas tripomatigotas de cultura celular de T. cruzi com células hospedeiras, com intuito de avaliar seu papel na modulação da resposta imune e sua influência sobre a dinâmica de infecção e permanência intracelular do parasito. Para isso, foram utilizadas duas cepas com diferentes características genéticas e de virulência (CL Brener e Dm28c), sendo que Dm28c é mais infectiva e possui maior taxa de liberação de TCTs a partir de células infectadas. Nós mostramos também que T. cruzi interage diferentemente com células de mioblasto e células de epitélio intestinal, o que poderia simular os ambientes encontrados pelo parasito no hospedeiro. As VEs derivadas da interação de T. cruzi com mioblasto e epitélio intestinal se diferem na ação sobre a infecção e na composição. Além disso, vimos que VEs possuem papel modulatório sobre células dendríticas e mostramos resultados iniciais do efeito das VEs sobre a infecção in vivo. Assim, estes resultados podem contribuir para a compreensão dos mecanismos de virulência e persistência do parasito, abrindo caminhos para os avanços no entendimento da Doença de Chagas.Abstract: The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, which was initially restricted to the Americas, but has spread throughout the world, reaching millions of individuals. During host infection, the different evolutionary forms of the parasite face different scenarios: while the metacyclic forms are responsible for the first contact with the host and must overcome the attack of the mammal's innate immune system, the blood trypomastigotes (or, experimentally, the culture-derived trypomastigotes) are responsible for the persistence of the infection and deal with an acquired immune response. For successful infection, T. cruzi has different mechanisms to evade the immune system and invade host cells, expressing different molecules on its surface and releasing extracellular vesicles (EVs). EVs are small nanoparticles composed of a lipid bilayer, with heterogeneous and dynamic size, content and membrane composition. Currently, EVs comprise microvesicles and exosomes, depending on their size and biogenesis. Our group showed that EVs derived from the interaction between T. cruzi and host cells have the capacity to inhibit the complement system and increase the invasion of metacyclic forms of T. cruzi to mammalian cells. Since the different infective forms of the parasite face diverse scenarios during infection and that EVs represent an important component of cellular communication between pathogens and the host, the present project aimed to characterize EVs derived from the interaction of tissue-derived trypomatigotes forms of T. cruzi with host cells, in order to evaluate its role in modulating the immune response and its influence on the dynamics of infection and intracellular permanence of the parasite. For this, two strains with distinct genetic and virulence characteristics were used (CL Brener and Dm28c), with Dm28c being more infective and having a higher TCTs release rate from infected cells. We also showed that T. cruzi interacts differently with myoblast cells and intestinal epithelial cells, which could simulate the environments found by the parasite in the host. EVs derived from the interaction between T. cruzi and myoblast or intestinal epithelium differ in their action during infection and composition. Furthermore, we saw that EVs have a modulatory role on dendritic cells and we showed initial results of the effect of EVs on in vivo infection. Thus, these results can contribute to the understanding of the mechanisms of virulence and persistence of the parasite, paving the way for advances in the understanding of Chagas disease

A new landscape of host–protozoa interactions involving the extracellular vesicles world

This version is free to view and download for private research and study only. Not for re-distribution, re-sale or use in derivative works. © Cambridge University Press 2018Extracellular vesicles (EVs) are released by a wide number of cells including blood cells, immune system cells, tumour cells, adult and embryonic stem cells. EVs are a heterogeneous group of vesicles (~30–1000 nm) including microvesicles and exosomes. The physiological release of EVs represents a normal state of the cell, raising a metabolic equilibrium between catabolic and anabolic processes. Moreover, when the cells are submitted to stress with different inducers or in pathological situations (malignancies, chronic diseases, infectious diseases.), they respond with an intense and dynamic release of EVs. The EVs released from stimulated cells vs those that are released constitutively may themselves differ, both physically and in their cargo. EVs contain protein, lipids, nucleic acids and biomolecules that can alter cell phenotypes or modulate neighbouring cells. In this review, we have summarized findings involving EVs in certain protozoan diseases. We have commented on strategies to study the communicative roles of EVs during host–pathogen interaction and hypothesized on the use of EVs for diagnostic, preventative and therapeutic purposes in infectious diseases. This kind of communication could modulate the innate immune system and reformulate concepts in parasitism. Moreover, the information provided within EVs could produce alternatives in translational medicine.Peer reviewedFinal Accepted Versio

Peptidylarginine Deiminase Inhibition Abolishes the Production of Large Extracellular Vesicles From

is a microaerophilic protozoan that is an important etiologic agent of diarrhea worldwide. There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which modulate the physiopathology of giardiasis. Here we describe new features of EV production, revealing its capacity to shed two different enriched EV populations: large (LEV) and small extracellular vesicles (SEV) and identified relevant adhesion functions associated with the larger population. Proteomic analysis revealed differences in proteins relevant for virulence and host-pathogen interactions between the two EV subsets, such as cytoskeletal and anti-oxidative stress response proteins in LEVS. We assessed the effect of two recently identified inhibitors of EV release in mammalian cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release from . The compounds were both able to effectively reduce EV shedding, the PAD-inhibitor specifically affecting the release of LEVs and reducing parasite attachment to host cells . Our results suggest that LEVs and SEVs have a different role in host-pathogen interaction, and that treatment with EV-inhibitors may be a novel treatment strategy for recurrent giardiasis. [Abstract copyright: Copyright © 2020 Gavinho, Sabatke, Feijoli, Rossi, da Silva, Evans-Osses, Palmisano, Lange and Ramirez.

Extracellular vesicles from Trypanosoma cruzi-dendritic cell interaction show modulatory properties and confer resistance to lethal infection as a cell-free based therapy strategy

Extracellular vesicles (EVs) include a heterogeneous group of particles. Microvesicles, apoptotic bodies and exosomes are the most characterized vesicles. They can be distinguished by their size, morphology, origin and molecular composition. To date, increasing studies demonstrate that EVs mediate intercellular communication. EVs reach considerable interest in the scientific community due to their role in diverse processes including antigenpresentation, stimulation of anti-tumoral immune responses, tolerogenic or inflammatory effects. In pathogens, EV shedding is well described in fungi, bacteria, protozoan and helminths parasites. For Trypanosoma cruzi EV liberation and protein composition was previously described. Dendritic cells (DCs), among other cells, are key players promoting the immune response against pathogens and also maintaining self-tolerance. In previous reports we have demonstrate that T. cruzi downregulates DCs immunogenicity in vitro and in vivo. Here we analyze EVs from the in vitro interaction between blood circulating trypomastigotes (Tp) and bone-marrow-derived DCs. We found that Tp incremented the number and the size of EVs in cultures with DCs. EVs displayed some exosome markers and intracellular RNA. Protein analysis demonstrated that the parasite changes the DC protein-EV profile. We observed that EVs from the interaction of Tp-DCs were easily captured by unstimulated-DCs in comparison with EVs from DCs cultured without the parasite, and also modified the activation status of LPS-stimulated DCs. Noteworthy, we found protection in animals treated with EVs-DCs+Tp and challenged with T. cruzi lethal infection. Our goal is to go deep into the molecular characterization of EVs from the DCs-Tp interaction, in order to identify mediators for therapeutic purposes

An induced population of Trypanosoma cruzi epimastigotes more resistant to complement lysis promotes a phenotype with greater differentiation, invasiveness, and release of extracellular vesicles

IntroductionChagas disease is a neglected tropical disease caused by Trypanosoma cruzi, which uses blood-feeding triatomine bugs as a vector to finally infect mammalian hosts. Upon entering the host, the parasite needs to effectively evade the attack of the complement system and quickly invade cells to guarantee an infection. In order to accomplish this, T. cruzi expresses different molecules on its surface and releases extracellular vesicles (EVs).MethodsHere, we have selected a population of epimastigotes (a replicative form) from T. cruzi through two rounds of exposure to normal human serum (NHS), to reach 30% survival (2R population). This 2R population was characterized in several aspects and compared to Wild type population.ResultsThe 2R population had a favored metacyclogenesis compared with wild-type (WT) parasites. 2R metacyclic trypomastigotes had a two-fold increase in resistance to complementmediated lysis and were at least three times more infective to eukaryotic cells, probably due to a higher GP82 expression in the resistant population. Moreover, we have shown that EVs from resistant parasites can transfer the invasive phenotype to the WT population. In addition, we showed that the virulence phenotype of the selected population remains in the trypomastigote form derived from cell culture, which is more infective and also has a higher rate of release of trypomastigotes from infected cells.ConclusionsAltogether, these data indicate that it is possible to select parasites after exposure to a particular stress factor and that the phenotype of epimastigotes remained in the infective stage. Importantly, EVs seem to be an important virulence fator increasing mechanism in this context of survival and persistence in the host

Trypanosoma cruzi interaction with host tissues modulate the composition of large extracellular vesicles

Abstract Trypanosoma cruzi is the protozoan that causes Chagas disease (CD), an endemic parasitosis in Latin America distributed around the globe. If CD is not treated in acute phase, the parasite remains silent for years in the host's tissues in a chronic form, which may progress to cardiac, digestive or neurological manifestations. Recently, studies indicated that the gastrointestinal tract represents an important reservoir for T. cruzi in the chronic phase. During interaction T. cruzi and host cells release extracellular vesicles (EVs) that modulates the immune system and infection, but the dynamics of secretion of host and parasite molecules through these EVs is not understood. Now, we used two cell lines: mouse myoblast cell line C2C12, and human intestinal epithelial cell line Caco-2to simulate the environments found by the parasite in the host. We isolated large EVs (LEVs) from the interaction of T. cruzi CL Brener and Dm28c/C2C12 and Caco-2 cells upon 2 and 24 h of infection. Our data showed that at two hours there is a strong cellular response mediated by EVs, both in the number, variety and enrichment/targeting of proteins found in LEVs for diverse functions. Qualitative and quantitative analysis showed that proteins exported in LEVs of C2C12 and Caco-2 have different patterns. We found a predominance of host proteins at early infection. The parasite-host cell interaction induces a switch in the functionality of proteins carried by LEVs and a heterogeneous response depending on the tissues analyzed. Protein–protein interaction analysis showed that cytoplasmic and mitochondrial homologues of the same parasite protein, tryparedoxin peroxidase, were differentially packaged in LEVs, also impacting the interacting molecule of this protein in the host. These data provide new evidence that the interaction with T. cruzi leads to a rapid tissue response through the release of LEVs, reflecting the enrichment of some proteins that could modulate the infection environment

Extracellular vesicles from Trypanosoma cruzi-dendritic cell interaction show modulatory properties and confer resistance to lethal infection as a cell-free based therapy strategy

Extracellular vesicles (EVs) include a heterogeneous group of particles. Microvesicles, apoptotic bodies and exosomes are the most characterized vesicles. They can be distinguished by their size, morphology, origin and molecular composition. To date, increasing studies demonstrate that EVs mediate intercellular communication. EVs reach considerable interest in the scientific community due to their role in diverse processes including antigen-presentation, stimulation of anti-tumoral immune responses, tolerogenic or inflammatory effects. In pathogens, EV shedding is well described in fungi, bacteria, protozoan and helminths parasites. For Trypanosoma cruzi EV liberation and protein composition was previously described. Dendritic cells (DCs), among other cells, are key players promoting the immune response against pathogens and also maintaining self-tolerance. In previous reports we have demonstrate that T. cruzi downregulates DCs immunogenicity in vitro and in vivo. Here we analyze EVs from the in vitro interaction between blood circulating trypomastigotes (Tp) and bone-marrow-derived DCs. We found that Tp incremented the number and the size of EVs in cultures with DCs. EVs displayed some exosome markers and intracellular RNA. Protein analysis demonstrated that the parasite changes the DC protein-EV profile. We observed that EVs from the interaction of Tp-DCs were easily captured by unstimulated-DCs in comparison with EVs from DCs cultured without the parasite, and also modified the activation status of LPS-stimulated DCs. Noteworthy, we found protection in animals treated with EVs-DCs+Tp and challenged with T. cruzi lethal infection. Our goal is to go deep into the molecular characterization of EVs from the DCs-Tp interaction, in order to identify mediators for therapeutic purposes.Fil: Gutierrez, Brenda Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Ancarola, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Volpato Rossi, Izadora. Universidade Federal do Paraná; BrasilFil: Marcilla, Antonio. Universidad de Valencia; EspañaFil: Ramirez, Marcel Ivan. Universidade Federal do Paraná; BrasilFil: Rosenzvit, Mara Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Cucher, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Poncini, Carolina Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

An induced population of Trypanosoma cruzi epimastigotes more resistant to complement lysis promotes a phenotype with greater differentiation, invasiveness, and release of extracellular vesicles

© 2022 Rossi, Nunes, Sabatke, Ribas, Winnischofer, Ramos, Inal and Ramirez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). https://creativecommons.org/licenses/by/4.0/Introduction: Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi, which uses blood-feeding triatomine bugs as a vector to finally infect mammalian hosts. Upon entering the host, the parasite needs to effectively evade the attack of the complement system and quickly invade cells to guarantee an infection. In order to accomplish this, T. cruzi expresses different molecules on its surface and releases extracellular vesicles (EVs). Methods: Here, we have selected a population of epimastigotes (a replicative form) from T. cruzi through two rounds of exposure to normal human serum (NHS), to reach 30% survival (2R population). This 2R population was characterized in several aspects and compared to Wild type population. Results: The 2R population had a favored metacyclogenesis compared with wild-type (WT) parasites. 2R metacyclic trypomastigotes had a two-fold increase in resistance to complementmediated lysis and were at least three times more infective to eukaryotic cells, probably due to a higher GP82 expression in the resistant population. Moreover, we have shown that EVs from resistant parasites can transfer the invasive phenotype to the WT population. In addition, we showed that the virulence phenotype of the selected population remains in the trypomastigote form derived from cell culture, which is more infective and also has a higher rate of release of trypomastigotes from infected cells. Conclusions: Altogether, these data indicate that it is possible to select parasites after exposure to a particular stress factor and that the phenotype of epimastigotes remained in the infective stage. Importantly, EVs seem to be an important virulence fator increasing mechanism in this context of survival and persistence in the host.Peer reviewe

Image_2_Extracellular vesicles from Trypanosoma cruzi-dendritic cell interaction show modulatory properties and confer resistance to lethal infection as a cell-free based therapy strategy.tiff

Extracellular vesicles (EVs) include a heterogeneous group of particles. Microvesicles, apoptotic bodies and exosomes are the most characterized vesicles. They can be distinguished by their size, morphology, origin and molecular composition. To date, increasing studies demonstrate that EVs mediate intercellular communication. EVs reach considerable interest in the scientific community due to their role in diverse processes including antigen-presentation, stimulation of anti-tumoral immune responses, tolerogenic or inflammatory effects. In pathogens, EV shedding is well described in fungi, bacteria, protozoan and helminths parasites. For Trypanosoma cruzi EV liberation and protein composition was previously described. Dendritic cells (DCs), among other cells, are key players promoting the immune response against pathogens and also maintaining self-tolerance. In previous reports we have demonstrate that T. cruzi downregulates DCs immunogenicity in vitro and in vivo. Here we analyze EVs from the in vitro interaction between blood circulating trypomastigotes (Tp) and bone-marrow-derived DCs. We found that Tp incremented the number and the size of EVs in cultures with DCs. EVs displayed some exosome markers and intracellular RNA. Protein analysis demonstrated that the parasite changes the DC protein-EV profile. We observed that EVs from the interaction of Tp-DCs were easily captured by unstimulated-DCs in comparison with EVs from DCs cultured without the parasite, and also modified the activation status of LPS-stimulated DCs. Noteworthy, we found protection in animals treated with EVs-DCs+Tp and challenged with T. cruzi lethal infection. Our goal is to go deep into the molecular characterization of EVs from the DCs-Tp interaction, in order to identify mediators for therapeutic purposes.</p

Image_3_Extracellular vesicles from Trypanosoma cruzi-dendritic cell interaction show modulatory properties and confer resistance to lethal infection as a cell-free based therapy strategy.tiff

Extracellular vesicles (EVs) include a heterogeneous group of particles. Microvesicles, apoptotic bodies and exosomes are the most characterized vesicles. They can be distinguished by their size, morphology, origin and molecular composition. To date, increasing studies demonstrate that EVs mediate intercellular communication. EVs reach considerable interest in the scientific community due to their role in diverse processes including antigen-presentation, stimulation of anti-tumoral immune responses, tolerogenic or inflammatory effects. In pathogens, EV shedding is well described in fungi, bacteria, protozoan and helminths parasites. For Trypanosoma cruzi EV liberation and protein composition was previously described. Dendritic cells (DCs), among other cells, are key players promoting the immune response against pathogens and also maintaining self-tolerance. In previous reports we have demonstrate that T. cruzi downregulates DCs immunogenicity in vitro and in vivo. Here we analyze EVs from the in vitro interaction between blood circulating trypomastigotes (Tp) and bone-marrow-derived DCs. We found that Tp incremented the number and the size of EVs in cultures with DCs. EVs displayed some exosome markers and intracellular RNA. Protein analysis demonstrated that the parasite changes the DC protein-EV profile. We observed that EVs from the interaction of Tp-DCs were easily captured by unstimulated-DCs in comparison with EVs from DCs cultured without the parasite, and also modified the activation status of LPS-stimulated DCs. Noteworthy, we found protection in animals treated with EVs-DCs+Tp and challenged with T. cruzi lethal infection. Our goal is to go deep into the molecular characterization of EVs from the DCs-Tp interaction, in order to identify mediators for therapeutic purposes.</p