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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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Genomic organization of the TcSMP family.A) Schematic representation of in silico chromosomes TcChr37-P, TcChr37-S and TcChr27-P showing the distribution of TcSMP genes (red boxes). The accession numbers of TcSMP genes in TriTrypDB are indicated above. B) Restriction Southern blot analysis of TcSMP loci. Genomic DNA of CLB was digested with different restriction enzymes, separated on a 0.8% agarose gel and transferred to nylon membrane. Autoradiogram obtained by hybridization with (32P) TcSMP probe. The enzymes are indicated above each lane. The colored boxes indicate the restriction fragments predicted in chromosomes TcChr37-P, TcChr37-S, and TcChr27-P. C) Karyotype mapping of TcSMP genes. Chromosomal bands of CLB, strain G and T. cruzi marinkellei were separated by PFGE, stained with ethidium bromide, transferred onto nylon membranes and hybridized with TcSMP probe. Numbers on the left correspond to the sizes (Mb) of Hansenula wingei chromosomes used as markers, and on the right the sizes of chromosomal bands hybridizing with (32P) TcSMP probe.
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pntd.0004216.g007: Genomic organization of the TcSMP family.A) Schematic representation of in silico chromosomes TcChr37-P, TcChr37-S and TcChr27-P showing the distribution of TcSMP genes (red boxes). The accession numbers of TcSMP genes in TriTrypDB are indicated above. B) Restriction Southern blot analysis of TcSMP loci. Genomic DNA of CLB was digested with different restriction enzymes, separated on a 0.8% agarose gel and transferred to nylon membrane. Autoradiogram obtained by hybridization with (32P) TcSMP probe. The enzymes are indicated above each lane. The colored boxes indicate the restriction fragments predicted in chromosomes TcChr37-P, TcChr37-S, and TcChr27-P. C) Karyotype mapping of TcSMP genes. Chromosomal bands of CLB, strain G and T. cruzi marinkellei were separated by PFGE, stained with ethidium bromide, transferred onto nylon membranes and hybridized with TcSMP probe. Numbers on the left correspond to the sizes (Mb) of Hansenula wingei chromosomes used as markers, and on the right the sizes of chromosomal bands hybridizing with (32P) TcSMP probe.

Mentions: T. cruzi (CLB) genomic sequences were assembled into 41 chromosome-sized scaffolds designated as TcChr1 to TcChr41 (T. cruzi in silico chromosomes) [51]. Due to the hybrid nature of clone CL Brener, the chromosome-sized scaffolds were designated S and P to denote the Esmeraldo and non-Esmeraldo haplotypes, respectively [51]. In silico, TcSMP genes were located in chromosomes TcChr37-S, TcChr37-P and TcChr27-P (Fig 7). The homologous chromosomes Tc-Chr37-S and TcChr37P contain a cluster of 3 and 5 TcSMP genes, respectively, while chromosome TcChr27-P contains only one copy. TcSMP genes in the cluster have the same transcriptional orientation (Fig 7A). TcSMP genes located on chromosome TcChr37-P share ≥92% identity with each other or with those in the homologous TcChr37S. Moreover, these sequences exhibit 82–86% identity with TcSMP (TcCLB.508173.120) located on chromosome TcChr27-P.


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)

Genomic organization of the TcSMP family.A) Schematic representation of in silico chromosomes TcChr37-P, TcChr37-S and TcChr27-P showing the distribution of TcSMP genes (red boxes). The accession numbers of TcSMP genes in TriTrypDB are indicated above. B) Restriction Southern blot analysis of TcSMP loci. Genomic DNA of CLB was digested with different restriction enzymes, separated on a 0.8% agarose gel and transferred to nylon membrane. Autoradiogram obtained by hybridization with (32P) TcSMP probe. The enzymes are indicated above each lane. The colored boxes indicate the restriction fragments predicted in chromosomes TcChr37-P, TcChr37-S, and TcChr27-P. C) Karyotype mapping of TcSMP genes. Chromosomal bands of CLB, strain G and T. cruzi marinkellei were separated by PFGE, stained with ethidium bromide, transferred onto nylon membranes and hybridized with TcSMP probe. Numbers on the left correspond to the sizes (Mb) of Hansenula wingei chromosomes used as markers, and on the right the sizes of chromosomal bands hybridizing with (32P) TcSMP probe.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004216.g007: Genomic organization of the TcSMP family.A) Schematic representation of in silico chromosomes TcChr37-P, TcChr37-S and TcChr27-P showing the distribution of TcSMP genes (red boxes). The accession numbers of TcSMP genes in TriTrypDB are indicated above. B) Restriction Southern blot analysis of TcSMP loci. Genomic DNA of CLB was digested with different restriction enzymes, separated on a 0.8% agarose gel and transferred to nylon membrane. Autoradiogram obtained by hybridization with (32P) TcSMP probe. The enzymes are indicated above each lane. The colored boxes indicate the restriction fragments predicted in chromosomes TcChr37-P, TcChr37-S, and TcChr27-P. C) Karyotype mapping of TcSMP genes. Chromosomal bands of CLB, strain G and T. cruzi marinkellei were separated by PFGE, stained with ethidium bromide, transferred onto nylon membranes and hybridized with TcSMP probe. Numbers on the left correspond to the sizes (Mb) of Hansenula wingei chromosomes used as markers, and on the right the sizes of chromosomal bands hybridizing with (32P) TcSMP probe.
Mentions: T. cruzi (CLB) genomic sequences were assembled into 41 chromosome-sized scaffolds designated as TcChr1 to TcChr41 (T. cruzi in silico chromosomes) [51]. Due to the hybrid nature of clone CL Brener, the chromosome-sized scaffolds were designated S and P to denote the Esmeraldo and non-Esmeraldo haplotypes, respectively [51]. In silico, TcSMP genes were located in chromosomes TcChr37-S, TcChr37-P and TcChr27-P (Fig 7). The homologous chromosomes Tc-Chr37-S and TcChr37P contain a cluster of 3 and 5 TcSMP genes, respectively, while chromosome TcChr27-P contains only one copy. TcSMP genes in the cluster have the same transcriptional orientation (Fig 7A). TcSMP genes located on chromosome TcChr37-P share ≥92% identity with each other or with those in the homologous TcChr37S. Moreover, these sequences exhibit 82–86% identity with TcSMP (TcCLB.508173.120) located on chromosome TcChr27-P.

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