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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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Modular architecture of TcSMP paralogs and orthologs.Left Panel) Schematic representation of TcSMP proteins from T. cruzi isolates (CLB, Sylvio X10/1 and Dm28c) and T. cruzi marinkellei. Right Panel) Schematic representation of TcSMP orthologs in T. brucei, T. brucei gambiense, T. congolense, T. vivax, T. rangeli and T. grayi. The amino acid sequence is represented by a black bar and the first and last residues are indicated below. The red vertical lines indicate the putative in frame initiator methionine residues. The positions of predicted signal peptide (SP), signal anchor (SA) and transmembrane domain (TM) are indicated by green, blue and orange lines, respectively. Sequences were identified by their entries in TriTrypDB, with the exception of those from T. rangeli and T. grayi, which were identified by GenBank accession numbers. T. cruzi sequences: TcCLB, clone CL Brener; TCSYLVIO, clone Sylvio X10/1; TCDM, clone Dm28c; Tc_MARK, T. cruzi marinkellei.
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pntd.0004216.g002: Modular architecture of TcSMP paralogs and orthologs.Left Panel) Schematic representation of TcSMP proteins from T. cruzi isolates (CLB, Sylvio X10/1 and Dm28c) and T. cruzi marinkellei. Right Panel) Schematic representation of TcSMP orthologs in T. brucei, T. brucei gambiense, T. congolense, T. vivax, T. rangeli and T. grayi. The amino acid sequence is represented by a black bar and the first and last residues are indicated below. The red vertical lines indicate the putative in frame initiator methionine residues. The positions of predicted signal peptide (SP), signal anchor (SA) and transmembrane domain (TM) are indicated by green, blue and orange lines, respectively. Sequences were identified by their entries in TriTrypDB, with the exception of those from T. rangeli and T. grayi, which were identified by GenBank accession numbers. T. cruzi sequences: TcCLB, clone CL Brener; TCSYLVIO, clone Sylvio X10/1; TCDM, clone Dm28c; Tc_MARK, T. cruzi marinkellei.

Mentions: TcSMP proteins have 2–3 hydrophobic domains at the N- and C-termini (Fig 1 and S5 Fig). Considering that the translation of TcSMP_L starts at the 3rd methionine, the first hydrophobic domain corresponds to a typical amino-terminal signal peptide with basic residues followed by a hydrophobic stretch with a putative cleavage site RSA-FF (Fig 1). TcSMP_S proteins have two transmembrane domains and the first one is located immediately after the initiator methionine, which was predicted to be a signal anchor [38]. An exception was Tc_MARK_3606 in which the first hydrophobic domain was predicted to be a signal peptide (Fig 2). A similar pattern is present in PSSA-2 of T. brucei (S5 Fig) which is attached to the plasma membrane by a stable transmembrane anchor characteristic of membrane proteins [25]. The C-terminus of TcSMP contains 4 proline residues centered on the YGQ motif that can also contribute to size differences among the TcSMP proteins (Fig 1). Interestingly, there are 8 tandem proline repeats on the cytoplasmic tail of PSSA-2 that are predicted to form tight helices [25]. TcSMP and PSSA-2 repeats share low degrees of similarity.


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)

Modular architecture of TcSMP paralogs and orthologs.Left Panel) Schematic representation of TcSMP proteins from T. cruzi isolates (CLB, Sylvio X10/1 and Dm28c) and T. cruzi marinkellei. Right Panel) Schematic representation of TcSMP orthologs in T. brucei, T. brucei gambiense, T. congolense, T. vivax, T. rangeli and T. grayi. The amino acid sequence is represented by a black bar and the first and last residues are indicated below. The red vertical lines indicate the putative in frame initiator methionine residues. The positions of predicted signal peptide (SP), signal anchor (SA) and transmembrane domain (TM) are indicated by green, blue and orange lines, respectively. Sequences were identified by their entries in TriTrypDB, with the exception of those from T. rangeli and T. grayi, which were identified by GenBank accession numbers. T. cruzi sequences: TcCLB, clone CL Brener; TCSYLVIO, clone Sylvio X10/1; TCDM, clone Dm28c; Tc_MARK, T. cruzi marinkellei.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004216.g002: Modular architecture of TcSMP paralogs and orthologs.Left Panel) Schematic representation of TcSMP proteins from T. cruzi isolates (CLB, Sylvio X10/1 and Dm28c) and T. cruzi marinkellei. Right Panel) Schematic representation of TcSMP orthologs in T. brucei, T. brucei gambiense, T. congolense, T. vivax, T. rangeli and T. grayi. The amino acid sequence is represented by a black bar and the first and last residues are indicated below. The red vertical lines indicate the putative in frame initiator methionine residues. The positions of predicted signal peptide (SP), signal anchor (SA) and transmembrane domain (TM) are indicated by green, blue and orange lines, respectively. Sequences were identified by their entries in TriTrypDB, with the exception of those from T. rangeli and T. grayi, which were identified by GenBank accession numbers. T. cruzi sequences: TcCLB, clone CL Brener; TCSYLVIO, clone Sylvio X10/1; TCDM, clone Dm28c; Tc_MARK, T. cruzi marinkellei.
Mentions: TcSMP proteins have 2–3 hydrophobic domains at the N- and C-termini (Fig 1 and S5 Fig). Considering that the translation of TcSMP_L starts at the 3rd methionine, the first hydrophobic domain corresponds to a typical amino-terminal signal peptide with basic residues followed by a hydrophobic stretch with a putative cleavage site RSA-FF (Fig 1). TcSMP_S proteins have two transmembrane domains and the first one is located immediately after the initiator methionine, which was predicted to be a signal anchor [38]. An exception was Tc_MARK_3606 in which the first hydrophobic domain was predicted to be a signal peptide (Fig 2). A similar pattern is present in PSSA-2 of T. brucei (S5 Fig) which is attached to the plasma membrane by a stable transmembrane anchor characteristic of membrane proteins [25]. The C-terminus of TcSMP contains 4 proline residues centered on the YGQ motif that can also contribute to size differences among the TcSMP proteins (Fig 1). Interestingly, there are 8 tandem proline repeats on the cytoplasmic tail of PSSA-2 that are predicted to form tight helices [25]. TcSMP and PSSA-2 repeats share low degrees of similarity.

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