Ca2+ signal induced by Trypanosoma cruzi metacyclic trypomastigote surface molecules implicated in mammalian cell invasion

ABSTRACT Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82-kDa surface glycoprotein (gp82) that has been implicated in host cell invasion. gp82-mediated interaction of metacyclic forms with target cells induces in both cells activation of the signal transduction pathways, leading to intracellular Ca2+ mobilization, which is required for parasite internalization. Noninfective epimastigotes do not express detectable levels of gp82 and are unable to induce a Ca2+ response. We stably transfected epimastigotes with a T. cruzi expression vector carrying the metacyclic stage gp82 cDNA. These transfectants produced a functional gp82, which bound to and triggered a Ca2+ response in HeLa cells, in the same manner as the metacyclic trypomastigote gp82. Such properties were not found in epimastigotes transfected with the plasmid vector alone. Epimastigotes expressing gp82 on the surface adhered to HeLa cells but were not internalized. Treatment of gp82-expressing epimastigotes with forskolin, an activator of adenylyl cyclase that increases the metacyclic trypomastigote entry into target cells, did not promote parasite internalization. P175, an intracellular tyrosine phosphorylated protein, which appears to play a role in gp82-dependent signaling cascade in metacyclic forms, was undetectable in epimastigotes, either transfected or not with pTEX-gp82. Overall, our results indicate that gp82 is required but not sufficient for target cell invasion.

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

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0004883 ◽

2016 ◽

Vol 10 (8) ◽

pp. e0004883 ◽

Cited By ~ 7

Author(s):

Tatiana Mordente Clemente ◽

Cristian Cortez ◽

Antônio da Silva Novaes ◽

Nobuko Yoshida

Keyword(s):

Trypanosoma Cruzi ◽

Host Cell ◽

Cell Invasion ◽

Host Cell Invasion ◽

Surface Molecules

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Actin Cytoskeleton-Dependent and -Independent Host Cell Invasion by Trypanosoma cruzi Is Mediated by Distinct Parasite Surface Molecules

Infection and Immunity ◽

10.1128/iai.00518-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5522-5528 ◽

Cited By ~ 38

Author(s):

Daniele Ferreira ◽

Mauro Cortez ◽

Vanessa D. Atayde ◽

Nobuko Yoshida

Keyword(s):

Trypanosoma Cruzi ◽

Actin Cytoskeleton ◽

Host Cell ◽

Cell Invasion ◽

Hela Cells ◽

Cytochalasin D ◽

Target Cells ◽

Host Cell Invasion ◽

Latrunculin B ◽

Surface Molecules

ABSTRACT The disassembly of host cell actin cytoskeleton as a facilitator of Trypanosoma cruzi invasion has been reported by some authors, while other workers claim that it instead inhibits internalization of the parasite. In this study we aimed at elucidating the basis of this discrepancy. We performed experiments with metacyclic trypomastigotes of T. cruzi strains G and CL, which differ markedly in infectivity and enter target cells by engaging the surface molecules gp35/50 and gp82, respectively, which have signaling activity. Treatment of HeLa cells with the F-actin-disrupting drug cytochalasin D or latrunculin B inhibited the invasion by strain G but not the invasion by strain CL. In contrast to cells penetrated by strain CL, which were previously shown to have a disrupted actin cytoskeleton architecture, no such alteration was observed in HeLa cells invaded by strain G, and parasites were found to be closely associated with target cell actin. Coinfection with enteroinvasive Escherichia coli (EIEC), which recruits host cell actin for internalization, drastically reduced entry of strain CL into HeLa cells but not entry of strain G. In contrast to gp82 in its recombinant form, which induces disruption of F-actin and inhibits EIEC invasion, purified mucin-like gp35/50 molecules promoted an increase in EIEC uptake by HeLa cells. These data, plus the finding that drugs that interfere with mammalian cell signaling differentially affect the internalization of metacyclic forms of strains G and CL, indicate that the host cell invasion mediated by gp35/50 is associated with signaling events that favor actin recruitment, in contrast to gp82-dependent invasion, which triggers the signaling pathways leading to disassembly of F-actin.

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

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.769722 ◽

2021 ◽

Vol 11 ◽

Author(s):

Leonardo Loch ◽

Thiago Souza Onofre ◽

João Paulo Ferreira Rodrigues ◽

Nobuko Yoshida

Keyword(s):

Trypanosoma Cruzi ◽

Host Cell ◽

Phospholipase C ◽

Western Blotting ◽

Cell Invasion ◽

Magnetic Beads ◽

Protein Profile ◽

Soluble Form ◽

Host Cell Invasion ◽

Surface Molecules

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.

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