Transmigration of Trypanosoma cruzi Trypomastigotes through 3D Spheroids Mimicking Host Tissues

Chagas´disease, caused by the kinetoplastid parasite Trypanosoma cruzi, presents a variety of chronic clinical manifestations whose determinants are still unknown but probably influenced by the host-parasite interplay established during the first stages of the infection, when bloodstream circulating trypomastigotes disseminate to different organs and tissues. After leaving the blood, trypomastigotes must migrate through tissues to invade cells and establish a chronic infection. How this process occurs remains unexplored.Three-dimensional (3D) cultures are physiologically relevant because mimic the microarchitecture of tissues and provide an environment similar to the encountered in natural infections. In this work, we combined the 3D culture technology with host-pathogen interaction, by studying transmigration of trypomastigotes into 3D spheroids. T. cruzi strains with similar infection dynamics in 2D monolayer cultures but with different in vivo behavior (CL Brener, virulent; SylvioX10 no virulent) presented different infection rates in spheroids (CL Brener ~40%, SylvioX10 <10%). Confocal microscopy images evidenced that trypomastigotes from CL Brener and other highly virulent strains presented a great ability to transmigrate inside 3D spheroids: as soon as 4 hours post infection parasites were found at 50 µm in depth inside the spheroids. CL Brener trypomastigotes were evenly distributed and systematically observed in the space between cells, suggesting a paracellular route of transmigration to deepen into the spheroids. On the other hand, poor virulent strains presented a weak migratory capacity and remained in the external layers of spheroids (<10µm) with a patch-like distribution pattern. The invasiveness -understood as the ability to transmigrate deep into spheroids-was not a transferable feature .

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Section: Parasites and Conditioned Mediamentioning

confidence: 99%

“…Infected spheroids were fixed by adding PFA to a 3.2% final concentration and incubated at 37°C for 1.5 h. Then, spheroids were washed with PBS as described previously [20] and mounted. Fluorescence images were acquired with a confocal Olympus FV1000 microscope.…”

Section: Parasite Invasiveness and Disseminationmentioning

confidence: 99%

Transmigration ofTrypanosoma cruzitrypomastigotes through3Dcultures resembling a physiological environment

Rodríguez

Rizzi

Caeiro

et al. 2020

Cellular Microbiology

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“…Culture-derived trypomastigotes were labeled with CellTrace™ CFSE as we previously described [20]. Trypomastigotes were alternatively labelled with CellTrace™ Far Red CTFR (5 µM), essentially with the same protocol with slight differences in the incubation time (20 min at 37°C, followed by the addition of 1 ml of complete medium and an additional incubation of 5 min at 37°C in the dark).…”

Section: Methodsmentioning

confidence: 99%

Transmigration of Trypanosoma cruzi trypomastigotes through 3D cultures resembling a physiological environment

Rodríguez

Rizzi

Caeiro

et al. 2019

Preprint

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AbstractChaga’ disease, caused by the kinetoplastid parasite Trypanosoma cruzi, presents a variety of chronic clinical manifestations whose determinants are still unknown but probably influenced by the host-parasite interplay established during the first stages of the infection, when bloodstream circulating trypomastigotes disseminate to different organs and tissues. After leaving the blood, trypomastigotes must migrate through tissues to invade cells and establish a chronic infection. How this process occurs remains unexplored. Three-dimensional (3D) cultures are physiologically relevant because mimic the microarchitecture of tissues and provide an environment similar to the encountered in natural infections. In this work, we combined the 3D culture technology with host-pathogen interaction, by studying transmigration of trypomastigotes into 3D spheroids. T. cruzi strains with similar infection dynamics in 2D monolayer cultures but with different in vivo behavior (CL Brener, virulent; SylvioX10 no virulent) presented different infection rates in spheroids (CL Brener ∼40%, SylvioX10 <10%). Confocal microscopy images evidenced that trypomastigotes from CL Brener and other highly virulent strains presented a great ability to transmigrate inside 3D spheroids: as soon as 4 hours post infection parasites were found at 50 µm in depth inside the spheroids. CL Brener trypomastigotes were evenly distributed and systematically observed in the space between cells, suggesting a paracellular route of transmigration to deepen into the spheroids. On the other hand, poor virulent strains presented a weak migratory capacity and remained in the external layers of spheroids (<10µm) with a patch-like distribution pattern. The invasiveness -understood as the ability to transmigrate deep into spheroids- was not a transferable feature between strains, neither by soluble or secreted factors nor by co-cultivation of trypomastigotes from invasive and non-invasive strains. We also studied the transmigration of recent T. cruzi isolates from children that were born congenitally infected, which showed a high migrant phenotype while an isolate form an infected mother (that never transmitted the infection to any of her 3 children) was significantly less migratory. Altogether, our results demonstrate that in a 3D microenvironment each strain presents a characteristic migration pattern and distribution of parasites in the spheroids that can be associated to their in vivo behavior. Certainly, the findings presented here could not have been studied with traditional 2D monolayer cultures.Author SummaryTrypanosoma cruzi is the protozoan parasite that causes Chaga’ disease, also known as American trypanosomiasis. Experimental models of the infection evidence that different strains of the parasite present different virulence in the host, which cannot be always reproduced in 2D monolayer cultures. Three dimensional (3D) cultures can be useful models to study complex host-parasite interactions because they mimic in vitro the microarchitecture of tissues and provide an environment similar to the encountered in natural infections. In particular, spheroids are small 3D aggregates of cells that interact with each other and with the extracellular matrix that they secrete resembling the original microenvironment both functionally and structurally. Spheroids have rarely been employed to explore infectious diseases and host-parasite interactions. In this work we studied how bloodstream trypomastigotes transmigrate through 3D spheroids mimicking the picture encountered by parasites in tissues soon after leaving circulation. We showed that the behavior of T. cruzi trypomastigotes in 3D cultures reflects their in vivo virulence: virulent strains transmigrate deeply into spheroids while non-virulent strains remain in the external layers of spheroids. Besides, this work demonstrates the usefulness of 3D cultures as an accurate in vitro model for the study of host-pathogen interactions that could not be addressed with conventional monolayer cultures.

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“…Another recent publication sheds light on the transmigration of T . cruzi trypomastigotes by employing a 3D spheroid model mimicking infection conditions in situ [ 55 ]. Apart from the added aspects of cell-to-cell interactions and the presence of an extracellular matrix (ECM), which are both missing in monolayer cultures, a unique feature of this system is the direct visualization of host and parasite cells by differential labeling with 2 fluorescent dyes.…”

Section: Review Of Existing In Vitro Models For Protozoan Parasitesmentioning

confidence: 99%

The Truman Show for protozoan parasites: A review of in vitro cultivation platforms

Sutrave

Richter

2021

PLoS Negl Trop Dis

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Protozoan parasites are responsible for severe disease and suffering in humans worldwide. Apart from disease transmission via insect vectors and contaminated soil, food, or water, transmission may occur congenitally or by way of blood transfusion and organ transplantation. Several recent outbreaks associated with fresh produce and potable water emphasize the need for vigilance and monitoring of protozoan parasites that cause severe disease in humans globally. Apart from the tropical parasite Plasmodium spp., other protozoa causing debilitating and fatal diseases such as Trypanosoma spp. and Naegleria fowleri need to be studied in more detail. Climate change and socioeconomic issues such as migration continue to be major drivers for the spread of these neglected tropical diseases beyond endemic zones. Due to the complex life cycles of protozoa involving multiple hosts, vectors, and stringent growth conditions, studying these parasites has been challenging. While in vivo models may provide insights into host–parasite interaction, the ethical aspects of laboratory animal use and the challenge of ready availability of parasite life stages underline the need for in vitro models as valid alternatives for culturing and maintaining protozoan parasites. To our knowledge, this review is the first of its kind to highlight available in vitro models for protozoa causing highly infectious diseases. In recent years, several research efforts using new technologies such as 3D organoid and spheroid systems for protozoan parasites have been introduced that provide valuable tools to advance complex culturing models and offer new opportunities toward the advancement of parasite in vitro studies. In vitro models aid scientists and healthcare providers in gaining insights into parasite infection biology, ultimately enabling the use of novel strategies for preventing and treating these diseases.

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Transmigration of Trypanosoma cruzi Trypomastigotes through 3D Spheroids Mimicking Host Tissues

Cited by 5 publications

References 36 publications

Transmigration ofTrypanosoma cruzitrypomastigotes through3Dcultures resembling a physiological environment

Transmigration ofTrypanosoma cruzitrypomastigotes through3Dcultures resembling a physiological environment

Transmigration of Trypanosoma cruzi trypomastigotes through 3D cultures resembling a physiological environment

The Truman Show for protozoan parasites: A review of in vitro cultivation platforms

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