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Repertoire, genealogy and genomic organization of cruzipain and homologous genes in Trypanosoma cruzi, T. cruzi-like and other trypanosome species.

Lima L, Ortiz PA, da Silva FM, Alves JM, Serrano MG, Cortez AP, Alfieri SC, Buck GA, Teixeira MM - PLoS ONE (2012)

Bottom Line: In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species.Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs.Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitologia, ICB, Universidade de São Paulo, São Paulo, São Paulo, Brasil.

ABSTRACT
Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.

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Comparative expression analysis of cruzipain and homologues in T. cruzi, T. cruzi marinkellei, T. dionisii and T. rangeli.(A) Northern blotting analysis of cruzipain transcripts from T. cruzi (Y) and cross-hybridization using the probe consisting of PCR-amplified catalytic domains of T. cruzi Y cruzipain labeled with 32P T. cruzi Y (TcY-CatL probe); agarose gel of RNA used for this analysis was stained with ethidium bromide (EtBr). (B) CATL proteolytic activities detected in epimastigote lysates of T. cruzi G, T. cruzi marinkellei, T. dionisii and T. rangeli. Activity banding profiles detected in gelatin gels, pH 5.0 and 5 mM DTT were inhibited in gel incubated with 10 µM E-64.
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pone-0038385-g006: Comparative expression analysis of cruzipain and homologues in T. cruzi, T. cruzi marinkellei, T. dionisii and T. rangeli.(A) Northern blotting analysis of cruzipain transcripts from T. cruzi (Y) and cross-hybridization using the probe consisting of PCR-amplified catalytic domains of T. cruzi Y cruzipain labeled with 32P T. cruzi Y (TcY-CatL probe); agarose gel of RNA used for this analysis was stained with ethidium bromide (EtBr). (B) CATL proteolytic activities detected in epimastigote lysates of T. cruzi G, T. cruzi marinkellei, T. dionisii and T. rangeli. Activity banding profiles detected in gelatin gels, pH 5.0 and 5 mM DTT were inhibited in gel incubated with 10 µM E-64.

Mentions: To assess the expression of cruzipain and homologous enzymes from virulent and non-virulent and human-infective or bat-restricted trypanosome species, developing or not within cells, total RNA from epimastigotes of Schizotrypanum species (T. cruzi Y, T. c. marinkellei and T. dionisii) and T. rangeli was compared by northern-blot hybridization using T. cruzi Y derived catalytic domain cruzipain probe that strongly cross-hybridized with RNA from other T. cruzi strains (G, CL and JJ) and from all other trypanosome species using low stringent conditions (data not shown). However, hybridization signals using more stringent conditions correlated very well with sequence identity displaying high cross-hybridization signal with transcripts of T. cruzi, moderate hybridization with T. c. marinkellei, weak hybridization signal with transcripts of T. dionisii and lack of hybrization with T. rangeli isolates. This analysis allowed estimation of transcripts of similar size (∼1.9 Kb) for all the Schizotrypanum species (Fig. 6). In agreement with the differential cross-hybridization among the transcripts of different species, we previously demonstrated that a probe consisting of T. rangeli catalytic domain of CATL (rangelipain) strongly hybridized with T. rangeli while cross-hybridization with Schizotrypanum species was very weak [54].


Repertoire, genealogy and genomic organization of cruzipain and homologous genes in Trypanosoma cruzi, T. cruzi-like and other trypanosome species.

Lima L, Ortiz PA, da Silva FM, Alves JM, Serrano MG, Cortez AP, Alfieri SC, Buck GA, Teixeira MM - PLoS ONE (2012)

Comparative expression analysis of cruzipain and homologues in T. cruzi, T. cruzi marinkellei, T. dionisii and T. rangeli.(A) Northern blotting analysis of cruzipain transcripts from T. cruzi (Y) and cross-hybridization using the probe consisting of PCR-amplified catalytic domains of T. cruzi Y cruzipain labeled with 32P T. cruzi Y (TcY-CatL probe); agarose gel of RNA used for this analysis was stained with ethidium bromide (EtBr). (B) CATL proteolytic activities detected in epimastigote lysates of T. cruzi G, T. cruzi marinkellei, T. dionisii and T. rangeli. Activity banding profiles detected in gelatin gels, pH 5.0 and 5 mM DTT were inhibited in gel incubated with 10 µM E-64.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369871&req=5

pone-0038385-g006: Comparative expression analysis of cruzipain and homologues in T. cruzi, T. cruzi marinkellei, T. dionisii and T. rangeli.(A) Northern blotting analysis of cruzipain transcripts from T. cruzi (Y) and cross-hybridization using the probe consisting of PCR-amplified catalytic domains of T. cruzi Y cruzipain labeled with 32P T. cruzi Y (TcY-CatL probe); agarose gel of RNA used for this analysis was stained with ethidium bromide (EtBr). (B) CATL proteolytic activities detected in epimastigote lysates of T. cruzi G, T. cruzi marinkellei, T. dionisii and T. rangeli. Activity banding profiles detected in gelatin gels, pH 5.0 and 5 mM DTT were inhibited in gel incubated with 10 µM E-64.
Mentions: To assess the expression of cruzipain and homologous enzymes from virulent and non-virulent and human-infective or bat-restricted trypanosome species, developing or not within cells, total RNA from epimastigotes of Schizotrypanum species (T. cruzi Y, T. c. marinkellei and T. dionisii) and T. rangeli was compared by northern-blot hybridization using T. cruzi Y derived catalytic domain cruzipain probe that strongly cross-hybridized with RNA from other T. cruzi strains (G, CL and JJ) and from all other trypanosome species using low stringent conditions (data not shown). However, hybridization signals using more stringent conditions correlated very well with sequence identity displaying high cross-hybridization signal with transcripts of T. cruzi, moderate hybridization with T. c. marinkellei, weak hybridization signal with transcripts of T. dionisii and lack of hybrization with T. rangeli isolates. This analysis allowed estimation of transcripts of similar size (∼1.9 Kb) for all the Schizotrypanum species (Fig. 6). In agreement with the differential cross-hybridization among the transcripts of different species, we previously demonstrated that a probe consisting of T. rangeli catalytic domain of CATL (rangelipain) strongly hybridized with T. rangeli while cross-hybridization with Schizotrypanum species was very weak [54].

Bottom Line: In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species.Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs.Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitologia, ICB, Universidade de São Paulo, São Paulo, São Paulo, Brasil.

ABSTRACT
Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.

Show MeSH
Related in: MedlinePlus