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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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Alignment of TcSMP amino acid sequences of T. cruzi—clone CL Brener (CLB).Sequences TcCLB.510129.20, TcCLB.510129.30, TcCLB.509639.10, TcCLB.507711.90, extended TcCLB.507711.100, extended TcCLB.507711.110 and TcCLB.508173.120 were deposited in the GenBank database by the T. cruzi Genome Project. Clones 23B, 24B and 24C (GenBank KJ682657, KJ682658 and KJ682659) were isolated in this work from genomic DNA (CLB) by PCR. Sequences were aligned by the MegAlign program (DNASTAR). Black, gray and light gray blocks indicate residues with 100%, 80% and 60% identity, respectively. Colored lines above the sequences denote the predicted signal peptide in green, transmembrane domains in orange and uncleaved anchor signal in blue. Red arrows indicate the putative initiator methionine. Yellow boxes correspond to peptides found in the proteomic analysis of T. cruzi membrane protein-enriched fractions [27]. Red boxes correspond to peptides identified in the T. cruzi secretome [43]. Blue boxes correspond to the YGQ motif present in Procyclic Surface Specific Antigen-2 (PSSA-2) of T. brucei.
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pntd.0004216.g001: Alignment of TcSMP amino acid sequences of T. cruzi—clone CL Brener (CLB).Sequences TcCLB.510129.20, TcCLB.510129.30, TcCLB.509639.10, TcCLB.507711.90, extended TcCLB.507711.100, extended TcCLB.507711.110 and TcCLB.508173.120 were deposited in the GenBank database by the T. cruzi Genome Project. Clones 23B, 24B and 24C (GenBank KJ682657, KJ682658 and KJ682659) were isolated in this work from genomic DNA (CLB) by PCR. Sequences were aligned by the MegAlign program (DNASTAR). Black, gray and light gray blocks indicate residues with 100%, 80% and 60% identity, respectively. Colored lines above the sequences denote the predicted signal peptide in green, transmembrane domains in orange and uncleaved anchor signal in blue. Red arrows indicate the putative initiator methionine. Yellow boxes correspond to peptides found in the proteomic analysis of T. cruzi membrane protein-enriched fractions [27]. Red boxes correspond to peptides identified in the T. cruzi secretome [43]. Blue boxes correspond to the YGQ motif present in Procyclic Surface Specific Antigen-2 (PSSA-2) of T. brucei.

Mentions: TcSMP genes shared 82–98% nucleotide identity (S1 Fig), whereas amino acid identity varied from 70–90% and 65–93% for TcSMP_L and TcSMP_S, respectively (Fig 1). Sequence alignment showed that TcSMP_L proteins share an N-terminal extension of 69 amino acids containing seven in frame putative initiator codons. The region immediately after the fourth ATG codon encodes a typical 25-amino acid signal peptide with a putative cleavage site between residues RSA-FF (Fig 1). Upstream sequences in the vicinity of the third ATG best fit the Kozak consensus sequence (S4 Fig). Among other aspects of the mRNA structure, the context surrounding the AUG codon can modulate the initiation of translation [40]. The Kozak sequence located upstream of the initiation codon is expected to facilitate ribosome binding and thus the beginning of protein synthesis. In view of this, we suggest that the translation of large sequences initiates at the fifth methionine. The presence of several initiation codons in the same reading frame is common among T. cruzi surface protein genes such as GP82 and GP90 [41, 42].


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)

Alignment of TcSMP amino acid sequences of T. cruzi—clone CL Brener (CLB).Sequences TcCLB.510129.20, TcCLB.510129.30, TcCLB.509639.10, TcCLB.507711.90, extended TcCLB.507711.100, extended TcCLB.507711.110 and TcCLB.508173.120 were deposited in the GenBank database by the T. cruzi Genome Project. Clones 23B, 24B and 24C (GenBank KJ682657, KJ682658 and KJ682659) were isolated in this work from genomic DNA (CLB) by PCR. Sequences were aligned by the MegAlign program (DNASTAR). Black, gray and light gray blocks indicate residues with 100%, 80% and 60% identity, respectively. Colored lines above the sequences denote the predicted signal peptide in green, transmembrane domains in orange and uncleaved anchor signal in blue. Red arrows indicate the putative initiator methionine. Yellow boxes correspond to peptides found in the proteomic analysis of T. cruzi membrane protein-enriched fractions [27]. Red boxes correspond to peptides identified in the T. cruzi secretome [43]. Blue boxes correspond to the YGQ motif present in Procyclic Surface Specific Antigen-2 (PSSA-2) of T. brucei.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004216.g001: Alignment of TcSMP amino acid sequences of T. cruzi—clone CL Brener (CLB).Sequences TcCLB.510129.20, TcCLB.510129.30, TcCLB.509639.10, TcCLB.507711.90, extended TcCLB.507711.100, extended TcCLB.507711.110 and TcCLB.508173.120 were deposited in the GenBank database by the T. cruzi Genome Project. Clones 23B, 24B and 24C (GenBank KJ682657, KJ682658 and KJ682659) were isolated in this work from genomic DNA (CLB) by PCR. Sequences were aligned by the MegAlign program (DNASTAR). Black, gray and light gray blocks indicate residues with 100%, 80% and 60% identity, respectively. Colored lines above the sequences denote the predicted signal peptide in green, transmembrane domains in orange and uncleaved anchor signal in blue. Red arrows indicate the putative initiator methionine. Yellow boxes correspond to peptides found in the proteomic analysis of T. cruzi membrane protein-enriched fractions [27]. Red boxes correspond to peptides identified in the T. cruzi secretome [43]. Blue boxes correspond to the YGQ motif present in Procyclic Surface Specific Antigen-2 (PSSA-2) of T. brucei.
Mentions: TcSMP genes shared 82–98% nucleotide identity (S1 Fig), whereas amino acid identity varied from 70–90% and 65–93% for TcSMP_L and TcSMP_S, respectively (Fig 1). Sequence alignment showed that TcSMP_L proteins share an N-terminal extension of 69 amino acids containing seven in frame putative initiator codons. The region immediately after the fourth ATG codon encodes a typical 25-amino acid signal peptide with a putative cleavage site between residues RSA-FF (Fig 1). Upstream sequences in the vicinity of the third ATG best fit the Kozak consensus sequence (S4 Fig). Among other aspects of the mRNA structure, the context surrounding the AUG codon can modulate the initiation of translation [40]. The Kozak sequence located upstream of the initiation codon is expected to facilitate ribosome binding and thus the beginning of protein synthesis. In view of this, we suggest that the translation of large sequences initiates at the fifth methionine. The presence of several initiation codons in the same reading frame is common among T. cruzi surface protein genes such as GP82 and GP90 [41, 42].

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