Characterization of a cDNA clone encoding the carboxy-terminal domain of a 90-kilodalton surface antigen of Trypanosoma cruzi metacyclic trypomastigotes

Franco, F R; Paranhos-Bacalla, Glaucia S.; Yamauchi, Luci M.; Yoshida, Nobuko; Silveira, José Franco da

doi:10.1128/iai.61.10.4196-4201.1993

Cited by 28 publications

(9 citation statements)

References 34 publications

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“…S3). To identify the MAb 5E7-reactive epitope, which is known to reside in the C-terminal domain of p90 ( 10 ), enzyme-linked immunosorbent assays (ELISAs) of inhibition of antibody binding were performed, using synthetic 20-mer peptides, with overlapping of 10 amino acid residues spanning the referred region ( Fig. 2D ).…”

Section: Resultsmentioning

confidence: 99%

“…Native gp90 was purified from detergent-soluble extract of G strain MT by affinity chromatography on immobilized MAb 1G7, as described previously ( 30 ). The recombinant protein containing the C-terminal domain of T. cruzi gp90 (GenBank accession number L11287 ) and the recombinant protein containing the full-length gp82 sequence (GenBank accession number L14824 ), both in fusion with glutathione S -transferase (GST), were produced in Escherichia coli as described previously ( 10 , 11 ). All steps for the purification of the recombinant proteins followed a previously described procedure ( 11 ), and the purified protein was analyzed by Coomassie blue staining of SDS-PAGE gels and by immunoblotting using monoclonal antibody directed to gp90 or gp82.…”

Section: Methodsmentioning

confidence: 99%

“…Two main metacyclic stage-specific surface molecules, gp82 and gp90, have been shown to play determinant roles in MT invasion of host cells in vitro as well as in oral T. cruzi infection in mice ( 5 – 8 ). gp82 and gp90 are members of the gp85/ trans -sialidase superfamily ( 9 , 10 ), which bind to host cells in a receptor-mediated manner and induce opposite effects. In contrast to gp82, which mediates MT internalization by inducing Ca 2+ signal in host cells, leading to actin cytoskeleton disruption ( 11 ), lysosome spreading, and exocytosis ( 12 , 13 ), events that contribute to parasitophorous vacuole formation ( 14 , 15 ), gp90 is devoid of such properties ( 16 , 17 ) and functions as a negative regulator of MT invasion ( 18 ).…”

Section: Introductionmentioning

confidence: 99%

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Inhibition of Host Cell Lysosome Spreading by Trypanosoma cruzi Metacyclic Stage-Specific Surface Molecule gp90 Downregulates Parasite Invasion

et al. 2017

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Successful infection by Trypanosoma cruzi, the agent of Chagas' disease, is critically dependent on host cell invasion by metacyclic trypomastigote (MT) forms. Two main metacyclic stage-specific surface molecules, gp82 and gp90, play determinant roles in target cell invasion in vitro and in oral T. cruzi infection in mice. The structure and properties of gp82, which is highly conserved among T. cruzi strains, are well known. Information on gp90 is still rather sparse. Here, we attempted to fill that gap. gp90, purified from poorly invasive G strain MT and expressing gp90 at high levels, inhibited HeLa cell lysosome spreading and the gp82-mediated internalization of a highly invasive CL strain MT expressing low levels of a diverse gp90 molecule. A recombinant protein containing the conserved C-terminal domain of gp90 exhibited the same properties as the native G strain gp90: it counteracted the host cell lysosome spreading induced by recombinant gp82 and exhibited an inhibitory effect on HeLa cell invasion by CL strain MT. Assays to identify the gp90 sequence associated with the property of downregulating MT invasion, using synthetic peptides spanning the gp90 C-terminal domain, revealed the sequence GVLYTADKEW. These data, plus the findings that lysosome spreading was induced upon HeLa cell interaction with CL strain MT, but not with G strain MT, and that in mixed infection CL strain MT internalization was inhibited by G strain MT, suggest that the inhibition of target cell lysosome spreading is the mechanism by which the gp90 molecule exerts its downregulatory role.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Inhibition of Host Cell Lysosome Spreading by Trypanosoma cruzi Metacyclic Stage-Specific Surface Molecule gp90 Downregulates Parasite Invasion

et al. 2017

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“…The recombinant gp90 protein, containing the C-terminal domain of T . cruzi gp90 (GenBank accession number L11287), and the recombinant protein containing the full-length gp82 sequence (GenBank accession number L14824) were produced in Escherichia coli as described [ 20 , 21 ]. All steps for the purification of the recombinant proteins followed a previously described procedure [ 21 ], and the purified protein was analyzed by Coomassie blue staining of SDS-PAGE gel and by immunoblotting using monoclonal antibody directed to gp90 or gp82.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2016

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BackgroundThe 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 FindingsMetacyclic 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/SignificanceOur 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.

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“…To ascertain that the correct protein was obtained, the purified preparations were analysed by silver staining of SDS‐PAGE gel and by immunoblotting using monoclonal antibody 3F6 or the polyclonal antibody directed to gp82. The recombinant protein T07, containing a sequence of MT surface molecule gp90 fused to GST (Franco et al ., 1993), was prepared in the same manner as J18.…”

Section: Methodsmentioning

confidence: 99%

Starvation and rapamycin differentially regulate host cell lysosome exocytosis and invasion by Trypanosoma cruzi metacyclic forms

Martins

Alves

Macedo

et al. 2011

Cellular Microbiology

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SummaryThe molecular mechanisms of host cell invasion by T. cruzi metacyclic trypomastigotes (MT), the developmental forms that initiate infection in the mammalian host, are only partially understood. Here we aimed at further identifying the target cell components involved in signalling cascades leading to MT internalization, and demonstrate for the first time the participation of mammalian target of rapamycin (mTOR). Treatment of human epithelial HeLa cells with mTOR inhibitor rapamycin reduced lysosomal exocytosis and MT invasion. Downregulation of phosphatidylinositol 3-kinase and protein kinase C also impaired exocytosis and MT internalization. The recombinant protein based on gp82, the MT surface molecule that mediates cell adhesion/invasion, induced exocytosis in HeLa cells. Such an effect has not previously been attributed to any T. cruzi surface molecule. Rapamycin treatment diminished gp82 binding as well. Cell invasion assays under conditions that promoted lysosome exocytosis, such as 1 h incubation in starvation medium PBS ++ , increased MT invasion, whereas pre-starvation of cells for 1-2 h had an opposite effect. In contrast to MT, invasion of tissue culture trypomastigotes (TCT) increased upon host cell pre-starvation or treatment with rapamycin, a novel finding that discloses quite distinctive features of the two infective forms in a key process for infection.

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Characterization of a cDNA clone encoding the carboxy-terminal domain of a 90-kilodalton surface antigen of Trypanosoma cruzi metacyclic trypomastigotes

Cited by 28 publications

References 34 publications

Inhibition of Host Cell Lysosome Spreading by Trypanosoma cruzi Metacyclic Stage-Specific Surface Molecule gp90 Downregulates Parasite Invasion

Inhibition of Host Cell Lysosome Spreading by Trypanosoma cruzi Metacyclic Stage-Specific Surface Molecule gp90 Downregulates Parasite Invasion

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

Starvation and rapamycin differentially regulate host cell lysosome exocytosis and invasion by Trypanosoma cruzi metacyclic forms

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