Limits...
Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion.

Martins NO, Souza RT, Cordero EM, Maldonado DC, Cortez C, Marini MM, Ferreira ER, Bayer-Santos E, Almeida IC, Yoshida N, Silveira JF - PLoS Negl Trop Dis (2015)

Bottom Line: TcSMP proteins were also located intracellularly likely associated with membrane-bound structures.We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells.TcSMP bound to mammalian cells and triggered Ca2+ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP, São Paulo, Brasil.

ABSTRACT

Background: The surface coat of Trypanosoma cruzi is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions.

Methodology/ principal findings: Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), which are conserved among different T. cruzi lineages and have orthologs in other Trypanosoma species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all T. cruzi developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the T. cruzi secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca2+ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion.

Conclusion/significance: We hypothesized that the productive interaction of T. cruzi with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by TcSMP may be additive to that triggered by the major surface molecule gp82, further increasing the host cell responses required for infection.

Show MeSH

Related in: MedlinePlus

Recombinant protein TcSMP properties in host cell binding, Ca2+ signaling, lysosome scattering and inhibition of T. cruzi metacyclic trypomastigote invasion.A) HeLa cells were incubated with the indicated proteins, at varying concentrations, and binding was evaluated as described in the methods section. Values are the means ± SD of triplicates of one representative assay out of three. B) HeLa cells were grown overnight in DMEM with 10% serum on ibidi multichamber dishes (Hi-Q4, ibidi), and the fluorescence intensity of Fluo-4 was determined as described in the methods section. Images show the basal fluorescence at time zero (left panel) and the maximum fluorescence after stimulation with 40 μg/mL purified TcSMP or GST (right panel). Note the increase in the fluorescence intensity after challenge with TcSMP but not with GST. Bar: 50 μm. C) HeLa cells were grown as in (B) and intracellular calcium was quantified after stimulation (black arrow) with recombinant protein TcSMP or GST. The graph shows the relative concentration of cytoplasmic Ca2+ at 400 sec after stimulation, expressed as the maximum peak of total fluorescence minus basal fluorescence at time zero. Thirty cells in three independent experiments were analyzed. Shown on the left side is the difference between the maximum calcium concentration at 400 sec after stimulation with TcSMP and GST (*p = 0.0005). D) HeLa cells were incubated for 30 min with 40 μg/mL of TcSMP or GST, and then processed for confocal fluorescence analysis using anti-LAMP2 antibody and Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA, under 63X objective. (Bar: 20 μm). Note the lysosome mobilization to the host cell periphery after treatment with TcSMP. E) HeLa cells were incubated with the indicated recombinant protein at 40 μg/mL. After 30 min, CL strain metacyclic trypomastigotes were added and incubation proceeded for 1 h before fixation and staining with Giemsa. The number of internalized parasites was counted in a total of 250 cells. Values are the means ± SD of three independent assays performed in duplicate. TcSMP, but not GST, significantly inhibited parasite invasion (*p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4643927&req=5

pntd.0004216.g005: Recombinant protein TcSMP properties in host cell binding, Ca2+ signaling, lysosome scattering and inhibition of T. cruzi metacyclic trypomastigote invasion.A) HeLa cells were incubated with the indicated proteins, at varying concentrations, and binding was evaluated as described in the methods section. Values are the means ± SD of triplicates of one representative assay out of three. B) HeLa cells were grown overnight in DMEM with 10% serum on ibidi multichamber dishes (Hi-Q4, ibidi), and the fluorescence intensity of Fluo-4 was determined as described in the methods section. Images show the basal fluorescence at time zero (left panel) and the maximum fluorescence after stimulation with 40 μg/mL purified TcSMP or GST (right panel). Note the increase in the fluorescence intensity after challenge with TcSMP but not with GST. Bar: 50 μm. C) HeLa cells were grown as in (B) and intracellular calcium was quantified after stimulation (black arrow) with recombinant protein TcSMP or GST. The graph shows the relative concentration of cytoplasmic Ca2+ at 400 sec after stimulation, expressed as the maximum peak of total fluorescence minus basal fluorescence at time zero. Thirty cells in three independent experiments were analyzed. Shown on the left side is the difference between the maximum calcium concentration at 400 sec after stimulation with TcSMP and GST (*p = 0.0005). D) HeLa cells were incubated for 30 min with 40 μg/mL of TcSMP or GST, and then processed for confocal fluorescence analysis using anti-LAMP2 antibody and Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA, under 63X objective. (Bar: 20 μm). Note the lysosome mobilization to the host cell periphery after treatment with TcSMP. E) HeLa cells were incubated with the indicated recombinant protein at 40 μg/mL. After 30 min, CL strain metacyclic trypomastigotes were added and incubation proceeded for 1 h before fixation and staining with Giemsa. The number of internalized parasites was counted in a total of 250 cells. Values are the means ± SD of three independent assays performed in duplicate. TcSMP, but not GST, significantly inhibited parasite invasion (*p < 0.05).

Mentions: As the TcSMP protein was detected on the T. cruzi cell surface (Fig 4), we examined whether it is implicated in parasite-host cell interactions. First, the target cell binding capacity of the recombinant TcSMP protein fused to GST was examined. HeLa cells, immobilized on the bottom of microtiter plates, were incubated with increasing concentrations of recombinant TcSMP or GST, and the bound protein was detected using antibodies directed against TcSMP or GST. Binding of TcSMP to HeLa cells was dose-dependent whereas GST failed to bind (Fig 5A). Next, we determined the ability of the TcSMP protein to trigger host cell Ca2+ signaling, which is required for T. cruzi internalization [44–46]. Recombinant TcSMP or GST, at 40 μg/mL, was added to HeLa cells loaded with the Ca2+ indicator fluo-4, and Ca2+ signal-inducing activity was monitored by fluorescence microscopy. TcSMP, but not GST, triggered an increase in the fluorescence intensity (Fig 5B). We also evaluated variation of the cytosolic free Ca2+ concentration after challenge with 40 μg/mL TcSMP or GST. The recombinant protein TcSMP induced a Ca2+ signal that, although moderate, was significant when compared with variation in the fluorescence intensity after stimulation with GST (Fig 5C). As the metacyclic stage surface molecule gp82, which mediates host cell invasion, binds to HeLa cells and induces Ca2+ signal [47] and lysosome scattering to the cell periphery followed by exocytosis [48], an event that contributes to parasitophorous vacuole biogenesis [49, 50], we tested whether the TcSMP protein could induce lysosome mobilization. HeLa cells were incubated for 30 min with 40 μg/mL recombinant TcSMP or GST and then processed for immunofluorescence to visualize lysosomes. Scattering of lysosomes from the perinuclear region to the cell periphery was induced by TcSMP protein but not by GST (Fig 5D). The finding that the TcSMP protein triggered Ca2+ signaling and lysosome scattering suggested that it could be involved in parasite internalization. To determine the involvement of TcSMP in host cell invasion, assays were performed by incubating CL strain metacyclic forms with HeLa cells for 1 h in the absence or presence of the recombinant TcSMP or GST, at 40 μg/mL. TcSMP protein, but not GST, significantly inhibited parasite invasion (Fig 5E). An additional experiment consisted in comparing the effects of TcSMP and gp82 on lysosome scattering and host cell invasion. HeLa cells were incubated for 30 min with TcSMP or the recombinant gp82, which is also fused to GST, and then processed for immunofluorescence. At 40 μg/mL, TcSMP induced lysosome-scattering comparable to that of gp82 at 20 μg/mL (Fig 6A). The effect of TcSMP at 20 μg/mL was much less pronounced, although it appeared to be higher than that of the GST control (Fig 6A). For cell invasion assays, HeLa cells were incubated for 1 h with CL strain metacyclic forms in the absence or presence of TcSMP or gp82 or in the presence of the two proteins at concentrations of 20 μg/mL and 40 μg/mL. Fig 6B shows that TcSMP significantly inhibited parasite internalization at 40 μg/mL but not at 20 μg/mL, in contrast to gp82, which exhibited an inhibitory effect at 20 μg/mL. The levels of inhibition by the combination of the two proteins were similar to that of gp82 alone (Fig 6B), indicating that the effects of TcSMP and gp82 are not additive.


Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion.

Martins NO, Souza RT, Cordero EM, Maldonado DC, Cortez C, Marini MM, Ferreira ER, Bayer-Santos E, Almeida IC, Yoshida N, Silveira JF - PLoS Negl Trop Dis (2015)

Recombinant protein TcSMP properties in host cell binding, Ca2+ signaling, lysosome scattering and inhibition of T. cruzi metacyclic trypomastigote invasion.A) HeLa cells were incubated with the indicated proteins, at varying concentrations, and binding was evaluated as described in the methods section. Values are the means ± SD of triplicates of one representative assay out of three. B) HeLa cells were grown overnight in DMEM with 10% serum on ibidi multichamber dishes (Hi-Q4, ibidi), and the fluorescence intensity of Fluo-4 was determined as described in the methods section. Images show the basal fluorescence at time zero (left panel) and the maximum fluorescence after stimulation with 40 μg/mL purified TcSMP or GST (right panel). Note the increase in the fluorescence intensity after challenge with TcSMP but not with GST. Bar: 50 μm. C) HeLa cells were grown as in (B) and intracellular calcium was quantified after stimulation (black arrow) with recombinant protein TcSMP or GST. The graph shows the relative concentration of cytoplasmic Ca2+ at 400 sec after stimulation, expressed as the maximum peak of total fluorescence minus basal fluorescence at time zero. Thirty cells in three independent experiments were analyzed. Shown on the left side is the difference between the maximum calcium concentration at 400 sec after stimulation with TcSMP and GST (*p = 0.0005). D) HeLa cells were incubated for 30 min with 40 μg/mL of TcSMP or GST, and then processed for confocal fluorescence analysis using anti-LAMP2 antibody and Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA, under 63X objective. (Bar: 20 μm). Note the lysosome mobilization to the host cell periphery after treatment with TcSMP. E) HeLa cells were incubated with the indicated recombinant protein at 40 μg/mL. After 30 min, CL strain metacyclic trypomastigotes were added and incubation proceeded for 1 h before fixation and staining with Giemsa. The number of internalized parasites was counted in a total of 250 cells. Values are the means ± SD of three independent assays performed in duplicate. TcSMP, but not GST, significantly inhibited parasite invasion (*p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643927&req=5

pntd.0004216.g005: Recombinant protein TcSMP properties in host cell binding, Ca2+ signaling, lysosome scattering and inhibition of T. cruzi metacyclic trypomastigote invasion.A) HeLa cells were incubated with the indicated proteins, at varying concentrations, and binding was evaluated as described in the methods section. Values are the means ± SD of triplicates of one representative assay out of three. B) HeLa cells were grown overnight in DMEM with 10% serum on ibidi multichamber dishes (Hi-Q4, ibidi), and the fluorescence intensity of Fluo-4 was determined as described in the methods section. Images show the basal fluorescence at time zero (left panel) and the maximum fluorescence after stimulation with 40 μg/mL purified TcSMP or GST (right panel). Note the increase in the fluorescence intensity after challenge with TcSMP but not with GST. Bar: 50 μm. C) HeLa cells were grown as in (B) and intracellular calcium was quantified after stimulation (black arrow) with recombinant protein TcSMP or GST. The graph shows the relative concentration of cytoplasmic Ca2+ at 400 sec after stimulation, expressed as the maximum peak of total fluorescence minus basal fluorescence at time zero. Thirty cells in three independent experiments were analyzed. Shown on the left side is the difference between the maximum calcium concentration at 400 sec after stimulation with TcSMP and GST (*p = 0.0005). D) HeLa cells were incubated for 30 min with 40 μg/mL of TcSMP or GST, and then processed for confocal fluorescence analysis using anti-LAMP2 antibody and Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA, under 63X objective. (Bar: 20 μm). Note the lysosome mobilization to the host cell periphery after treatment with TcSMP. E) HeLa cells were incubated with the indicated recombinant protein at 40 μg/mL. After 30 min, CL strain metacyclic trypomastigotes were added and incubation proceeded for 1 h before fixation and staining with Giemsa. The number of internalized parasites was counted in a total of 250 cells. Values are the means ± SD of three independent assays performed in duplicate. TcSMP, but not GST, significantly inhibited parasite invasion (*p < 0.05).
Mentions: As the TcSMP protein was detected on the T. cruzi cell surface (Fig 4), we examined whether it is implicated in parasite-host cell interactions. First, the target cell binding capacity of the recombinant TcSMP protein fused to GST was examined. HeLa cells, immobilized on the bottom of microtiter plates, were incubated with increasing concentrations of recombinant TcSMP or GST, and the bound protein was detected using antibodies directed against TcSMP or GST. Binding of TcSMP to HeLa cells was dose-dependent whereas GST failed to bind (Fig 5A). Next, we determined the ability of the TcSMP protein to trigger host cell Ca2+ signaling, which is required for T. cruzi internalization [44–46]. Recombinant TcSMP or GST, at 40 μg/mL, was added to HeLa cells loaded with the Ca2+ indicator fluo-4, and Ca2+ signal-inducing activity was monitored by fluorescence microscopy. TcSMP, but not GST, triggered an increase in the fluorescence intensity (Fig 5B). We also evaluated variation of the cytosolic free Ca2+ concentration after challenge with 40 μg/mL TcSMP or GST. The recombinant protein TcSMP induced a Ca2+ signal that, although moderate, was significant when compared with variation in the fluorescence intensity after stimulation with GST (Fig 5C). As the metacyclic stage surface molecule gp82, which mediates host cell invasion, binds to HeLa cells and induces Ca2+ signal [47] and lysosome scattering to the cell periphery followed by exocytosis [48], an event that contributes to parasitophorous vacuole biogenesis [49, 50], we tested whether the TcSMP protein could induce lysosome mobilization. HeLa cells were incubated for 30 min with 40 μg/mL recombinant TcSMP or GST and then processed for immunofluorescence to visualize lysosomes. Scattering of lysosomes from the perinuclear region to the cell periphery was induced by TcSMP protein but not by GST (Fig 5D). The finding that the TcSMP protein triggered Ca2+ signaling and lysosome scattering suggested that it could be involved in parasite internalization. To determine the involvement of TcSMP in host cell invasion, assays were performed by incubating CL strain metacyclic forms with HeLa cells for 1 h in the absence or presence of the recombinant TcSMP or GST, at 40 μg/mL. TcSMP protein, but not GST, significantly inhibited parasite invasion (Fig 5E). An additional experiment consisted in comparing the effects of TcSMP and gp82 on lysosome scattering and host cell invasion. HeLa cells were incubated for 30 min with TcSMP or the recombinant gp82, which is also fused to GST, and then processed for immunofluorescence. At 40 μg/mL, TcSMP induced lysosome-scattering comparable to that of gp82 at 20 μg/mL (Fig 6A). The effect of TcSMP at 20 μg/mL was much less pronounced, although it appeared to be higher than that of the GST control (Fig 6A). For cell invasion assays, HeLa cells were incubated for 1 h with CL strain metacyclic forms in the absence or presence of TcSMP or gp82 or in the presence of the two proteins at concentrations of 20 μg/mL and 40 μg/mL. Fig 6B shows that TcSMP significantly inhibited parasite internalization at 40 μg/mL but not at 20 μg/mL, in contrast to gp82, which exhibited an inhibitory effect at 20 μg/mL. The levels of inhibition by the combination of the two proteins were similar to that of gp82 alone (Fig 6B), indicating that the effects of TcSMP and gp82 are not additive.

Bottom Line: TcSMP proteins were also located intracellularly likely associated with membrane-bound structures.We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells.TcSMP bound to mammalian cells and triggered Ca2+ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP, São Paulo, Brasil.

ABSTRACT

Background: The surface coat of Trypanosoma cruzi is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions.

Methodology/ principal findings: Here, we described a novel family of T. cruzi surface membrane proteins (TcSMP), which are conserved among different T. cruzi lineages and have orthologs in other Trypanosoma species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all T. cruzi developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the T. cruzi secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca2+ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion.

Conclusion/significance: We hypothesized that the productive interaction of T. cruzi with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by TcSMP may be additive to that triggered by the major surface molecule gp82, further increasing the host cell responses required for infection.

Show MeSH
Related in: MedlinePlus