Proteomic characterization of larval and adult developmental stages in Echinococcus granulosus reveals novel insight into host–parasite interactions

Cui, Shu-Jian; Xu, Leilei; Zhang, Ting; Xu, Ming; Yao, Jun; Fang, Changming; Feng, Zijian; Yang, Pengyuan; Hu, Wei; Liu, Feng

doi:10.1016/j.jprot.2013.04.013

Cited by 83 publications

(70 citation statements)

References 60 publications

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“…EgAgB3 was the most abundant, followed by EgAgB1 with a moderate gene expression level and EgAgB5 displaying a low gene expression level in PS samples but a slightly increased expression level in PSP samples. Previous proteomic analyses have detected the presence of the EgAgB1 subunit in protoscoleces [28] as well as in the hydatid fluid and germinal layer [29]. Our results were also consistent with the EgAgB1-5 expression data where CYP-1 was used as a reference gene in E. multilocularis [21].…”

Section: Discussionsupporting

confidence: 91%

“…However, there are very few publications that validate the transcriptomic data. A more comprehensive overview considering the available protein expression data for E. granulosus [28],[29] could help to obtain and understand the correlations between transcription and translation pathways. In our analysis, we focused on the identification of genes that are stably expressed (normalizer or reference genes) in PS and PSP experimental conditions of E. granulosus s.s. (G1) and E. ortleppi .…”

Section: Discussionmentioning

confidence: 99%

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Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

Ferreira

Zaha

2014

PLoS ONE

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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 (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-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-1α and TBP were the most stable genes for both species. Interestingly, βACT-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-1α 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-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α 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.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

Ferreira

Zaha

2014

PLoS ONE

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“…MS database searching was performed as previously described, with some modifications (22). Peak lists were first generated from raw files with ProteoWizard v3.0 at default settings.…”

Section: Methodsmentioning

confidence: 99%

Affinity Purification of the Hepatitis C Virus Replicase Identifies Valosin-Containing Protein, a Member of the ATPases Associated with Diverse Cellular Activities Family, as an Active Virus Replication Modulator

Fang

Zou

et al. 2016

J Virol

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Like almost all of the positive-strand RNA viruses, hepatitis C virus (HCV) induces host intracellular membrane modification to form the membrane-bound viral replication complex (RC), within which viral replicases amplify the viral RNA genome. Despite accumulated information about how HCV co-opts host factors for viral replication, our knowledge of the molecular mechanisms by which viral proteins hijack host factors for replicase assembly has only begun to emerge. Purification of the viral replicase and identification of the replicase-associated host factors to dissect their roles in RC biogenesis will shed light on the molecular mechanisms of RC assembly. To purify the viral replicase in the context of genuine viral replication, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into HCV NS5A and NS5B. After solubilizing the replicon cells, we purified the viral replicase by two-step affinity purification and identified the associated host factors by mass spectrometry. We identified valosin-containing protein (VCP), a member of the ATPases associated with diverse cellular activities (AAA؉ATPase) family, as an active viral replication modulator whose ATPase activity is required for viral replication. A transient replication assay indicated that VCP is involved mainly in viral genome amplification. VCP associated with viral replicase and colocalized with a viral RC marker. Further, in an HCV replicase formation surrogate system, abolishing VCP function resulted in aberrant distribution of HCV NS5A. We propose that HCV may co-opt a host AAA؉ATPase for its replicase assembly. IMPORTANCEAlmost all of the positive-strand RNA viruses share a replication strategy in which viral proteins modify host membranes to form the membrane-associated viral replicase. Viruses hijack host factors to facilitate this energy-unfavorable process. Understanding of this fundamental process is hampered by the challenges of purifying the replicase because of the technical difficulties involved. In this study, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into two replicase components. Using this dual-affinity-tagged replicon system, we purified the viral replicase and identified valosin-containing protein (VCP) AAA؉ATPase as a pivotal viral replicase-associated host factor that is required for viral genome replication. Abolishing VCP function resulted in aberrant viral protein distribution. We propose that HCV hijacks a host AAA؉ATPase for its replicase assembly. Understanding the molecular mechanism of VCP regulates viral replicase assembly may lead to novel antiviral strategies targeting the most conserved viral replication step.T he positive-strand RNA viruses are the largest class of viruses and include many medically and economically important pathogens, including hepatitis C virus (HCV); picornaviruses, which cause hand, foot, and mouth disease; and flaviviruses, such as the West N...

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“…Recent taxonomic studies have suggested E. granulosus in 4 distinct groups, including E. granulosus sensu stricto (G1-G3 genotypes), Echinococcus equinus (G4), Echinococcus ortleppi (G5), and Echinococcus canadensis (G6-G10) [11]. The identification of E. granulosus strains has been carried out in different laboratories using various analytical methods (morphology, physiology, biochemistry, and molecular genetics), and all of them were proved to be useful, particularly when used together [12,13,14,15,16]. The possible susceptibility factors for genetic variation that occur in E. granulosus may influence many different phenotypic features such as the life cycle patterns, development rate, host specificity, geographical distribution, transmission dynamics, infectivity, antigenicity, control of diseases, and chemotherapeutic agents.…”

Section: Introductionmentioning

confidence: 99%

Genetic Diversity ofEchinococcus granulosus in Center of Iran

et al. 2014

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Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.

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Proteomic characterization of larval and adult developmental stages in Echinococcus granulosus reveals novel insight into host–parasite interactions

Cited by 83 publications

References 60 publications

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

Affinity Purification of the Hepatitis C Virus Replicase Identifies Valosin-Containing Protein, a Member of the ATPases Associated with Diverse Cellular Activities Family, as an Active Virus Replication Modulator

Genetic Diversity ofEchinococcus granulosus in Center of Iran

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