Evolutionary fates of universal stress protein paralogs in Platyhelminthes

Background Universal stress proteins (USPs) are present in all domains of life. Their expression is upregulated in response to a large variety of stress conditions. The functional diversity found in this protein family, paired with the sequence degeneration of the characteristic ATP-binding motif, suggests a complex evolutionary pattern for the paralogous USP-encoding genes. In this work, we investigated the origin, genomic organization, expression patterns and evolutionary history of the USP gene family in species of the phylum Platyhelminthes. Results Our data showed a cluster organization, a lineage-specific distribution, and the presence of several pseudogenes among the USP gene copies identified. The absence of a well conserved -CCAATCA- motif in the promoter region was positively correlated with low or null levels of gene expression, and with amino acid changes within the ligand binding motifs. Despite evidence of the pseudogenization of various USP genes, we detected an important functional divergence at several residues, mostly located near sites that are critical for ligand interaction. Conclusions Our results provide a broad framework for the evolution of the USP gene family, based on the emergence of new paralogs that face very contrasting fates, including pseudogenization, subfunctionalization or neofunctionalization. This framework aims to explain the sequence and functional diversity of this gene family, providing a foundation for future studies in other taxa in which USPs occur

Validação de genes normalizadores em Echinococcus granulosus S.S (G1) e Echinococcus ortleppi para estudos de expressão gênica através de PCR em tempo real

Recentemente, uma quantidade significativa de dados de sequência (tanto genômicas como transcritômicas) para Echinococcus spp. foi publicado, facilitando a análise de genes expressos durante um estágio especifico ou envolvidos no desenvolvimento do parasito. Para realizar uma análise adequada de quantificação da expressão gênica, o uso de genes de referência validados é fortemente recomendado. Portanto, o objetivo deste trabalho foi identificar e validar genes de referência para permitir a normalização confiável da expressão gênica de determinados genes de interesse em Echinococcus granulosus sensu stricto (s.s.) (G1) e Echinococcus ortleppi durante o desenvolvimento inicial da forma pré-adulta do parasito. Protoescólices não tratados (PS) e protoescólices tratados com pepsina (PSP) do metacestódeo de E. granulosus s.s. (G1) e E. ortleppi foram usados para extração de RNA total e análise da expressão gênica. A estabilidade na expressão gênica de onze genes candidatos (TUB, NDUFV2, RPL13, TBP, CYP, RNApol sub2, EF-1α, βACT, GAPDH, ETIF4 e ERK) foi avaliada usando os programas geNorm, NormFinder e RefFinder. Nossos dados de RT-qPCR mostraram uma alta correlação com dados recentemente publicados de RNA-seq. Em relação à estabilidade na expressão, EF-1α e TBP foram os genes mais estáveis para ambas as espécies. Entretanto, βACT (gene comumente utilizado como normalizador), GAPDH e ETIF4 (previamente identificados como genes housekeeping) não tiveram um comportamento estável em nossas condições de ensaio. Desta maneira, propomos o uso de EF-1α como gene de referência para estúdios que envolvam análises de expressão gênica no inicio do desenvolvimento da forma pré-adulta de E. granulosus s.s. (G1) e E. ortleppi. Para demonstrar sua aplicabilidade, EF-1α foi usado como gene normalizador na quantificação relativa de transcritos para genes que codificam as proteínas ribossômicas L10, L14, e S15, e para as subunidades do antígeno B de E. granulosus. O mesmo gene de referência EF-1α poderia ser usado em estudos com outras espécies de Echinococcus sensu lato. Este é o primeiro relato que valida genes de referência adequados para a classe Cestoda, phyllum Platyhelminthes, portanto fornecendo uma base para futuras validações em espécies de cestódeos epidemiologicamente importantes, como aquelas do gênero Taenia

Validação de genes normalizadores em Echinococcus granulosus S.S (G1) e Echinococcus ortleppi para estudos de expressão gênica através de PCR em tempo real

Recentemente, uma quantidade significativa de dados de sequência (tanto genômicas como transcritômicas) para Echinococcus spp. foi publicado, facilitando a análise de genes expressos durante um estágio especifico ou envolvidos no desenvolvimento do parasito. Para realizar uma análise adequada de quantificação da expressão gênica, o uso de genes de referência validados é fortemente recomendado. Portanto, o objetivo deste trabalho foi identificar e validar genes de referência para permitir a normalização confiável da expressão gênica de determinados genes de interesse em Echinococcus granulosus sensu stricto (s.s.) (G1) e Echinococcus ortleppi durante o desenvolvimento inicial da forma pré-adulta do parasito. Protoescólices não tratados (PS) e protoescólices tratados com pepsina (PSP) do metacestódeo de E. granulosus s.s. (G1) e E. ortleppi foram usados para extração de RNA total e análise da expressão gênica. A estabilidade na expressão gênica de onze genes candidatos (TUB, NDUFV2, RPL13, TBP, CYP, RNApol sub2, EF-1α, βACT, GAPDH, ETIF4 e ERK) foi avaliada usando os programas geNorm, NormFinder e RefFinder. Nossos dados de RT-qPCR mostraram uma alta correlação com dados recentemente publicados de RNA-seq. Em relação à estabilidade na expressão, EF-1α e TBP foram os genes mais estáveis para ambas as espécies. Entretanto, βACT (gene comumente utilizado como normalizador), GAPDH e ETIF4 (previamente identificados como genes housekeeping) não tiveram um comportamento estável em nossas condições de ensaio. Desta maneira, propomos o uso de EF-1α como gene de referência para estúdios que envolvam análises de expressão gênica no inicio do desenvolvimento da forma pré-adulta de E. granulosus s.s. (G1) e E. ortleppi. Para demonstrar sua aplicabilidade, EF-1α foi usado como gene normalizador na quantificação relativa de transcritos para genes que codificam as proteínas ribossômicas L10, L14, e S15, e para as subunidades do antígeno B de E. granulosus. O mesmo gene de referência EF-1α poderia ser usado em estudos com outras espécies de Echinococcus sensu lato. Este é o primeiro relato que valida genes de referência adequados para a classe Cestoda, phyllum Platyhelminthes, portanto fornecendo uma base para futuras validações em espécies de cestódeos epidemiologicamente importantes, como aquelas do gênero Taenia

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (bTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1a, bACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1a and TBP were the most stable genes for both species. Interestingly, bACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1a as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1a was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1a reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (bTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1a, bACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1a and TBP were the most stable genes for both species. Interestingly, bACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1a as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1a was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1a reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus

Rapid detection of Echinococcus species by a high-resolution melting (HRM) approach

Background: High-resolution melting (HRM) provides a low-cost, fast and sensitive scanning method that allows the detection of DNA sequence variations in a single step, which makes it appropriate for application in parasite identification and genotyping. The aim of this work was to implement an HRM-PCR assay targeting part of the mitochondrial cox1 gene to achieve an accurate and fast method for Echinococcus spp. differentiation. Findings: For melting analysis, a total of 107 samples from seven species were used in this study. The species analyzed included Echinococcus granulosus (n = 41) and Echinococcus ortleppi (n = 50) from bovine, Echinococcus vogeli (n = 2) from paca, Echinococcus oligarthra (n = 3) from agouti, Echinococcus multilocularis (n = 6) from monkey and Echinococcus canadensis (n = 2) and Taenia hydatigena (n = 3) from pig. DNA extraction was performed, and a 444-bp fragment of the cox1 gene was amplified. Two approaches were used, one based on HRM analysis, and a second using SYBR Green Tm-based. In the HRM analysis, a specific profile for each species was observed. Although some species exhibited almost the same melting temperature (Tm) value, the HRM profiles could be clearly discriminated. The SYBR Green Tm-based analysis showed differences between E. granulosus and E. ortleppi and between E. vogeli and E. oligarthra. Conclusions: In this work, we report the implementation of HRM analysis to differentiate species of the genus Echinococcus using part of the mitochondrial gene cox1. This method may be also potentially applied to identify other species belonging to the Taeniidae family

Rapid detection of Echinococcus species by a high-resolution melting (HRM) approach

Background: High-resolution melting (HRM) provides a low-cost, fast and sensitive scanning method that allows the detection of DNA sequence variations in a single step, which makes it appropriate for application in parasite identification and genotyping. The aim of this work was to implement an HRM-PCR assay targeting part of the mitochondrial cox1 gene to achieve an accurate and fast method for Echinococcus spp. differentiation. Findings: For melting analysis, a total of 107 samples from seven species were used in this study. The species analyzed included Echinococcus granulosus (n = 41) and Echinococcus ortleppi (n = 50) from bovine, Echinococcus vogeli (n = 2) from paca, Echinococcus oligarthra (n = 3) from agouti, Echinococcus multilocularis (n = 6) from monkey and Echinococcus canadensis (n = 2) and Taenia hydatigena (n = 3) from pig. DNA extraction was performed, and a 444-bp fragment of the cox1 gene was amplified. Two approaches were used, one based on HRM analysis, and a second using SYBR Green Tm-based. In the HRM analysis, a specific profile for each species was observed. Although some species exhibited almost the same melting temperature (Tm) value, the HRM profiles could be clearly discriminated. The SYBR Green Tm-based analysis showed differences between E. granulosus and E. ortleppi and between E. vogeli and E. oligarthra. Conclusions: In this work, we report the implementation of HRM analysis to differentiate species of the genus Echinococcus using part of the mitochondrial gene cox1. This method may be also potentially applied to identify other species belonging to the Taeniidae family