The Formin-Homology Protein SmDia Interacts with the Src Kinase SmTK and the GTPase SmRho1 in the Gonads of Schistosoma mansoni

BACKGROUND:Schistosomiasis (bilharzia) is a parasitic disease of worldwide significance affecting human and animals. As schistosome eggs are responsible for pathogenesis, the understanding of processes controlling gonad development might open new perspectives for intervention. The Src-like tyrosine-kinase SmTK3 of Schistosoma mansoni is expressed in the gonads, and its pharmacological inhibition reduces mitogenic activity and egg production in paired females in vitro. Since Src kinases are important signal transduction proteins it is of interest to unravel the signaling cascades SmTK3 is involved in to understand its cellular role in the gonads. METHODOLOGY AND RESULTS:Towards this end we established and screened a yeast two-hybrid (Y2H) cDNA library of adult S. mansoni with a bait construct encoding the SH3 (src homology) domain and unique site of SmTK3. Among the binding partners found was a diaphanous homolog (SmDia), which was characterized further. SmDia is a single-copy gene transcribed throughout development with a bias towards male transcription. Its deduced amino acid sequence reveals all diaphanous-characteristic functional domains. Binding studies with truncated SmDia clones identified SmTK3 interaction sites demonstrating that maximal binding efficiency depends on the N-terminal part of the FH1 (formin homology) domain and the inter-domain region of SmDia located upstream of FH1 in combination with the unique site and the SH3 domain of SmTK3, respectively. SmDia also directly interacted with the GTPase SmRho1 of S. mansoni. In situ hybridization experiments finally demonstrated that SmDia, SmRho1, and SmTK3 are transcribed in the gonads of both genders. CONCLUSION:These data provide first evidence for the existence of two cooperating pathways involving Rho and Src that bridge at SmDia probably organizing cytoskeletal events in the reproductive organs of a parasite, and beyond that in gonads of eukaryotes. Furthermore, the FH1 and inter domain region of SmDia have been discovered as binding sites for the SH3 and unique site domains of SmTK3, respectively

Gender and stage-specific expression of SmDia compared to PDI.

<p>Semi-quantitative RT-PCR analysis (A), densitometric quantification (B), and real-time PCR analysis (C). RT-PCR and qPCR experiments were performed with total RNA from adult worms (females or males) and free-living larval stages (miracidia and cercariae). A: Using SmDia gene-specific primers (SIV5′/SIV3′), RT-PCR products of the expected size of 0.6 kb were obtained from each template, males (Ma), females (Fe), cercariae (Ce), or miracidia (Mi). For control, RNA from mixed worm pools (mixed-sex adult worms and larvae) was used without initial cDNA synthesis (RT⁻). As an additional negative control, PCR was done without any template (H₂O). In the reactions for quantification, primers against the constitutively transcribed protein disulfide-isomerase gene (PDI, 33) were used. B: Result of the densitometric quantification using the PDI RT-PCR products for normalization (n = 1). C: qPCR results, again taking PDI as reference (n = 3).</p

Contribution of the unique site on the binding of SmTK3 to different SmDia deletion constructs.

<p>A comparative β-Gal liquid assay showing the relative binding strength of 8 partial, N-terminally shortened SmDia clones of different length found in the Y2H library (S439-S440; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006998#pone-0006998-t001" target="_blank">table 1</a>), and two further deletion constructs generated by <i>in vitro</i> mutagenesis (ID1 and ID2) with the TK3-SH3- and TK3-US-SH3 bait-constructs of SmTK3 (n = 6). Indicated are the individual N-terminal end positions (arrows) of the different SmDia clones relative to the SmDia amino acid sequence (bottom; amino acid positions are given). Furthermore, the relative position of the PxxP core motif, the inter-domain region (ID) α-helix as predicted by computer analyses, as well as the 4 α-helices (17–20) within the DIM domain are specified. Light grey bars: interactions of the 10 SmDia clones with TK3-SH3; dark grey bars: interactions of the 10 SmDia clones with TK3-US-SH3.</p

Tissue-specific transcription of SmDia and SmRho1 in adult <i>S. mansoni.</i>

<p><i> In situ</i> hybridization experiments to localize SmDia (A) and SmRho1 (B) using DIG-labeled antisense- and sense-RNA probes of SmDia (fragment S4, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006998#pone-0006998-g003" target="_blank">Fig. 3</a>), or SmRho1 (nearly the complete cDNA, 564 bp). A: SmDia transcripts were detected in the cytoplasm of vitelline cells (v) and in the oocytes within the ovary (o) of the female, in the testes (t) of the male, and in the parenchyma (p) and the subtegumental (s) regions of both genders. Within the ovary signal intensity was stronger in mature oocytes (mo) compared to immature oocytes (io). No signal was found in the gastrodermis (ga) or in the tegument (te) of both genders. As negative control sense transcripts of SmDia (fragment S4) were used, which provided no signal. c, gynaecophoric canal; h, head sucker; g, gut. B: SmRho1 transcripts were also detected in the cytoplasm of vitelline cells and oocytes of the female, in the testes of the male, and in parenchyma and subtegumental regions of both genders. SmRho1 sense transcripts provided no signal (control).</p

Northern-blot analyses of SmDia transcripts.

<p>Northern-blot analyses with RNA (left side) of adult schistosomes (mixed-sex). As probe for the filter, a radioactively labeled part of SmDia (fragments S3, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006998#pone-0006998-g004" target="_blank">Fig. 4A</a>) was used (right side). Sizes (kb) are indicated. M = RNA molecular weight marker.</p

SmDia/SmRho1-interaction in yeast.

<p>Qualitative (A, B) and quantitative (C) Y2H analyses of SmDia-SmRho1 interaction. As bait, the RBD domain of SmDia was cloned into the bait vector pGBKT7 in fusion with the Gal4 DNA-binding domain (pGBKT7-DiaRBD). As prey, a mutated form of SmRho1 was generated lacking the CAAX-box at its C-terminal end and cloned into the pACT2 prey vector. Subsequently, this construct was employed to generate two variants containing point mutations at positions 15 and 64 (G15V or Q64L), respectively. A: Yeast cells were transformed with the pGBKT7-DiaRBD and the SmRho1 variants RhoWt (wildtype; 1), RhoG15V (2), or RhoQ64L (3) in pACT2. Transformed yeast cells were selected for the presence of the plasmids and for reporter gene expression (Trp⁻/Leu⁻/Ade⁻/His⁻). B: Control, the same transformed yeast cells grew on media selective for the presence of the plasmids (Trp⁻/Leu⁻). C: The β-Gal liquid assays (n = 2) demonstrated a reduced binding affinity for SmRho1 wildtype compared to the mutant forms RhoG15V or RhoQ64L, as indicated in A.</p

Schematic structure of SmDia and multi-alignment of different Dia homologs.

<p>A: Diagram of the structure of SmDia, which consists of an N-terminal Rho GTPase binding-domain (RBD), an armadillo repeat/dimerization region (ARR/DIM; formerly called FH3 domain), the inter-domain (ID) region, the forming-homology domains 1–2 (FH-domains 1–2), and a diaphanous autoregulatory domain (D) at the C-terminus. Above the diagram, nucleotide positions are indicated. Within the DIM region there are 4 α-helices (17–20). The predicted α-helix (α) within the ID region is depicted. At the 5′-part of the FH1 domain a PxxP motif occurs, which is necessary for binding to the SH3 domain of Src kinases. Below the diagram, PCR primers and their positions are indicated (g5′, SIV5′, SIV3′, SIII5′, SIII3′) and the probes (S4, S3) used for Southern-, or Northern-blot analyses (see text for details). B: Multialignment of the SmDia amino acid sequence (Dia_Sm; FN179407) with diaphanous sequences from mouse (Diap3_Mm; NP_062644), human (DIAPH3_Hs; AAW78862), and <i>Drosophila</i> (Dia_Dm; AAA67715). Boxed grey areas indicate domains characteristic for diaphanous proteins such as the terminal Rho GTPase binding-domain (RBD), armadillo repeat region (ARR), the dimerization region (DIM), the forming-homology domains 1 (FH1) and 2 (FH2), and the diaphanous autoregulatory domain (DAD). In addition, the 4 α-helices within the DIM domain (α17–α20), the predicted α-helix within the ID region (sinuous line). Furthermore, the start points of the partial, N-terminally shortened clones (S439–S440) identified during the Y2H screening as well as the deletions constructs generated by <i>in vitro</i> mutagenesis (ID1 and ID2) are shown. These clones were used for SmDia/SmTK3 binding-studies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006998#pone-0006998-g007" target="_blank">Fig. 7</a>). The small box within the FH1 domain of SmDia (Dia_Sm) indicates the putative PxxP-motif region.</p

Model of signaling pathways involving Rho, Src, and Dia in cytoskeleton organization.

<p>Based on the existing model of Tominaga et al. (2000) and Grosse et al. (2003) (21, 39), we suggest the existence of a minimum of two conserved cooperative signal-transduction pathways in <i>S. mansoni</i> involving the small GTPase SmRho1 and the Src kinase SmTK3 that bridge at SmDia to organize cytoskeletal events in the reproductive organs of males and females (figure modified from 39).</p

Identified downstream binding partners of SmTK3.

<p>Clones identified by Y2H analyses using the SmTK3 baits TK3-SH3 or TK3-US-SH3. The appropriate clone numbers are given as well as their insert sizes (bp), and the levels of homology of their sequences (E-values). * = fully sequenced clones.</p>*<p>completely sequenced clones.</p

Comparative liquid β-Gal assays to determine the relative binding strengths of potential SmTK3 interaction partners.

<p>Comparative β-Gal liquid assays were performed to determine the relative binding strength of the identified interaction partners (n = 6). A representative member of each clone group was re-transformed in yeast cells (AH109) together with TK3-SH3 (light grey) or TK3-US-SH3 (dark grey). The tested clones were (from left to right): diaphanous (SmDia), the eukaryotic translation initiation factor (eIF4γ2b), the BAF60 subunit of the SWI/SNF complex (SWI/SNF-BAF60), the YME1-like metalloprotease (YME1-homologue), the mRNA (guanine-7) methyl transferase (RNA methyl TF), the quinolinate phosphoribosyl-transferase (phosphoribosyl TF), the SH3 binding protein (SH3 domain BP), the Smad protein (Smad2/3), and pericentrin B. As negative control, yeast cells were transformed with TK3-US-SH3 only (bait only).</p