Shedding of Trypanosoma cruzi Surface Molecules That Regulate Host Cell Invasion Involves Phospholipase C and Increases Upon Sterol Depletion.

Leonardo Loch,Thiago Souza Onofre,João Paulo Ferreira Rodrigues,Nobuko Yoshida

doi:10.3389/fcimb.2021.769722

Leonardo Loch, Thiago Souza Onofre + Show 2 more

Open Access

https://doi.org/10.3389/fcimb.2021.769722

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Abstract

Metacyclic trypomastigote (MT) forms of Trypanosoma cruzi have been shown to release into medium gp82 and gp90, the stage-specific surface molecules that regulate host cell invasion, either in vesicles or in soluble form. Here, we found that during interaction of poorly invasive G strain with the host cell, gp82 and gp90 were released in vesicle-like forms, whereas no such release by highly invasive CL strain was observed. Shedding of vesicles of varying sizes by CL and G strains was visualized by scanning electron microscopy, and the protein profile of conditioned medium (CM) of the two strains was similar, but the content of gp82 and gp90 differed, with both molecules being detected in G strain as bands of high intensity in Western blotting, whereas in CL strain, they were barely detectable. Confocal images revealed a distinct distribution of gp82 and gp90 on MT surface of CL and G strains. In cell invasion assays, addition of G strain CM resulted in decreased CL strain internalization. Depletion of gp82 in G strain CM, by treatment with specific mAb-coupled magnetic beads, increased its inhibitory effect on CL strain invasion, in contrast to CM depleted in gp90. The effect of cholesterol-depleting drug methyl-β-cyclodextrin (MβCD) on gp82 and gp90 release by MTs was also examined. G strain MTs, untreated or treated with MβCD, were incubated in serum-containing medium or in nutrient-depleted PBS++, and the CM generated under these conditions was analyzed by Western blotting. In PBS++, gp82 and gp90 were released at lower levels by untreated MTs, as compared with MβCD-treated parasites. CM from untreated and MβCD-treated G strain, generated in PBS++, inhibited CL strain internalization. Treatment of CL strain MTs with MβCD resulted in increased gp82 and gp90 shedding and in decreased host cell invasion. The involvement of phospholipase C (PLC) on gp82 and gp90 shedding was also investigated. The CM from G strain MTs pretreated with specific PLC inhibitor contained lower levels of gp82 and gp90, as compared with untreated parasites. Our results contribute to shed light on the mechanism by which T. cruzi releases surface molecules implicated in host cell invasion.

Highlights

Secreted signaling molecules play essential roles in intercellular communication
A previous study has shown that gp82 and gp90 are released into medium in considerable amounts by G strain, either in vesicles or in soluble form, whereas release by CL strain Metacyclic trypomastigote (MT) is minimal (Clemente et al, 2016)
Both G and CL strain MTs induced the spreading of lysosomes and accumulation at the cell edges, an event known to be stimulated by gp82 (Cortez et al, 2016), and was evident in large multinucleated cells (Figure 1)

Summary

Introduction

Secreted signaling molecules play essential roles in intercellular communication. As a means of communication between cells, extracellular vesicles (EVs), which are lipid bound particles containing proteins, lipids, and nucleic acids, have gained prominence and are recognized to mediate the regulation of physiological functions and to be involved in pathological processes (Yáñez-Mó et al, 2015; Zaborowski et al, 2015). As regards T. cruzi, the agent of Chagas disease, it was reported three decades ago that the tissue culture-derived trypomastigote (TCT) forms spontaneously shed the entire set of surface polypeptides into the culture medium, mostly as plasma membrane vesicles (Gonçalves et al, 1991). It was shown that, upon injection into mice, TCT-shed vesicles increase heart parasitism and generate an intense inflammatory response (Torrecilhas et al, 2009). As regards TCT, EVs derived from different T. cruzi strains were shown to trigger differential innate and chronic immune responses (Nogueira et al, 2015). Quantitative and qualitative differences in TCT-shed EVs and secreted proteins from different T. cruzi strains, revealed by proteomic analysis, were suggested to correlate with infectivity/virulence during the host–parasite interaction (Ribeiro et al, 2018). In Toll-like-receptor 2transfected Chinese hamster ovary (CHO) cells, an increase in the percentage of TCT-infected cells was observed upon incubation with TCT-shed EVs (Cronemberger-Andrade et al, 2020)

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