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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.

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Cellular distribution of TcSMP proteins in different stages of the T. cruzi life cycle.Indirect immunofluorescence with anti-TcSMP antibodies in permeabilized (left) or non-permeabilized (right) T. cruzi (CL strain). T. cruzi developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). Labeling with DAPI and TcSMP proteins is shown in blue and green, respectively. Bar: 10 μm.
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pntd.0004216.g004: Cellular distribution of TcSMP proteins in different stages of the T. cruzi life cycle.Indirect immunofluorescence with anti-TcSMP antibodies in permeabilized (left) or non-permeabilized (right) T. cruzi (CL strain). T. cruzi developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). Labeling with DAPI and TcSMP proteins is shown in blue and green, respectively. Bar: 10 μm.

Mentions: Live and permeabilized parasites were analyzed by indirect immunofluorescence, using anti-TcSMP antibodies (Fig 4). TcSMP distribution varied from dispersed throughout the cytosol in permeabilized parasites to punctate and concentrated in discrete spots on the cell surface of live parasites. From these results, we conclude that TcSMP proteins are located on the surface as well as intracellularly in T. cruzi developmental forms. Surface proteins follow the parasite’s secretory pathway through the endoplasmic reticulum (ER), where the signal peptide is removed before being addressed to the cell membrane. To confirm that the signal peptide directs TcSMP to the ER, parasites expressing TcSMP-GFP were incubated with anti-BIP, an endoplasmic reticulum marker. Overlapping of confocal images obtained from each fluorescence channel showed co-localized pixels between TcSMP-GFP and anti-BIP (S2B Fig). This result suggests that TcSMP goes to the ER and then it is addressed to the cell surface membrane. The PSSA-2 protein was located on the surface of T. brucei procyclic forms of parasites transfected with the complete ORF protein [26]. We confirmed the presence of TcSMP and PSSA-2 on the cell surface of T. cruzi tissue culture trypomastigotes (TCT) and T. brucei procyclic forms by flow cytometry analysis, labeling live and permeabilized parasites with anti-TcSMP antibodies. S6 Fig shows the labeling of live T. cruzi TCT forms and T. brucei procyclic forms with anti-TcSMP. Approximately 90%-92% of live T. cruzi and T. brucei parasites labeled with anti-TcSMP antibodies exhibited greater fluorescence intensity than those incubated with pre-immune serum. Besides indicate that anti-TcSMP antibodies recognized an ortholog in the T. brucei surface membrane, these results confirm that TcSMP is located on the cell surface of T. cruzi tissue culture trypomastigotes (TCT).


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)

Cellular distribution of TcSMP proteins in different stages of the T. cruzi life cycle.Indirect immunofluorescence with anti-TcSMP antibodies in permeabilized (left) or non-permeabilized (right) T. cruzi (CL strain). T. cruzi developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). Labeling with DAPI and TcSMP proteins is shown in blue and green, respectively. Bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004216.g004: Cellular distribution of TcSMP proteins in different stages of the T. cruzi life cycle.Indirect immunofluorescence with anti-TcSMP antibodies in permeabilized (left) or non-permeabilized (right) T. cruzi (CL strain). T. cruzi developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). Labeling with DAPI and TcSMP proteins is shown in blue and green, respectively. Bar: 10 μm.
Mentions: Live and permeabilized parasites were analyzed by indirect immunofluorescence, using anti-TcSMP antibodies (Fig 4). TcSMP distribution varied from dispersed throughout the cytosol in permeabilized parasites to punctate and concentrated in discrete spots on the cell surface of live parasites. From these results, we conclude that TcSMP proteins are located on the surface as well as intracellularly in T. cruzi developmental forms. Surface proteins follow the parasite’s secretory pathway through the endoplasmic reticulum (ER), where the signal peptide is removed before being addressed to the cell membrane. To confirm that the signal peptide directs TcSMP to the ER, parasites expressing TcSMP-GFP were incubated with anti-BIP, an endoplasmic reticulum marker. Overlapping of confocal images obtained from each fluorescence channel showed co-localized pixels between TcSMP-GFP and anti-BIP (S2B Fig). This result suggests that TcSMP goes to the ER and then it is addressed to the cell surface membrane. The PSSA-2 protein was located on the surface of T. brucei procyclic forms of parasites transfected with the complete ORF protein [26]. We confirmed the presence of TcSMP and PSSA-2 on the cell surface of T. cruzi tissue culture trypomastigotes (TCT) and T. brucei procyclic forms by flow cytometry analysis, labeling live and permeabilized parasites with anti-TcSMP antibodies. S6 Fig shows the labeling of live T. cruzi TCT forms and T. brucei procyclic forms with anti-TcSMP. Approximately 90%-92% of live T. cruzi and T. brucei parasites labeled with anti-TcSMP antibodies exhibited greater fluorescence intensity than those incubated with pre-immune serum. Besides indicate that anti-TcSMP antibodies recognized an ortholog in the T. brucei surface membrane, these results confirm that TcSMP is located on the cell surface of T. cruzi tissue culture trypomastigotes (TCT).

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