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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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Expression levels of allele-specific genes encoding the identified conserved and polymorphic peptides.(A) Peptide C6_30_cons. (B) Peptide A6_30_col. (C) Peptide B2_30_y. (D) Peptide B9_30_cl. INDEX was calculated by the copy number from the mRNA level of each gene normalized by the GAPDH mRNA level. C-, negative control. T, trypomastigote. A, amastigote. E, epimastigote. The alleles were classified based on the annotation of the hybrid CL Brener genome.
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pntd-0002524-g005: Expression levels of allele-specific genes encoding the identified conserved and polymorphic peptides.(A) Peptide C6_30_cons. (B) Peptide A6_30_col. (C) Peptide B2_30_y. (D) Peptide B9_30_cl. INDEX was calculated by the copy number from the mRNA level of each gene normalized by the GAPDH mRNA level. C-, negative control. T, trypomastigote. A, amastigote. E, epimastigote. The alleles were classified based on the annotation of the hybrid CL Brener genome.

Mentions: Although all peptides are derived from the CL Brener genome, the sera from mice infected with this strain had lower antibody affinities for the A6_30_col and B2_30_y peptides than did the sera from mice infected with the Colombiana or Y strain (Figure 2). Because CL Brener is a hybrid strain [34], [42], [45], the polymorphic epitopes encoded by its pairs of alleles may have distinct expression levels that could explain the differences in their reactivity. To investigate this further, we designed allele-specific primers for the genes that encode the epitopes to evaluate their expression levels in the trypomastigote and amastigote forms, the parasite stages found in mammalian hosts (Figure 5). As expected based on the T. cruzi phylogeny [46], Y expressed only the Esmo-like variants, and Colombiana expressed only the Non-Esmo variants, except for the B9_30 transcript. CL Brener expressed both alleles of all genes, except for the B9_30 transcript. The conserved peptide was expressed by both the Esmo and Non-Esmo haplotypes of CL Brener (Figure 5A). The polymorphic Non-Esmo peptide A6_30_col was expressed by the Colombiana and CL Brener strains (Figure 5B), and CL Brener also expressed the Esmo-like allele for this peptide. The opposite profile was observed for the polymorphic Esmo B2_30_y peptide, whereby only the Y and CL Brener strains expressed the Esmo-like allele and CL Brener also expressed the Non-Esmo allele of this peptide (Figure 5C). CL Brener only expressed the Esmo-like variant of the B9_30_CL epitope, and its level of expression was approximately 5 times higher than in the Y strain (Figure 5D).


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)

Expression levels of allele-specific genes encoding the identified conserved and polymorphic peptides.(A) Peptide C6_30_cons. (B) Peptide A6_30_col. (C) Peptide B2_30_y. (D) Peptide B9_30_cl. INDEX was calculated by the copy number from the mRNA level of each gene normalized by the GAPDH mRNA level. C-, negative control. T, trypomastigote. A, amastigote. E, epimastigote. The alleles were classified based on the annotation of the hybrid CL Brener genome.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002524-g005: Expression levels of allele-specific genes encoding the identified conserved and polymorphic peptides.(A) Peptide C6_30_cons. (B) Peptide A6_30_col. (C) Peptide B2_30_y. (D) Peptide B9_30_cl. INDEX was calculated by the copy number from the mRNA level of each gene normalized by the GAPDH mRNA level. C-, negative control. T, trypomastigote. A, amastigote. E, epimastigote. The alleles were classified based on the annotation of the hybrid CL Brener genome.
Mentions: Although all peptides are derived from the CL Brener genome, the sera from mice infected with this strain had lower antibody affinities for the A6_30_col and B2_30_y peptides than did the sera from mice infected with the Colombiana or Y strain (Figure 2). Because CL Brener is a hybrid strain [34], [42], [45], the polymorphic epitopes encoded by its pairs of alleles may have distinct expression levels that could explain the differences in their reactivity. To investigate this further, we designed allele-specific primers for the genes that encode the epitopes to evaluate their expression levels in the trypomastigote and amastigote forms, the parasite stages found in mammalian hosts (Figure 5). As expected based on the T. cruzi phylogeny [46], Y expressed only the Esmo-like variants, and Colombiana expressed only the Non-Esmo variants, except for the B9_30 transcript. CL Brener expressed both alleles of all genes, except for the B9_30 transcript. The conserved peptide was expressed by both the Esmo and Non-Esmo haplotypes of CL Brener (Figure 5A). The polymorphic Non-Esmo peptide A6_30_col was expressed by the Colombiana and CL Brener strains (Figure 5B), and CL Brener also expressed the Esmo-like allele for this peptide. The opposite profile was observed for the polymorphic Esmo B2_30_y peptide, whereby only the Y and CL Brener strains expressed the Esmo-like allele and CL Brener also expressed the Non-Esmo allele of this peptide (Figure 5C). CL Brener only expressed the Esmo-like variant of the B9_30_CL epitope, and its level of expression was approximately 5 times higher than in the Y strain (Figure 5D).

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