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Identification of strain-specific B-cell epitopes in Trypanosoma cruzi using genome-scale epitope prediction and high-throughput immunoscreening with peptide arrays.

Mendes TA, Reis Cunha JL, de Almeida Lourdes R, Rodrigues Luiz GF, Lemos LD, dos Santos AR, da Câmara AC, Galvão LM, Bern C, Gilman RH, Fujiwara RT, Gazzinelli RT, Bartholomeu DC - PLoS Negl Trop Dis (2013)

Bottom Line: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs.Four peptides were tested against a panel of chagasic patients using ELISA.A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.

ABSTRACT

Background: The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA.

Methodology: By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain.

Findings and conclusions: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.

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Mapping of antibody binding sites on the conserved and polymorphic epitopes by Ala-Scan.Reactivity of peptide C6_30_cons against the sera from mice infected with Colombiana (A), Y (B), and CL Brener (C). Reactivity of peptide A6_30_col against the sera from mice infected with the Colombiana clone (D). Reactivity of peptide B2_30_y against the sera from mice infected with the Y strain (E). Reactivity of peptide B9_30_cl against the sera from mice infected with the CL Brener clone (F). 1, original peptide. RI, relative intensity calculated by the ratio between the reactivity of the peptide with a specific amino acid substitution and the original peptide. The dotted line indicates half of the intensity value obtained with the original peptide. The red letters represent amino acid substitutions that reduce the reactivity to at least half of the value obtained with the original peptide. The blue squares show the conserved amino acids critical for antibody binding with the sera from mice infected with different T. cruzi strains.
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pntd-0002524-g003: Mapping of antibody binding sites on the conserved and polymorphic epitopes by Ala-Scan.Reactivity of peptide C6_30_cons against the sera from mice infected with Colombiana (A), Y (B), and CL Brener (C). Reactivity of peptide A6_30_col against the sera from mice infected with the Colombiana clone (D). Reactivity of peptide B2_30_y against the sera from mice infected with the Y strain (E). Reactivity of peptide B9_30_cl against the sera from mice infected with the CL Brener clone (F). 1, original peptide. RI, relative intensity calculated by the ratio between the reactivity of the peptide with a specific amino acid substitution and the original peptide. The dotted line indicates half of the intensity value obtained with the original peptide. The red letters represent amino acid substitutions that reduce the reactivity to at least half of the value obtained with the original peptide. The blue squares show the conserved amino acids critical for antibody binding with the sera from mice infected with different T. cruzi strains.

Mentions: We next analyzed the polymorphisms of the epitopes identified in this study and predicted their reactivity with sera from individuals infected with T. cruzi strains representative of each DTU (TcI to TcVI). To this end, we first subjected the peptide sequences to an AlaScan analysis [44] to identify the amino acid residues critical to antibody binding. We found that the pattern GXXXXMRQNE in the carboxy-terminal region of conserved peptide C6_30_cons is important for the interaction with the antibodies generated in infection caused by the three T. cruzi strains (Figures 3A, B, and C). As for the polymorphic epitopes, the patterns PPXDXSLXXP in peptide A6_30_col (Figure 3D), QPQPXPQXXXQP in B2_30_y (Figure 3E), and DEXXXXG in B9_30_cl (Figure 3F) are critical for binding with the antibodies generated by Colombiana, Y, and CL Brener infections, respectively.


Identification of strain-specific B-cell epitopes in Trypanosoma cruzi using genome-scale epitope prediction and high-throughput immunoscreening with peptide arrays.

Mendes TA, Reis Cunha JL, de Almeida Lourdes R, Rodrigues Luiz GF, Lemos LD, dos Santos AR, da Câmara AC, Galvão LM, Bern C, Gilman RH, Fujiwara RT, Gazzinelli RT, Bartholomeu DC - PLoS Negl Trop Dis (2013)

Mapping of antibody binding sites on the conserved and polymorphic epitopes by Ala-Scan.Reactivity of peptide C6_30_cons against the sera from mice infected with Colombiana (A), Y (B), and CL Brener (C). Reactivity of peptide A6_30_col against the sera from mice infected with the Colombiana clone (D). Reactivity of peptide B2_30_y against the sera from mice infected with the Y strain (E). Reactivity of peptide B9_30_cl against the sera from mice infected with the CL Brener clone (F). 1, original peptide. RI, relative intensity calculated by the ratio between the reactivity of the peptide with a specific amino acid substitution and the original peptide. The dotted line indicates half of the intensity value obtained with the original peptide. The red letters represent amino acid substitutions that reduce the reactivity to at least half of the value obtained with the original peptide. The blue squares show the conserved amino acids critical for antibody binding with the sera from mice infected with different T. cruzi strains.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814679&req=5

pntd-0002524-g003: Mapping of antibody binding sites on the conserved and polymorphic epitopes by Ala-Scan.Reactivity of peptide C6_30_cons against the sera from mice infected with Colombiana (A), Y (B), and CL Brener (C). Reactivity of peptide A6_30_col against the sera from mice infected with the Colombiana clone (D). Reactivity of peptide B2_30_y against the sera from mice infected with the Y strain (E). Reactivity of peptide B9_30_cl against the sera from mice infected with the CL Brener clone (F). 1, original peptide. RI, relative intensity calculated by the ratio between the reactivity of the peptide with a specific amino acid substitution and the original peptide. The dotted line indicates half of the intensity value obtained with the original peptide. The red letters represent amino acid substitutions that reduce the reactivity to at least half of the value obtained with the original peptide. The blue squares show the conserved amino acids critical for antibody binding with the sera from mice infected with different T. cruzi strains.
Mentions: We next analyzed the polymorphisms of the epitopes identified in this study and predicted their reactivity with sera from individuals infected with T. cruzi strains representative of each DTU (TcI to TcVI). To this end, we first subjected the peptide sequences to an AlaScan analysis [44] to identify the amino acid residues critical to antibody binding. We found that the pattern GXXXXMRQNE in the carboxy-terminal region of conserved peptide C6_30_cons is important for the interaction with the antibodies generated in infection caused by the three T. cruzi strains (Figures 3A, B, and C). As for the polymorphic epitopes, the patterns PPXDXSLXXP in peptide A6_30_col (Figure 3D), QPQPXPQXXXQP in B2_30_y (Figure 3E), and DEXXXXG in B9_30_cl (Figure 3F) are critical for binding with the antibodies generated by Colombiana, Y, and CL Brener infections, respectively.

Bottom Line: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs.Four peptides were tested against a panel of chagasic patients using ELISA.A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.

ABSTRACT

Background: The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA.

Methodology: By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain.

Findings and conclusions: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.

Show MeSH
Related in: MedlinePlus