Validation of Suitable Reference Genes for Expression Normalization in <i>Echinococcus</i> spp. Larval Stages

<div><p>In recent years, a significant amount of sequence data (both genomic and transcriptomic) for <i>Echinococcus</i> 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 <i>Echinococcus granulosus</i> sensu stricto (s.s.) (G1) and <i>Echinococcus ortleppi</i> upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from <i>E. granulosus</i> s.s. (G1) and <i>E. ortleppi</i> metacestode were used. The gene expression stability of eleven candidate reference genes (<i>βTUB</i>, <i>NDUFV2</i>, <i>RPL13</i>, <i>TBP</i>, <i>CYP-1</i>, <i>RPII</i>, <i>EF</i>-<i>1α</i>, <i>βACT-1</i>, <i>GAPDH</i>, <i>ETIF4A-III</i> and <i>MAPK3</i>) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, <i>EF-1α</i> and <i>TBP</i> were the most stable genes for both species. Interestingly, <i>βACT-1</i> (the most commonly used reference gene), and <i>GAPDH</i> and <i>ETIF4A-III</i> (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of <i>EF-1α</i> as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for <i>E. granulosus</i> s.s. and <i>E. ortleppi</i>. To demonstrate its applicability, <i>EF-1α</i> was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same <i>EF-1α</i> reference gene may be used in studies with other <i>Echinococcus</i> 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 <i>Taenia</i> genus.</p></div

Gene expression stability (left) and determination of the optimal number of reference genes (right) by geNorm.

<p>The top charts correspond to <i>E. granulosus</i> s.s. (G1), and the bottom charts correspond to <i>E. ortleppi</i>. The dotted line represents the cut-off of 0.15 proposed by geNorm authors to determine the optimal number of reference genes to be used.</p

Assessment of transcript abundance and comparison with RNA-seq data.

<p>Note the similarity in the boxplot distribution for each gene and the high dispersal values for several genes, such as <i>βTUB</i> and <i>RPII</i>, in both species. The mean Cq value for each gene (including both PS and PSP experimental groups) in <i>E. ortleppi</i> was compared with the published RNA-seq data for <i>E. granulosus</i> spp. protoscolex (FPKM from the Cuffdiff program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102228#pone.0102228-Tsai1" target="_blank">[9]</a>; RPKM from the ERANGE program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102228#pone.0102228-Zheng1" target="_blank">[8]</a>) as shown in the top right. Due to a high standard deviation, the <i>βTUB</i> gene was excluded in the qPCR vs. RNA-seq comparison.</p

Gene expression stability for <i>E. granulosus</i> s.s. (G1) (top) and <i>E. ortleppi</i> (bottom) as assessed by RefFinder.

<p>doi:10.1371/journal.pone.0102228.t003</p

Details of each primer designed for the candidate reference genes and target genes.

a<p>Values correspond to the amplification efficiency average of the samples (both PS and PSP groups) used in the gene expression experiments and obtained with the LinRegPCR software.</p>b<p>Amplification products confirmed by sequencing.</p>c<p>N/C = not calculated.</p

Descriptions of candidate reference genes.

a<p>Databases and accession numbers used in this work.</p>b<p>Accession number obtained from GeneDB database after the <i>E. granulosus</i> genome annotation.</p

Relative mRNA expression of the selected target genes using <i>EF-1α</i> as reference gene.

<p>The constitutive (<i>ELP</i>) and differential (<i>RPs15</i> and <i>RPL14</i>) gene expression between both PS and PSP conditions are shown in (A). Here, the PS treatment of the <i>EgELP</i> gene was used as calibrator to obtain the 2^−ΔΔCT values and, subsequently, the bar graph bar. <i>E. ortleppi</i> was used only for the <i>ELP</i> gene (<i>EoELP</i>) expression analysis, and <i>E. granulosus</i> was used to analyze the <i>ELP</i> gene (<i>EgELP</i>) and all other target genes. Results for the <i>EgAgB1-5</i> genes (B) are consistent with previous works in <i>Echinococcus</i> spp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102228#pone.0102228-Zheng1" target="_blank">[8]</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102228#pone.0102228-Mamuti1" target="_blank">[21]</a>, where <i>EgAgB1</i> and <i>EgAgB3</i> are the most abundant in protoscoleces, and <i>EgAgB5</i> exhibit a very low gene expression. The increase of <i>EgAgB1</i> and the decline of <i>EgAgB2</i>, <i>EgAgB3</i> and <i>EgAgB4</i> genes in the PSP group is comparable to previously described for the immature adult form of <i>E. multilocularis</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102228#pone.0102228-Mamuti1" target="_blank">[21]</a>. The statistical significance between EgAgB subunits in both PS and PSP groups (*) as well as only for PSP (¤) or only for PS (§) are shown in the inset. Here, the PS treatment of the EgAgB3 subunit was used as a calibrator. A significance level of α = 0.05 was considered.</p

Additional file 3: Figure S1. of Evolutionary fates of universal stress protein paralogs in Platyhelminthes

Phylogenetic tree generated using the aLRT-SH method with PhyML. Figure S2. Phylogenetic tree generated using Bayesian Inference with BEAST v1.8.4. Figure S3. EgrG_08736 3D protein modeling. Figure S4. Protein alignment used to generate the phylogenetic trees. Figure S5. Positive selection analysis in Cestoda species. (PPTX 8391 kb

Additional file 2: of Evolutionary fates of universal stress protein paralogs in Platyhelminthes

Protein and upstream sequences. (TXT 117 kb

Antigen B from <i>Echinococcus granulosus</i> enters mammalian cells by endocytic pathways

<div><p>Background</p><p>Cystic hydatid disease is a zoonosis caused by the larval stage (hydatid) of <i>Echinococcus granulosus</i> (Cestoda, Taeniidae). The hydatid develops in the viscera of intermediate host as a unilocular structure filled by the hydatid fluid, which contains parasitic excretory/secretory products. The lipoprotein Antigen B (AgB) is the major component of <i>E</i>. <i>granulosus</i> metacestode hydatid fluid. Functionally, AgB has been implicated in immunomodulation and lipid transport. However, the mechanisms underlying AgB functions are not completely known.</p><p>Methodology/Principal findings</p><p>In this study, we investigated AgB interactions with different mammalian cell types and the pathways involved in its internalization. AgB uptake was observed in four different cell lines, NIH-3T3, A549, J774 and RH. Inhibition of caveolae/raft-mediated endocytosis causes about 50 and 69% decrease in AgB internalization by RH and A549 cells, respectively. Interestingly, AgB colocalized with the raft endocytic marker, but also showed a partial colocalization with the clathrin endocytic marker. Finally, AgB colocalized with an endolysosomal tracker, providing evidence for a possible AgB destination after endocytosis.</p><p>Conclusions/Significance</p><p>The results indicate that caveolae/raft-mediated endocytosis is the main route to AgB internalization, and that a clathrin-mediated entry may also occur at a lower frequency. A possible fate for AgB after endocytosis seems to be the endolysosomal system. Cellular internalization and further access to subcellular compartments could be a requirement for AgB functions as a lipid carrier and/or immunomodulatory molecule, contributing to create a more permissive microenvironment to metacestode development and survival.</p></div