Correction: Venus Kinase Receptors Control Reproduction in the Platyhelminth Parasite <i>Schistosoma mansoni</i>

Correction: Venus Kinase Receptors Control Reproduction in the Platyhelminth Parasite <i>Schistosoma mansoni</i

Analysis of signaling pathways triggered by SmVKR activation in <i>Xenopus</i> oocytes.

<p>SmVKR1-Myc and SmVKR2-Myc proteins were expressed in <i>Xenopus</i> oocytes for 5 h in ND96 incubation medium with or without their respective ligands (L-Arg 1 µM and Ca²⁺ 1 mM). <b>A</b>-Oocyte lysates were analyzed by Western blot to detect the phosphorylation state of Akt, S6K, ERK2, JNK and p38γ following SmVKR1 and SmVKR2 activation by L-Arg and Ca²⁺ respectively. As a control, SmVKR1-expressing oocytes were stimulated by progesterone (PG), the natural hormonal stimulus for oocyte maturation. Results show that both SmVKR1 and SmVKR2 activate Akt and Erk2 pathways. S6K can be phosphorylated by both receptors, but more importantly by SmVKR2. Only SmVKR1 can trigger JNK activation. The p38γ pathway, is activated in control PG-stimulated oocytes, but not under activation of SmVKR1 or SmVKR2. <b>B</b>-Immunoprecipitation of oocyte extracts by anti-Myc antibodies allowed us to confirm the expression of receptors (WB anti-Myc) and their activation/phosphorylation (WB anti-pY20) in the presence of their respective ligands.</p

Morphological analysis of reproductive organs from worms treated with dsSmvkr1 or dsSmvkr2 RNA.

<p>CLSM images of whole-mount preparations of <i>S. mansoni</i> worm couples stained with carmine red. Worms were treated exactly as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004138#ppat-1004138-g002" target="_blank">Fig. 2</a> with dsSmvkr1 (A, B), dsSmvkr2 (C, D), dsSmvkr1 and dsSmvkr2 (E, F) or control dsLuc (G, H) RNA. The morphology of female (left) and male (right) reproductive organs was analyzed. io: immature oocytes, mo: mature oocytes, ot: ootype, sv: sperm vesicle, t: testes. Scale bar: 20 µm.</p

Ligand determination of SmVKR1 and SmVKR2. Importance of a conserved Ser residue of the VFT domain in amino-acid binding and activation of the receptors.

<p>A) Dose-effect of selected amino acids on the activation of SmVKR1 and potential to induce GVBD in <i>Xenopus</i> oocytes. L-Arg is the most potent activator of SmVKR1 and is active at 1 µM. B) Dose-effect of Ca²⁺ on the capacity of SmVKR2 to induce GVBD. C) Importance of Ser₄₆₆ on the potential of L-Arg to activate SmVKR1. SmVKR1 mutated on its Ser₄₆₆ residue present in the VFT domain requires 10³ fold higher amounts of L-Arg to be activated. The mutation of Ser₄₁₀ in the VFT of SmVKR2 (which can be activated by 1 mM L-Arg in the absence of Ca²⁺) also abolishes its capacity to be activated by L-Arg, confirming the importance of this Ser residue highly conserved in the amino acid binding motif of VFT <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004138#ppat.1004138-Ahier2" target="_blank">[28]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004138#ppat.1004138-Acher1" target="_blank">[33]</a>. All experiments have been repeated three times and the mean percentages of GVBD are indicated. D) Western blot analysis of membrane extracts of oocytes expressing wild-type and mutated SmVKR proteins confirming the expression of the receptors and indicating their level of phosphorylation (activation) following L-Arg binding. SmVKR1S₄₆₆A is not recognized by anti-phosphotyrosine antibodies in the presence of L-Arg 1 µM and SmVKR2S₄₁₀A is no more phosphorylated with L-Arg even added at 10 mM.</p

Efficiency of SmVKR1 and SmVKR2 knock-down by RNA interference in adult worm pairs.

<p>Worm couples were electroporated and incubated for 5 days either with dsSmvkr1 (20 µg) or dsSmvkr2 (20 µg) or with both of them (10 µg each) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004138#s4" target="_blank">Materials and Methods</a>. Control worms were treated in the same conditions with irrelevant dsLuc RNA (20 µg). Levels of <i>Smvkr1</i> (A) and <i>Smvkr2</i> (B) transcripts were determined by quantitative RT-PCR in each worm sample. RNAi treatment with dsSmvkr1 or with dsSmvkr2 results in specific reduction of <i>Smvkr1</i> and <i>Smvkr2</i> transcripts respectively. Treatment with both dsSmvkr1 and dsSmvkr2 affects the expression of both <i>Smvkr1</i> and <i>Smvkr2</i> genes. For graphical representation, the ΔΔCt method was used to evaluate the relative expression of transcripts in interfered parasites compared to control dsLuc-treated parasites. Statistical analysis was performed using the Student's t-test and values are expressed as mean+/− SEM of three determinations (** p≤0.01, *** p≤0.001).</p

SmVKR1 and SmVKR2 expression patterns in adult worms.

<p>A: Quantification of <i>Smvkr1</i> and <i>Smvkr2</i> transcripts in adult female and male worms by quantitative RT-PCR. Tubulin transcripts were used as internal controls for each condition. For graphical representation, values were expressed as relative fold-difference using the ΔΔCt method and the <i>Smvkr1</i> ΔCt value in males as reference. Values are expressed as the mean (+/- SEM) of three determinations. B: Localization in sections of paired adult worms of <i>Smvkr1</i> (a, c, e) and <i>Smvkr2</i> (b, d, f) transcripts by <i>in situ</i> hybridization. <i>Smvkr1</i> transcripts were detected in mature oocytes (a) whereas <i>Smvkr2</i> transcripts were detected in immature oocytes and in the area surrounding the ootype (b). No expression of <i>Smvkr1</i> (c) and <i>Smvkr2</i> (d) was detected in the vitellarium. Sense probes of <i>Smvkr1</i> and <i>Smvkr2</i> were used respectively as controls in e and f. Abbreviations: io: immature oocytes, mo: mature oocytes, ot: ootype, v: vitellarium. Scale bar: 100µm.</p

Comparison of qPCR and microarray results of genes involved in eggshell-formation processes.

<p>Summary of the log₂ratio (treated/control) obtained by qPCR and microarray analyses of genes coding for proteins proven or hypothesised to be involved eggshell-formation processes following treatment of paired female schistosomes with either 300 nM TRIKI, 4.5 µM Herb A, or the combination of both inhibitors. The investigated genes were the eggshell precursor proteins Smp14 and Smp48 as well as a predicted eggshell precursor protein (precursor), the tyrosinase 1 (SmTYR1), and a gene similar to fs800. For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.</p

Comparison of qPCR and microarray results following TRIKI-treatment.

<p>Transcriptional changes from the TRIKI-treatment of adult schistosome females detected in qPCR and microarray data were calculated as log₂ratios (treated/control). The investigated genes were SmTβRI, SmActRIIb, a protein similar to fs800, eggshell precursor, and Na/K-pump as representative for enhanced transcription within the microarray data set (A). The selected genes representing the set of genes with repressed transcription within the microarray data set (B) were calmodulin-4, tetraspanin 18, SmSmad 4, and snurp. For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.</p

Visual model of the inhibitor effects on transcription of genes involved in eggshell-formation.

<p>The inhibition of Src kinases targeted by Herb A and TβRI by TRIKI revealed an influence of both inhibitors on the transcription of genes known to play roles in eggshell formation such as Smp14, Smp48, eggshell precursor protein, SmTYR1 and fs800. Compared to TRIKI treatment, however, a stronger effect on transcription was observed when adult schistosome couples were treated with Herb A. From this we conclude cooperative signal- transduction activities during eggshell-formation with a more dominant role of Src kinases in promoting the expression of involved genes.</p

Comparison of qPCR and microarray results following Herbimycin A-treatment.

<p>Transcriptional changes from the Herb A-treatment of adult schistosome females detected in qPCR and microarray data were calculated as log₂ratios (treated/control). According to the microarray analysis SmActRIIb, calmodulin-4, and hsp70 showed enhanced transcription (A), whereas tetraspanin-1, SmSmad 4, and Smp48 were detected as transcriptionally repressed (B). For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.</p