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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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Western blotting analysis of TcSMP proteins in the different developmental stages of T. cruzi and in the procyclic form of T. brucei rhodesiense.A) Samples of T. cruzi (CL strain) (1 x 107 cells/slot) and T. brucei (5 x 106 cells/slot) were lysed in Laemmli sample buffer. Proteins were separated by electrophoresis on SDS- polyacrylamide gel (10%), transferred to a nitrocellulose membrane and reacted against anti-TcSMP polyclonal antibodies. Antibody against a constitutively expressed protein (tubulin) was used as a loading control. T. cruzi developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). T. brucei rhodesiense procyclic forms. B) Two μg of the recombinant TcSMP-GST purified protein was separated by SDS-PAGE and reacted against anti-TcSMP and anti-GST antibodies. The recombinant protein is coded by a 311-bp fragment of TcSMP in phase with GST (see methods and S4 Fig). Molecular mass markers in kilodaltons (kDa) are indicated on the left and reactive proteins molecular mass on the right.
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pntd.0004216.g003: Western blotting analysis of TcSMP proteins in the different developmental stages of T. cruzi and in the procyclic form of T. brucei rhodesiense.A) Samples of T. cruzi (CL strain) (1 x 107 cells/slot) and T. brucei (5 x 106 cells/slot) were lysed in Laemmli sample buffer. Proteins were separated by electrophoresis on SDS- polyacrylamide gel (10%), transferred to a nitrocellulose membrane and reacted against anti-TcSMP polyclonal antibodies. Antibody against a constitutively expressed protein (tubulin) was used as a loading control. T. cruzi developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). T. brucei rhodesiense procyclic forms. B) Two μg of the recombinant TcSMP-GST purified protein was separated by SDS-PAGE and reacted against anti-TcSMP and anti-GST antibodies. The recombinant protein is coded by a 311-bp fragment of TcSMP in phase with GST (see methods and S4 Fig). Molecular mass markers in kilodaltons (kDa) are indicated on the left and reactive proteins molecular mass on the right.

Mentions: Expression of TcSMP was analyzed using polyclonal antibodies raised against a recombinant protein carrying a small fragment of TcSMP that encodes a predicted transmembrane helix flanked by cytoplasmic and extracellular hydrophilic regions (see S5 Fig). Western blot analysis of whole parasite extracts using mouse anti-TcSMP polyclonal antibodies detected a ~43 kDa protein band expressed in all parasite developmental forms, which is the predicted molecular mass for TcSMP_S (Fig 3). Assuming that TcSMP_L has a signal peptide that is removed after trafficking through the endoplasmic reticulum, all members of this family can be expected to generate proteins of the same molecular mass as TcSMP_S. An additional ~70 kDa band consistently reacted against anti-TcSMP antibody; this size is close to that of two of these proteins together. Fragoso et al. [26] found some evidence that PSSA-2 forms homodimers or multidimers. As with its ortholog, there are few cysteine residues in the TcSMP protein sequence that could be responsible for the formation of disulfide bridges. Although the sample buffer contains 2-mercaptoethanol or dithiothreitol (DTT), our protein samples were not prepared with antioxidants; hence, intermolecular disulfide bonds could be reconstituted during electrophoresis. Interestingly, when we performed a western blot containing the recombinant TcSMP protein excised from a single 38 kDa band of bacterial extract, two larger bands (~80 kDa and ~160 kDa) reacted against anti-TcSMP antibody after SDS-PAGE (Fig 3). It is possible that the larger bands are dimer/multidimers and that this could be the stable conformation of native TcSMP. Whilst some evidence supports the hypothesis that this band may be a dimer, further experiments should be carried out to prove this hypothesis.


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)

Western blotting analysis of TcSMP proteins in the different developmental stages of T. cruzi and in the procyclic form of T. brucei rhodesiense.A) Samples of T. cruzi (CL strain) (1 x 107 cells/slot) and T. brucei (5 x 106 cells/slot) were lysed in Laemmli sample buffer. Proteins were separated by electrophoresis on SDS- polyacrylamide gel (10%), transferred to a nitrocellulose membrane and reacted against anti-TcSMP polyclonal antibodies. Antibody against a constitutively expressed protein (tubulin) was used as a loading control. T. cruzi developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). T. brucei rhodesiense procyclic forms. B) Two μg of the recombinant TcSMP-GST purified protein was separated by SDS-PAGE and reacted against anti-TcSMP and anti-GST antibodies. The recombinant protein is coded by a 311-bp fragment of TcSMP in phase with GST (see methods and S4 Fig). Molecular mass markers in kilodaltons (kDa) are indicated on the left and reactive proteins molecular mass on the right.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004216.g003: Western blotting analysis of TcSMP proteins in the different developmental stages of T. cruzi and in the procyclic form of T. brucei rhodesiense.A) Samples of T. cruzi (CL strain) (1 x 107 cells/slot) and T. brucei (5 x 106 cells/slot) were lysed in Laemmli sample buffer. Proteins were separated by electrophoresis on SDS- polyacrylamide gel (10%), transferred to a nitrocellulose membrane and reacted against anti-TcSMP polyclonal antibodies. Antibody against a constitutively expressed protein (tubulin) was used as a loading control. T. cruzi developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). T. brucei rhodesiense procyclic forms. B) Two μg of the recombinant TcSMP-GST purified protein was separated by SDS-PAGE and reacted against anti-TcSMP and anti-GST antibodies. The recombinant protein is coded by a 311-bp fragment of TcSMP in phase with GST (see methods and S4 Fig). Molecular mass markers in kilodaltons (kDa) are indicated on the left and reactive proteins molecular mass on the right.
Mentions: Expression of TcSMP was analyzed using polyclonal antibodies raised against a recombinant protein carrying a small fragment of TcSMP that encodes a predicted transmembrane helix flanked by cytoplasmic and extracellular hydrophilic regions (see S5 Fig). Western blot analysis of whole parasite extracts using mouse anti-TcSMP polyclonal antibodies detected a ~43 kDa protein band expressed in all parasite developmental forms, which is the predicted molecular mass for TcSMP_S (Fig 3). Assuming that TcSMP_L has a signal peptide that is removed after trafficking through the endoplasmic reticulum, all members of this family can be expected to generate proteins of the same molecular mass as TcSMP_S. An additional ~70 kDa band consistently reacted against anti-TcSMP antibody; this size is close to that of two of these proteins together. Fragoso et al. [26] found some evidence that PSSA-2 forms homodimers or multidimers. As with its ortholog, there are few cysteine residues in the TcSMP protein sequence that could be responsible for the formation of disulfide bridges. Although the sample buffer contains 2-mercaptoethanol or dithiothreitol (DTT), our protein samples were not prepared with antioxidants; hence, intermolecular disulfide bonds could be reconstituted during electrophoresis. Interestingly, when we performed a western blot containing the recombinant TcSMP protein excised from a single 38 kDa band of bacterial extract, two larger bands (~80 kDa and ~160 kDa) reacted against anti-TcSMP antibody after SDS-PAGE (Fig 3). It is possible that the larger bands are dimer/multidimers and that this could be the stable conformation of native TcSMP. Whilst some evidence supports the hypothesis that this band may be a dimer, further experiments should be carried out to prove this hypothesis.

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