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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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Related in: MedlinePlus

Synteny of TcSMP genes among T. brucei, T. cruzi and T. grayi.Genomic regions around the T. cruzi (CLB) TcSMP paralogs and T. brucei and T. grayi orthologs are shown. Analyses were conducted via TBLASTN using the Artemis Comparison Tool (ACT) [37] with an E value of 500. Comparison among TcChr37 (‘‘P” chromosome assigned to the non-Esmeraldo haplotype and ‘‘S” to the Esmeraldo haplotype), T. brucei chromosome Tb10 and the contig Tgr_12_V1 of T. grayi. Homologous genes are connected by colored lines. The matches and reverse matches are represented in red and in blue, respectively. Grey blocks represent each chromosome. Chromosome markers are drawn in the sense (+) and antisense (-) strands. The numbers indicate the location on chromosomes: T. brucei TREU927 (CHR10—position: 2729733–2761348 nt), CLB T. cruzi (TcChr37-S—positions: 848335–873376 nt and TcChr37-P 848287–882361 nt) and contig of T. grayi (Tgr_12_V1 –position: 1–64810 nt). The locations of the TcSMP paralogs and T. brucei and T. grayi orthologs are depicted in chromosomes by red blocks. Abbreviations: TcSMP (TcSMP—T. cruzi/ PSSA-2—T. brucei); HIRA (HIRA-interacting protein 5, putative); ubiqui (ubiquitin-like modifier-activating enzyme ATG7, putative); RNA-bp (RNA-binding protein, putative); ubiqui E1 (ubiquitin activating E1 enzyme, putative); surf glyco (surface glycoprotein, putative); PRC (paraflagellar rod component, putative); HP (hypothetical protein, conserved); S-methyl (S-methyl-5thioribose kinase); POT (proton-dependent oligopeptide transporter, POT family); tetratrico (tetratricopeptide domain 4); 60S (putative 60S ribosomal protein L6); sterol C (sterol C-24 reductase); IFT 57 (predicted: intraflagellar transporter protein 57 homolog); tRNA-s lig (tRNA-splicing ligase RtcB); PP 2A (protein phosphatase 2A regulatory subunit).
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pntd.0004216.g008: Synteny of TcSMP genes among T. brucei, T. cruzi and T. grayi.Genomic regions around the T. cruzi (CLB) TcSMP paralogs and T. brucei and T. grayi orthologs are shown. Analyses were conducted via TBLASTN using the Artemis Comparison Tool (ACT) [37] with an E value of 500. Comparison among TcChr37 (‘‘P” chromosome assigned to the non-Esmeraldo haplotype and ‘‘S” to the Esmeraldo haplotype), T. brucei chromosome Tb10 and the contig Tgr_12_V1 of T. grayi. Homologous genes are connected by colored lines. The matches and reverse matches are represented in red and in blue, respectively. Grey blocks represent each chromosome. Chromosome markers are drawn in the sense (+) and antisense (-) strands. The numbers indicate the location on chromosomes: T. brucei TREU927 (CHR10—position: 2729733–2761348 nt), CLB T. cruzi (TcChr37-S—positions: 848335–873376 nt and TcChr37-P 848287–882361 nt) and contig of T. grayi (Tgr_12_V1 –position: 1–64810 nt). The locations of the TcSMP paralogs and T. brucei and T. grayi orthologs are depicted in chromosomes by red blocks. Abbreviations: TcSMP (TcSMP—T. cruzi/ PSSA-2—T. brucei); HIRA (HIRA-interacting protein 5, putative); ubiqui (ubiquitin-like modifier-activating enzyme ATG7, putative); RNA-bp (RNA-binding protein, putative); ubiqui E1 (ubiquitin activating E1 enzyme, putative); surf glyco (surface glycoprotein, putative); PRC (paraflagellar rod component, putative); HP (hypothetical protein, conserved); S-methyl (S-methyl-5thioribose kinase); POT (proton-dependent oligopeptide transporter, POT family); tetratrico (tetratricopeptide domain 4); 60S (putative 60S ribosomal protein L6); sterol C (sterol C-24 reductase); IFT 57 (predicted: intraflagellar transporter protein 57 homolog); tRNA-s lig (tRNA-splicing ligase RtcB); PP 2A (protein phosphatase 2A regulatory subunit).

Mentions: In contrast to T. cruzi, mammal-dwelling African trypanosomes and reptilian trypanosomes appear to have reduced the TcSMP repertoire to a minimum. While T. cruzi CLB harbors 9 TcSMP genes, T. brucei and Trypanosoma grayi genomes are limited to one ortholog. We compared the corresponding syntenic regions around the TcSMP genes in the genomes of T. cruzi CLB, T. brucei and T. grayi, this last was isolated from African crocodiles (Fig 8). The analysis was performed using a region of approximately 30 kb from chromosome Chr10 of T. brucei, TcChr37-S and TcChr37-P of T. cruzi and contig Tgr_12_V1 of T. grayi. This region exhibits a conserved synteny, the same genes are located in these genomic surroundings in trypanosomes. The conserved genome structure was punctuated by structural divergence, including the insertion/deletion of individual genes and intergenic regions. The bottom of Fig 8 shows the synteny between the chromosome TcChr37P of T. cruzi and the T. grayi contig. While conserved synteny relationships between T. cruzi and T. grayi can be defined, the exact orientation of T. grayi genes in relation to T. cruzi cannot be inferred because the genome of T. grayi has not yet been assembled. The maintenance of this chromosome region during trypanosome evolution suggests that its genomic organization may be functionally important.


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)

Synteny of TcSMP genes among T. brucei, T. cruzi and T. grayi.Genomic regions around the T. cruzi (CLB) TcSMP paralogs and T. brucei and T. grayi orthologs are shown. Analyses were conducted via TBLASTN using the Artemis Comparison Tool (ACT) [37] with an E value of 500. Comparison among TcChr37 (‘‘P” chromosome assigned to the non-Esmeraldo haplotype and ‘‘S” to the Esmeraldo haplotype), T. brucei chromosome Tb10 and the contig Tgr_12_V1 of T. grayi. Homologous genes are connected by colored lines. The matches and reverse matches are represented in red and in blue, respectively. Grey blocks represent each chromosome. Chromosome markers are drawn in the sense (+) and antisense (-) strands. The numbers indicate the location on chromosomes: T. brucei TREU927 (CHR10—position: 2729733–2761348 nt), CLB T. cruzi (TcChr37-S—positions: 848335–873376 nt and TcChr37-P 848287–882361 nt) and contig of T. grayi (Tgr_12_V1 –position: 1–64810 nt). The locations of the TcSMP paralogs and T. brucei and T. grayi orthologs are depicted in chromosomes by red blocks. Abbreviations: TcSMP (TcSMP—T. cruzi/ PSSA-2—T. brucei); HIRA (HIRA-interacting protein 5, putative); ubiqui (ubiquitin-like modifier-activating enzyme ATG7, putative); RNA-bp (RNA-binding protein, putative); ubiqui E1 (ubiquitin activating E1 enzyme, putative); surf glyco (surface glycoprotein, putative); PRC (paraflagellar rod component, putative); HP (hypothetical protein, conserved); S-methyl (S-methyl-5thioribose kinase); POT (proton-dependent oligopeptide transporter, POT family); tetratrico (tetratricopeptide domain 4); 60S (putative 60S ribosomal protein L6); sterol C (sterol C-24 reductase); IFT 57 (predicted: intraflagellar transporter protein 57 homolog); tRNA-s lig (tRNA-splicing ligase RtcB); PP 2A (protein phosphatase 2A regulatory subunit).
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004216.g008: Synteny of TcSMP genes among T. brucei, T. cruzi and T. grayi.Genomic regions around the T. cruzi (CLB) TcSMP paralogs and T. brucei and T. grayi orthologs are shown. Analyses were conducted via TBLASTN using the Artemis Comparison Tool (ACT) [37] with an E value of 500. Comparison among TcChr37 (‘‘P” chromosome assigned to the non-Esmeraldo haplotype and ‘‘S” to the Esmeraldo haplotype), T. brucei chromosome Tb10 and the contig Tgr_12_V1 of T. grayi. Homologous genes are connected by colored lines. The matches and reverse matches are represented in red and in blue, respectively. Grey blocks represent each chromosome. Chromosome markers are drawn in the sense (+) and antisense (-) strands. The numbers indicate the location on chromosomes: T. brucei TREU927 (CHR10—position: 2729733–2761348 nt), CLB T. cruzi (TcChr37-S—positions: 848335–873376 nt and TcChr37-P 848287–882361 nt) and contig of T. grayi (Tgr_12_V1 –position: 1–64810 nt). The locations of the TcSMP paralogs and T. brucei and T. grayi orthologs are depicted in chromosomes by red blocks. Abbreviations: TcSMP (TcSMP—T. cruzi/ PSSA-2—T. brucei); HIRA (HIRA-interacting protein 5, putative); ubiqui (ubiquitin-like modifier-activating enzyme ATG7, putative); RNA-bp (RNA-binding protein, putative); ubiqui E1 (ubiquitin activating E1 enzyme, putative); surf glyco (surface glycoprotein, putative); PRC (paraflagellar rod component, putative); HP (hypothetical protein, conserved); S-methyl (S-methyl-5thioribose kinase); POT (proton-dependent oligopeptide transporter, POT family); tetratrico (tetratricopeptide domain 4); 60S (putative 60S ribosomal protein L6); sterol C (sterol C-24 reductase); IFT 57 (predicted: intraflagellar transporter protein 57 homolog); tRNA-s lig (tRNA-splicing ligase RtcB); PP 2A (protein phosphatase 2A regulatory subunit).
Mentions: In contrast to T. cruzi, mammal-dwelling African trypanosomes and reptilian trypanosomes appear to have reduced the TcSMP repertoire to a minimum. While T. cruzi CLB harbors 9 TcSMP genes, T. brucei and Trypanosoma grayi genomes are limited to one ortholog. We compared the corresponding syntenic regions around the TcSMP genes in the genomes of T. cruzi CLB, T. brucei and T. grayi, this last was isolated from African crocodiles (Fig 8). The analysis was performed using a region of approximately 30 kb from chromosome Chr10 of T. brucei, TcChr37-S and TcChr37-P of T. cruzi and contig Tgr_12_V1 of T. grayi. This region exhibits a conserved synteny, the same genes are located in these genomic surroundings in trypanosomes. The conserved genome structure was punctuated by structural divergence, including the insertion/deletion of individual genes and intergenic regions. The bottom of Fig 8 shows the synteny between the chromosome TcChr37P of T. cruzi and the T. grayi contig. While conserved synteny relationships between T. cruzi and T. grayi can be defined, the exact orientation of T. grayi genes in relation to T. cruzi cannot be inferred because the genome of T. grayi has not yet been assembled. The maintenance of this chromosome region during trypanosome evolution suggests that its genomic organization may be functionally important.

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