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Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of Trypanosoma cruzi reveal an undetected level of complexity.

Freitas LM, dos Santos SL, Rodrigues-Luiz GF, Mendes TA, Rodrigues TS, Gazzinelli RT, Teixeira SM, Fujiwara RT, Bartholomeu DC - PLoS ONE (2011)

Bottom Line: We demonstrated that all seven groups represented in the array are antigenic.A highly reactive peptide occurs in sixty TcS proteins including members of two new groups and may contribute to the known cross-reactivity of T. cruzi epitopes during infection.Taken together, our results contribute to a better understanding of the real complexity of the TcS family and open new avenues for investigating novel roles of this family during T. cruzi infection.

View Article: PubMed Central - PubMed

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

ABSTRACT
The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a highly debilitating human pathology that affects millions of people in the Americas. The sequencing of this parasite's genome reveals that trans-sialidase/trans-sialidase-like (TcS), a polymorphic protein family known to be involved in several aspects of T. cruzi biology, is the largest T. cruzi gene family, encoding more than 1,400 genes. Despite the fact that four TcS groups are well characterized and only one of the groups contains active trans-sialidases, all members of the family are annotated in the T. cruzi genome database as trans-sialidase. After performing sequence clustering analysis with all TcS complete genes, we identified four additional groups, demonstrating that the TcS family is even more heterogeneous than previously thought. Interestingly, members of distinct TcS groups show distinctive patterns of chromosome localization. Members of the TcSgroupII, which harbor proteins involved in host cell attachment/invasion, are preferentially located in subtelomeric regions, whereas members of the largest and new TcSgroupV have internal chromosomal locations. Real-time RT-PCR confirms the expression of genes derived from new groups and shows that the pattern of expression is not similar within and between groups. We also performed B-cell epitope prediction on the family and constructed a TcS specific peptide array, which was screened with sera from T. cruzi-infected mice. We demonstrated that all seven groups represented in the array are antigenic. A highly reactive peptide occurs in sixty TcS proteins including members of two new groups and may contribute to the known cross-reactivity of T. cruzi epitopes during infection. Taken together, our results contribute to a better understanding of the real complexity of the TcS family and open new avenues for investigating novel roles of this family during T. cruzi infection.

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Expression profile of TcS genes by qRT-PCR.Relative quantity (Rq) calculations were based on specific standard curves for each TcS gene. Rq values of each cDNA sample (TcS Rq) were normalized with the GAPDH gene (GAPDH Rq), a gene constitutively expressed throughout the parasite life cycle. Alpha-tubulin and amastin were used as controls for genes more expressed in epimastigote and amastigote stages.
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pone-0025914-g005: Expression profile of TcS genes by qRT-PCR.Relative quantity (Rq) calculations were based on specific standard curves for each TcS gene. Rq values of each cDNA sample (TcS Rq) were normalized with the GAPDH gene (GAPDH Rq), a gene constitutively expressed throughout the parasite life cycle. Alpha-tubulin and amastin were used as controls for genes more expressed in epimastigote and amastigote stages.

Mentions: To characterize the expression profile of TcS genes belonging to distinct groups, we have performed real-time RT-PCR using member-specific primers, designed as described in the material and methods section. The expression of 12 TcS genes derived from six TcS groups was evaluated throughout the three parasite developmental stages using GAPDH mRNA levels, whose expression is constitutive throughout the parasite life cycle, for internal normalization (Figure 5). As a control, we used primers to amplify the cDNAs from the alpha-tubulin and amastin genes, whose mRNA levels we have previously shown to be up-regulated in epimastigotes and amastigotes, respectively [24], [31]. The majority of the TcS transcripts are expressed in trypomastigotes and/or amastigote forms. Interestingly, within a group, the expression profile may be highly variable. For example, the TcS5 gene that belongs to TcSgroupII is highly expressed in trypomastigote forms, whereas the TcS27 from the same group shows a much lower level of expression in the trypomastigote and amastigote forms and is barely detected in epimastigotes. Also, TcS9 and TcS33 from TcSgroupIV are more expressed in trypomastigotes and amastigotes; however, TcS34, which is from the same group, is scarcely expressed in all the development stages. The new groups also display a variable expression profile. A very low level of expression was verified for the two genes analyzed from TcSgroupV in all the developmental stages (Figure 5) as well as in the blood trypomastigotes (data not shown). On the other hand, the gene TcS32 from TcSgroupVII is more expressed in the trypomastigotes. The two members of TcSgroupVIII show a variable expression profile, with TcS24 more expressed in trypomastigotes and TcS25 more expressed in amastigotes.


Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of Trypanosoma cruzi reveal an undetected level of complexity.

Freitas LM, dos Santos SL, Rodrigues-Luiz GF, Mendes TA, Rodrigues TS, Gazzinelli RT, Teixeira SM, Fujiwara RT, Bartholomeu DC - PLoS ONE (2011)

Expression profile of TcS genes by qRT-PCR.Relative quantity (Rq) calculations were based on specific standard curves for each TcS gene. Rq values of each cDNA sample (TcS Rq) were normalized with the GAPDH gene (GAPDH Rq), a gene constitutively expressed throughout the parasite life cycle. Alpha-tubulin and amastin were used as controls for genes more expressed in epimastigote and amastigote stages.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198458&req=5

pone-0025914-g005: Expression profile of TcS genes by qRT-PCR.Relative quantity (Rq) calculations were based on specific standard curves for each TcS gene. Rq values of each cDNA sample (TcS Rq) were normalized with the GAPDH gene (GAPDH Rq), a gene constitutively expressed throughout the parasite life cycle. Alpha-tubulin and amastin were used as controls for genes more expressed in epimastigote and amastigote stages.
Mentions: To characterize the expression profile of TcS genes belonging to distinct groups, we have performed real-time RT-PCR using member-specific primers, designed as described in the material and methods section. The expression of 12 TcS genes derived from six TcS groups was evaluated throughout the three parasite developmental stages using GAPDH mRNA levels, whose expression is constitutive throughout the parasite life cycle, for internal normalization (Figure 5). As a control, we used primers to amplify the cDNAs from the alpha-tubulin and amastin genes, whose mRNA levels we have previously shown to be up-regulated in epimastigotes and amastigotes, respectively [24], [31]. The majority of the TcS transcripts are expressed in trypomastigotes and/or amastigote forms. Interestingly, within a group, the expression profile may be highly variable. For example, the TcS5 gene that belongs to TcSgroupII is highly expressed in trypomastigote forms, whereas the TcS27 from the same group shows a much lower level of expression in the trypomastigote and amastigote forms and is barely detected in epimastigotes. Also, TcS9 and TcS33 from TcSgroupIV are more expressed in trypomastigotes and amastigotes; however, TcS34, which is from the same group, is scarcely expressed in all the development stages. The new groups also display a variable expression profile. A very low level of expression was verified for the two genes analyzed from TcSgroupV in all the developmental stages (Figure 5) as well as in the blood trypomastigotes (data not shown). On the other hand, the gene TcS32 from TcSgroupVII is more expressed in the trypomastigotes. The two members of TcSgroupVIII show a variable expression profile, with TcS24 more expressed in trypomastigotes and TcS25 more expressed in amastigotes.

Bottom Line: We demonstrated that all seven groups represented in the array are antigenic.A highly reactive peptide occurs in sixty TcS proteins including members of two new groups and may contribute to the known cross-reactivity of T. cruzi epitopes during infection.Taken together, our results contribute to a better understanding of the real complexity of the TcS family and open new avenues for investigating novel roles of this family during T. cruzi infection.

View Article: PubMed Central - PubMed

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

ABSTRACT
The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a highly debilitating human pathology that affects millions of people in the Americas. The sequencing of this parasite's genome reveals that trans-sialidase/trans-sialidase-like (TcS), a polymorphic protein family known to be involved in several aspects of T. cruzi biology, is the largest T. cruzi gene family, encoding more than 1,400 genes. Despite the fact that four TcS groups are well characterized and only one of the groups contains active trans-sialidases, all members of the family are annotated in the T. cruzi genome database as trans-sialidase. After performing sequence clustering analysis with all TcS complete genes, we identified four additional groups, demonstrating that the TcS family is even more heterogeneous than previously thought. Interestingly, members of distinct TcS groups show distinctive patterns of chromosome localization. Members of the TcSgroupII, which harbor proteins involved in host cell attachment/invasion, are preferentially located in subtelomeric regions, whereas members of the largest and new TcSgroupV have internal chromosomal locations. Real-time RT-PCR confirms the expression of genes derived from new groups and shows that the pattern of expression is not similar within and between groups. We also performed B-cell epitope prediction on the family and constructed a TcS specific peptide array, which was screened with sera from T. cruzi-infected mice. We demonstrated that all seven groups represented in the array are antigenic. A highly reactive peptide occurs in sixty TcS proteins including members of two new groups and may contribute to the known cross-reactivity of T. cruzi epitopes during infection. Taken together, our results contribute to a better understanding of the real complexity of the TcS family and open new avenues for investigating novel roles of this family during T. cruzi infection.

Show MeSH
Related in: MedlinePlus