Análise comparativa do transcritoma de dois estágios no desenvolvimento do parasito cestódeo Mesocestoides corti

O desenvolvimento em cestódeos abrange mudanças fisiológicas e morfológicas complexas. Com o intuito de ganhar conhecimento nos mecanismos moleculares que regulam este processo, foi realizada uma análise exaustiva da expressão de microRNAs e genes em dois estágios do desenvolvimento (tetratirídeo e verme estrobilado) do cestódeo Mesocestoides corti. Utilizando uma abordagem de sequenciamento de alto rendimento, foi identificada evidencia transcricional para 42 loci de microRNAs, assim como microRNAs diferencialmente expressos ou estágios específicos. Além disso, foi demonstrado que a uridilação é um mecanismo de modificação pós-transcricional diferencial presente nos microRNAs de M. corti. O conjunto completo de microRNAs de M. corti identificado representa 33 famílias únicas, e confirma a notável perda de famílias de microRNAs conservadas dentro do grupo de platelmintos parasitos. O transcritôma de M. corti foi obtido utilizando uma abordagem baseada em sequenciamento de RNA. Foram evidenciados 19,053 transcritos, incluindo isoformas e genes previamente não anotados. Foram identificados um total de 66 transcritos específicos de tetratirídeo e 136 específicos de verme estrobilado, assim como genes diferencialmente expressos (342 e 559 respectivamente), sugerindo regulação de caraterísticas específicas de cada estágio. A analise de expressão diferencial e enriquecimento de termos GO refletem os processos biológicos associados a cada estágio, sendo o tetratirídeo capaz de reproduzirse assexuadamente e migrar através dos tecidos do hospedeiro intermediário, e o verme estrobilado de maturar e reproduzir-se sexuadamente no hospedeiro definitivo. Globalmente, os resultados apresentados fornecem uma plataforma para estudos de novos mecanismos moleculares da regulação gênica póstranscricional baseados em microRNAs em cestódeos, necessários para a elucidação de aspectos do desenvolvimento da complexa biologia destes parasitos. Além disso, este trabalho significativamente contribui ao conhecimento do perfil de expressão gênica de M. corti aumentando o número de transcritos sequenciados identificados através da anotação funcional de vários genes.Cestode development involves complex morphological and physiological changes. To gain knowledge in the molecular pathways that regulate this process, a comprehensive analysis of microRNAs and genes expressed in two developmental stages (tetrathyridium and strobilated worm) of the model cestode Mesocestoides corti was performed. Using a high-throughput sequencing approach, transcriptional evidence of 42 microRNA loci, as well as differentially expressed and stage-specific miRNAs between these developmental stages were found. Moreover, it was shown that uridylation is a differential mechanism of post-transcriptional modification of M. corti microRNAs. The whole set of M. corti microRNAs represent 33 unique miRNA families, and confirm the remarkable loss of conserved miRNA families within platyhelminth parasites. The M. corti transcriptome was obtained through a RNA-seq-based approach. Transcriptional evidence for 19,053 transcripts was found, including isoforms and previously not annotated genes. A total of 66 tetrathyridium and 136 strobilatedspecific transcripts were found, as well as differentially expressed genes (342 and 559, respectively), suggesting regulation of stage-specific features. Differential expression and GO term enrichment analysis reflects the biological processes associated with each stage, the tetrathyridium stage being able to reproduce asexually and actively migrate through the intermediate host tissue; and the strobilated worm that undergoes sexual maduration and reproduction in the definitive host. Overall, the presented results provide a valuable platform to studies aiming to identify and characterize novel miRNA-based molecular mechanisms of post-transcriptional gene regulation in cestodes, necessary for the elucidation of developmental aspects of the complex biology of these parasites. In addition, this work significantly contributes to the knowledge of the gene expression profile of M. corti by increasing the number of sequenced transcripts identified and through functional annotation of several genes

Identification of Thioredoxin Glutathione Reductase Inhibitors That Kill Cestode and Trematode Parasites

Parasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR) is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms) and trematoda (flukes), while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 µM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites

Contribución a la dilucidación de los mecanismos proteolíticos que operan en la digestión intestinal del trematodo parásito Fasciola hepatica

Orientador: Dr. Carlos Carmona, co-orientador Dr. José F. Tor

Análise comparativa do transcritoma de dois estágios no desenvolvimento do parasito cestódeo Mesocestoides corti

O desenvolvimento em cestódeos abrange mudanças fisiológicas e morfológicas complexas. Com o intuito de ganhar conhecimento nos mecanismos moleculares que regulam este processo, foi realizada uma análise exaustiva da expressão de microRNAs e genes em dois estágios do desenvolvimento (tetratirídeo e verme estrobilado) do cestódeo Mesocestoides corti. Utilizando uma abordagem de sequenciamento de alto rendimento, foi identificada evidencia transcricional para 42 loci de microRNAs, assim como microRNAs diferencialmente expressos ou estágios específicos. Além disso, foi demonstrado que a uridilação é um mecanismo de modificação pós-transcricional diferencial presente nos microRNAs de M. corti. O conjunto completo de microRNAs de M. corti identificado representa 33 famílias únicas, e confirma a notável perda de famílias de microRNAs conservadas dentro do grupo de platelmintos parasitos. O transcritôma de M. corti foi obtido utilizando uma abordagem baseada em sequenciamento de RNA. Foram evidenciados 19,053 transcritos, incluindo isoformas e genes previamente não anotados. Foram identificados um total de 66 transcritos específicos de tetratirídeo e 136 específicos de verme estrobilado, assim como genes diferencialmente expressos (342 e 559 respectivamente), sugerindo regulação de caraterísticas específicas de cada estágio. A analise de expressão diferencial e enriquecimento de termos GO refletem os processos biológicos associados a cada estágio, sendo o tetratirídeo capaz de reproduzirse assexuadamente e migrar através dos tecidos do hospedeiro intermediário, e o verme estrobilado de maturar e reproduzir-se sexuadamente no hospedeiro definitivo. Globalmente, os resultados apresentados fornecem uma plataforma para estudos de novos mecanismos moleculares da regulação gênica póstranscricional baseados em microRNAs em cestódeos, necessários para a elucidação de aspectos do desenvolvimento da complexa biologia destes parasitos. Além disso, este trabalho significativamente contribui ao conhecimento do perfil de expressão gênica de M. corti aumentando o número de transcritos sequenciados identificados através da anotação funcional de vários genes.Cestode development involves complex morphological and physiological changes. To gain knowledge in the molecular pathways that regulate this process, a comprehensive analysis of microRNAs and genes expressed in two developmental stages (tetrathyridium and strobilated worm) of the model cestode Mesocestoides corti was performed. Using a high-throughput sequencing approach, transcriptional evidence of 42 microRNA loci, as well as differentially expressed and stage-specific miRNAs between these developmental stages were found. Moreover, it was shown that uridylation is a differential mechanism of post-transcriptional modification of M. corti microRNAs. The whole set of M. corti microRNAs represent 33 unique miRNA families, and confirm the remarkable loss of conserved miRNA families within platyhelminth parasites. The M. corti transcriptome was obtained through a RNA-seq-based approach. Transcriptional evidence for 19,053 transcripts was found, including isoforms and previously not annotated genes. A total of 66 tetrathyridium and 136 strobilatedspecific transcripts were found, as well as differentially expressed genes (342 and 559, respectively), suggesting regulation of stage-specific features. Differential expression and GO term enrichment analysis reflects the biological processes associated with each stage, the tetrathyridium stage being able to reproduce asexually and actively migrate through the intermediate host tissue; and the strobilated worm that undergoes sexual maduration and reproduction in the definitive host. Overall, the presented results provide a valuable platform to studies aiming to identify and characterize novel miRNA-based molecular mechanisms of post-transcriptional gene regulation in cestodes, necessary for the elucidation of developmental aspects of the complex biology of these parasites. In addition, this work significantly contributes to the knowledge of the gene expression profile of M. corti by increasing the number of sequenced transcripts identified and through functional annotation of several genes

Identification and profiling of microRNAs in two developmental stages of the model cestode parasite Mesocestoides corti

MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as metabolism and development. The particular developmental characteristics of cestode parasites highlight the importance of studying miRNA gene regulation in these organisms. Here, we performed a comprehensive analysis of miRNAs in two developmental stages of the model cestode Mesocestoides corti. Using a high-throughput sequencing approach, we found transcriptional evidence of 42 miRNA loci in tetrathyridia larvae and strobilated worms. Tetrathyridium and strobilated worm-specific miRNAs were found, as well as differentialy expressed miRNAs between these developmental stages, suggesting miRNA regulation of stage-specific features. Moreover, it was shown that uridylation is a differential mechanism of post-transcriptional modification of M. corti miRNAs. The whole set of M. corti miRNAs represent 33 unique miRNA families, and confirm the remarkable loss of conserved miRNA families within platyhelminth parasites, reflecting their relatively low morphological complexity and high adaptation to parasitism. Overall, the presented results provide a valuable platform to studies aiming to identify and characterize novel miRNA-based molecular mechanisms of post-transcriptional gene regulation in cestodes, necessary for the elucidation of developmental aspects of the complex biology of these parasites.Fil: Basika, Tatiana. Universidade Federal do Rio Grande do Sul; BrasilFil: Macchiaroli, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Cucher, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Espínola, Sergio Martín. Universidade Federal do Rio Grande do Sul; BrasilFil: Kamenetzky, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Zaha, Arnaldo. Universidade Federal do Rio Grande do Sul; BrasilFil: Rosenzvit, Mara Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Ferreira, Henrique B.. Universidade Federal do Rio Grande do Sul; Brasi

Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity

Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L.

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity