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Surface Molecules Released by Trypanosoma cruzi Metacyclic Forms Downregulate Host Cell Invasion.

ABSTRACT: BACKGROUND:The question whether metacylic trypomastigote (MT) forms of different T. cruzi strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using T. cruzi strains that differ widely in the ability to invade cells. METHODOLOGY/PRINCIPAL FINDINGS:Metacyclic forms were incubated at 37°C for 1 h in complete D10 medium or in nutrient-deprived PBS containing Ca2+ and Mg2+ (PBS++). The conditioned medium (CM), collected after parasite centrifugation, was used for cell invasion assays and Western blot analysis, using monoclonal antibodies directed to gp82 and gp90, the MT surface molecules that promote and negatively regulate invasion, respectively. CM of poorly invasive G strain (G-CM) contained high amounts of gp90 and gp82, either in vesicles or as soluble molecules. CM of highly invasive CL strain (CL-CM) contained gp90 and gp82 at very low levels. HeLa cells were incubated for 1 h with CL strain MT in D10, in absence or in the presence of G-CM or CL-CM. Parasite invasion was significantly inhibited by G-CM, but not by CL-CM. As G strain MT invasion rate in D10 is very low, assays with this strain were performed in PBS++, which induces invasion-promoting lysosome-spreading. G-CM, but not CL-CM, significantly inhibited G strain internalization, effect that was counteracted by preincubating G-CM with an anti-gp90 monoclonal antibody or anti-gp82 polyclonal antibody that do not recognize live MT. G strain CM generated in PBS++ contained much lower amounts of gp90 and gp82 as compared to CM produced in D10, and exhibited lower inhibitory effect on host cell invasion. CONCLUSION/SIGNIFICANCE:Our data suggest that the surface molecules spontaneously released by MT impair parasite-host cell interaction, gp82 presumably competing with the molecule expressed on MT surface for the host cell receptor, and gp90 further contributing to down modulate invasion.

SUBMITTER: Clemente TM

PROVIDER: S-EPMC4970754 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Surface Molecules Released by Trypanosoma cruzi Metacyclic Forms Downregulate Host Cell Invasion.

Clemente Tatiana Mordente TM Cortez Cristian C Novaes Antônio da Silva Ada S Yoshida Nobuko N

PLoS neglected tropical diseases 20160802 8

<h4>Background</h4>The question whether metacylic trypomastigote (MT) forms of different T. cruzi strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using T. cruzi strains that differ widely in the ability to invade cells.<h4>Methodology/principal findings</h4>Metacyclic forms were incubated at 37°C for 1 h in complete D10 medium or in nutrient-deprived PBS containing Ca2+ and Mg2+ (PBS++). The condi ...[more]

PMID: 27483135

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Project description: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.

Project description:BackgroundSevere changes in temperature can affect the behavior and ecology of some infectious agents. Trypanosoma cruzi is a protozoan that causes Chagas disease. This parasite has high genetic variability and can be divided into six discrete typing units (DTUs). Trypanosoma cruzi also has a complex life-cycle, which includes the process of metacyclogenesis when non-infective epimastigote forms are differentiated into infective metacyclic trypomastigotes (MT). Studies in triatomines have shown that changes in temperature also affect the number and viability of MT.MethodsThe objective of this study was to evaluate how temperature affects the transcriptional profiles of T. cruzi I and II (TcI and TcII) MT by exposing parasites to two temperatures (27 °C and 28 °C) and comparing those to normal culture conditions at 26 °C. Subsequently, RNA-seq was conducted and differentially expressed genes were quantified and associated to metabolic pathways.ResultsA statistically significant difference was observed in the number of MT between the temperatures evaluated and the control, TcII DTU was not strongly affected to exposure to high temperatures compared to TcI. Similar results were found when we analyzed gene expression in this DTU, with the greatest number of differentially expressed genes being observed at 28 °C, which could indicate a dysregulation of different signaling pathways under this temperature. Chromosome analysis indicated that chromosome 1 harbored the highest number of changes for both DTUs for all thermal treatments. Finally, gene ontology (GO) analyses showed a decrease in the coding RNAs involved in the regulation of processes related to the metabolism of lipids and carbohydrates, the evasion of oxidative stress, and proteolysis and phosphorylation processes, and a decrease in RNAs coding to ribosomal proteins in TcI and TcII, along with an increase in the expression of surface metalloprotease GP63 in TcII.ConclusionsSlight temperature shifts lead to increased cell death of metacyclic trypomastigotes because of the deregulation of gene expression of different processes essential for the TcI and TcII DTUs of T. cruzi.

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Surface Molecules Released by Trypanosoma cruzi Metacyclic Forms Downregulate Host Cell Invasion.

Publications

Surface Molecules Released by Trypanosoma cruzi Metacyclic Forms Downregulate Host Cell Invasion.

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