Characterization of the Cell Adhesion Site of<i>Trypanosoma cruzi</i>Metacyclic Stage Surface Glycoprotein gp82

Manque, Patrício; Eichinger, Daniel; Juliano, María A.; Juliano, Luiz; Araya, Jorge E.; Yoshida, Nobuko

doi:10.1128/iai.68.2.478-484.2000

Cited by 39 publications

(42 citation statements)

References 37 publications

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“…To determine whether the cDNA-encoded recombinant protein which binds to mammalian cells in the same manner as the native gp90 (17) was capable of inhibiting the adhesion of gp90 to host cells, assays were carried out by incubating HeLa cells for 1 h at 37°C with the native gp90 at 20 g/ml in the absence or presence of various concentrations of gp90 recombinant protein (r-gp90) fused to GST. As controls, HeLa cells were incubated with the native gp90 plus GST or J18b at 40 g/ml the (J18b is the GST-fused recombinant protein containing the C-teminal domain of metacyclic stage surface molecule gp82, which binds to HeLa cells through a sequence that is not represented in rgp90) (10). Binding of gp90 to HeLa cells was revealed by MAb 1G7, which recognizes the native molecule but not rgp90.…”

Section: Resultsmentioning

confidence: 99%

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Targeted Reduction in Expression ofTrypanosoma cruziSurface Glycoprotein gp90 Increases Parasite Infectivity

Málaga

Yoshida

2001

Infect Immun

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A previous study had shown that the expression of gp90, a stage-specific surface glycoprotein of Trypanosoma cruzi metacyclic trypomastigotes, is inversely correlated with the parasite's ability to invade mammalian cells. By using antisense oligonucleotides complementary to a region of the gp90 gene implicated in host cell adhesion, we investigated whether the selective inhibition of gp90 synthesis affected the capacity of metacyclic forms to enter target cells. Parasites were incubated for 24 h with 20 M PS1, a phosphorothioate oligonucleotide based on a sequence of the gp90 coding strand; PS2, the antisense counterpart of PS1; or PO2, the unmodified version of PS2 containing phosphodiester linkages, and the expression of surface molecules was analyzed by flow cytometry and immunoblotting using specific monoclonal antibodies. PS2 but not PS1 or PO2 inhibited the expression of gp90. Inhibition by PS2 was dose dependent. Northern blot analysis revealed that steady-state gp90 mRNA levels were diminished in PS2-treated parasites compared to untreated controls. Treatment with PS2 did not affect the expression of other metacyclic stage surface glycoproteins involved in parasite-host cell interaction, such as gp82 and the mucin-like gp35/50. Expression of gp90 was also inhibited by other phosphorothioate oligonucleotides targeted to the gp90 gene (PS4, PS5, PS6, and PS7) but not by PS3, with the same base composition as PS2 but a mismatched sequence. Parasites treated with PS2, PS4, or PS5 entered HeLa cells in significantly higher numbers than untreated controls, whereas the invasive capacity of PS1-and PS3-treated parasites was unchanged, confirming the inverse association between infectivity and gp90 expression.

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Section: Resultsmentioning

confidence: 99%

“…We have recently mapped the host cell binding site of gp82 (10). It is located in the C-terminal domain of the molecule, in a region not equivalent to the gp90 C-terminal domain, which contains the host cell binding site.…”

Section: Discussionmentioning

confidence: 99%

Targeted Reduction in Expression ofTrypanosoma cruziSurface Glycoprotein gp90 Increases Parasite Infectivity

Málaga

Yoshida

2001

Infect Immun

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“…An additional experiment confirmed that the FN-binding site of gp82 is not nested in the central domain of the molecule. Inhibition binding assays, performed by incubating FN-coated plates with the recombinant gp82 in the presence of individual 20-mer synthetic peptides with an overlap of 10 residues, spanning the gp82 central domain (32), revealed that none of the 10 peptides tested was capable of significantly inhibiting gp82 binding to FN (data not shown).…”

Section: Fn Inhibits T Cruzi Mt Entry Into Host Cellsmentioning

confidence: 99%

Fibronectin-Degrading Activity of Trypanosoma cruzi Cysteine Proteinase Plays a Role in Host Cell Invasion

et al. 2014

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Trypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insectstage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. Using T. cruzi CL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the major T. cruzi cysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-␤1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase of T. cruzi metacyclic forms, through its fibronectin-degrading activity, is implicated in host cell invasion. E xtracellular matrix (ECM) proteins, which serve as substrates for diverse adhesion molecules and are involved in many important physiological processes, also may mediate cell attachment and/or invasion of pathogenic microorganisms. Among these molecules, fibronectin (FN) has been reported to play a role in adherence to and invasion of host cells by bacteria such as Staphylococcus aureus, Streptococcus pyogenes, and Campylobacter jejuni (1-6). The interaction of fibronectin with Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, also has been described. T. cruzi infection is initiated by metacyclic trypomastigote (MT) from the insect vector. This parasite form is responsible for the initial T. cruzi-host cell interaction upon entering the mammalian host through the skin or by the oral route. Following MT internalization in a membrane-bound vacuole and escape to the cytoplasm, the parasite differentiates into amastigote form. After several rounds of replication, amastigotes transform into trypomastigotes that are released in the circulation upon host cell rupture. Experiments with tissue culture trypomastigote (TCT), which corresponds to the bloodstream trypomastigote, revealed the involvement of fibronectin in target cell adhesion/invasion. The treatment of 3T3 fibroblasts or rat peritoneal macrophages, or TCT, with human plasma FN increased parasite-cell association (7), and binding ...

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“…gp82 is a glycoprotein containing N-linked oligosaccharide (22), and the carbohydrate portion is not involved in cell adhesion and invasion (25). Its cell binding site is a peptide sequence, possibly formed by juxtaposition of two charged sequences separated by a hydrophobic stretch (18). Although gp82 is expressed in both strains, evidence shows that strain G metacyclic forms preferentially use gp35/50 to interact with the host cell, possibly because the cell adhesion capacity of gp35/50 is higher than that of gp82 (25).…”

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Actin Cytoskeleton-Dependent and -Independent Host Cell Invasion byTrypanosoma cruziIs Mediated by Distinct Parasite Surface Molecules

et al. 2006

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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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Characterization of the Cell Adhesion Site ofTrypanosoma cruziMetacyclic Stage Surface Glycoprotein gp82

Cited by 39 publications

References 37 publications

Targeted Reduction in Expression ofTrypanosoma cruziSurface Glycoprotein gp90 Increases Parasite Infectivity

Targeted Reduction in Expression ofTrypanosoma cruziSurface Glycoprotein gp90 Increases Parasite Infectivity

Fibronectin-Degrading Activity of Trypanosoma cruzi Cysteine Proteinase Plays a Role in Host Cell Invasion

Actin Cytoskeleton-Dependent and -Independent Host Cell Invasion byTrypanosoma cruziIs Mediated by Distinct Parasite Surface Molecules

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