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

Abstract

<jats:title>Abstract</jats:title><jats:p>Chaga’ disease, caused by the kinetoplastid parasite <jats:italic>Trypanosoma cruzi</jats:italic>, 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. <jats:italic>T. cruzi</jats:italic> strains with similar infection dynamics in 2D monolayer cultures but with different <jats:italic>in vivo</jats:italic> behavior (CL Brener, virulent; SylvioX10 no virulent) presented different infection rates in spheroids (CL Brener ∼40%, SylvioX10 &lt;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 (&lt;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 <jats:italic>T. cruzi</jats:italic> 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 <jats:italic>in vivo</jats:italic> behavior. Certainly, the findings presented here could not have been studied with traditional 2D monolayer cultures.</jats:p><jats:sec><jats:title>Author Summary</jats:title><jats:p><jats:italic>Trypanosoma cruzi</jats:italic> 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 <jats:italic>in vitro</jats:italic> 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 <jats:italic>T. cruzi</jats:italic> trypomastigotes in 3D cultures reflects their <jats:italic>in vivo</jats:italic> 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 <jats:italic>in vitro</jats:italic> model for the study of host-pathogen interactions that could not be addressed with conventional monolayer cultures.</jats:p></jats:sec>

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

“…The 3D HeLa cell spheroids were labeled with the red fluorescence protein (RFP) and the trypanomastigotes were labeled with carboxyfluorescein succinimidyl ester (CFSE) to visualize transmigration of trypomastigotes within the spheroids using confocal microscopy. A follow-up study by Rodriguez and colleagues [ 56 ] published in 2020 showed that T . cruzi trypomastigotes were less infective in HeLaR2 spheroids than in 2D monolayers and concluded that 3D spheroids provide an optimal physiological environment for studying host–parasite interactions.…”