Schistosoma mansoni TGF-beta receptor II: role in host ligand-induced regulation of a schistosome target gene

Abstract

Members of transforming growth factor-beta (TGF-beta) superfamily play pivotal roles in development in multicellular organisms. We report the functional characterization of the Schistosoma mansoni type II receptor (SmTbetaRII). Mining of the S. mansoni expressed sequence tag (EST) database identified an EST clone that shows homology to the kinase domain of type II receptors from different species. The amplified EST sequence was used as a probe to isolate a cDNA clone spanning the entire coding region of a type II serine/threonine kinase receptor. The interaction of SmTbetaRII with SmTbetaRI was elucidated and shown to be dependent on TGF-beta ligand binding. Furthermore, in the presence of human TGF-beta1, SmTbetaRII was able to activate SmTbetaRI, which in turn activated SmSmad2 and promoted its interaction with SmSmad4, proving the transfer of the signal from the receptor complex to the Smad proteins. Gynaecophoral canal protein (GCP), whose expression in male worms is limited to the gynaecophoric canal, was identified as a potential TGF-beta target gene in schistosomes. Knocking down the expression of SmTbetaRII using short interfering RNA molecules (siRNA) resulted in a concomitant reduction in the expression of GCP. These data provide evidence for the direct involvement of SmTbetaRII in mediating TGF-beta-induced activation of the TGF-beta target gene, SmGCP, within schistosome parasites. The results also provide additional evidence for a role for the TGF-beta signaling pathway in male-induced female reproductive development.

Figure 1. Genomic Structure of S. mansoni TGF-β Type II Receptor Gene

A schematic representation of the genomic locus of S. mansoni TGF-β receptor II (middle) and the two alternatively spliced transcripts, SmTβRII (top) and SmRK2 (bottom). The sizes of introns are represented in kb and that of exons (represented as cylinders) are in bp. The red exon is unique for SmRK2 and the blue exon is unique for SmTβRII. Block arrows represent the two receptor isoforms, SmRK2 (red) and SmTβRII (blue) with locations of start and stop codons indicated.

Figure 2. Identification of Native TGF-β Receptor II in NP-40 Extract of S. mansoni Adult Worm Pairs

Coomassie blue–stained SDS gel (panel A), and Western blot analysis of NP-40 schistosome extract using an IgG fraction of pre-immune (panel B) and SmTβRII-immunized (panel C) rabbit sera. Arrows 1 and 2 point to 62 and 92 kDa isoforms, respectively.

Figure 3. Whole-Mount S. mansoni Adult Worm Immunofluorescence

S. mansoni adult worms were probed with anti-SmTβRII rabbit IgG and pre-immune rabbit IgG (Figure S2), followed by biotin-conjugated anti-rabbit IgG. Reactive complexes were detected using streptavidin Alexa Fluor 647 conjugate and analyzed with a Bio-Rad MRC1024 confocal laser microscope. Anti-SmTβRII reactivity is shown in a live male worm (♂) (panels A–C) in different laser sections in tubercles (T) (panel B) and gynaecophoric canal (G) (panel C). Specific surface fluorescence is also shown in live female worms (♀) (panels F and I), whereas green fluorescence fields show the non-specific auto-fluorescence in vitellaria (V), oviduct (OvD) (panel E), and ova (Ov) (panels E and H). An acetone-fixed male worm (♂) shows anti-SmTβRII reactivity in the gynaecophoric canal, oral (Os) and ventral suckers (Vs) (panel K), and in esophagus (O) (panel L). Panels A, D, G, and J are phase-contrast fields of the fluorescent fields B and C, E and F, H and I, and K and L, respectively.

Figure 4. Semi-Quantitative RT-PCR Analysis of SmTβRII

Top panel shows agarose gel separation of the PCR products of SmTβRII (middle), SmTβRI (bottom) and the constitutively transcribed control, α-tubulin (top). Lanes are numbered and the respective stages are listed at the bottom of the bar graph. The bar graph representation shows the percentage values of the optical densities in pixels of the PCR bands of SmTβRII (gray bars) and SmTβRI (white bars) compared to the corresponding band of α-tubulin control from the same stage. Values were calculated from three independent PCR amplifications (Error bars represent standard deviation values). Samples included in the assay were the hepato-pancreas regions of the intermediate host; uninfected and 30-d–infected B. glabrata snails, which represent different stages of daughter sporocysts; parasite eggs obtained from the liver of Syrian golden hamsters infected with 5,000 cercariae; cercariae shed and collected from infected snails; and stages representing different time points during schistosome development in mammalian host, obtained by perfusion of infected hamsters for the specified time (15–45-d-old worms). Adult male and female worms, separated after perfusion, were also included in the assay. Samples were subjected to total RNA extraction and cDNA synthesis, followed by PCR amplification using specific primer pairs.

Figure 5. Localization of SmTβRII mRNA Transcripts in Tissue Sections of S. mansoni Adult Worms by FISH

Column A represent phase-contrast fields, Column B represent non-specific autofluorescence fields observed in vitelline lobules (V) using green fluorescence filter (522 nm) and Column C shows specific probe reactivity as represented by far-red fluorescence using 680 nm filter. Row (I) shows sections of a male (♂) and a female (♀) worm probed with the positive control cRNA probe (the antisense strand of eggshell protein P14). Specific fluorescence could be observed in the vitellaria (V) of the female worm. As expected, no specific fluorescence could be observed in male worm sections. Specific reactivity of SmTβRII antisense probe could be seen in vitelline cells (V) and gut epithelial cells (G) in a female worm section (panel IIC) and in subtegumental cells (STC) in a male worm section (panel IIIC). No significant fluorescence could be seen in the negative control reaction using SmTβRII sense cRNA strand (unpublished data).

Figure 6. In Vitro Interaction of Amino- (N-) Terminal Domains of SmTβRI and SmTβRII

Interaction of the in vitro translated non-labeled N- terminal domain of SmTβRII with ³⁵S-labeled N-terminal domain of SmTβRI, in the presence or absence of different TGF-β ligands (panels A and B) or varied amounts of different TGF-β₁ (panel B). Ligand concentrations (in nM) are shown at the top of each lane. Reactions were precipitated using S-protein agarose beads (EMD Biosciences, Novagen). Precipitated products were separated by SDS-PAGE and subjected to autofluorography. In vitro translated products (5% and 20 %) of input radiolabeled products are shown in the left lane of panels A and B, respectively. Background precipitation of ³⁵S-labeled-SmTβRI-N by S-protein agarose beads is shown in lane 2, panel A. The background reactivity was removed in later experiments by pre-clearing ³⁵S-SmTβRI-N by treatment with the S-protein agarose beads (panel B) prior to use in interaction assay. Percentage values of precipitated reactive radiolabeled product of each reaction are shown at the bottom of each lane.

Figure 7. The Transduction of TGF-β Signal to SmSmad2 via Activated SmTβRII/SmTβRI Receptor Complex, In Vitro: Co-immunoprecipitation of SmSmad2-MH2 and SmSmad4

³⁵S-labeled, in vitro translated products of SmSmad2-MH2 (panel A) and SmSmad2-MH2/AAA (panel B) were incubated with SmSmad4 in the presence of SmTβR-I (wt) and SmTβR-II in the presence or absence of TGF-β1 (1.0 nM) or BMP2 (5.0 nM). Radiolabeled, in vitro translated products were co-precipitated with SmSmad4, using anti-SmSmad4-linker IgG and Protein A Sepharose beads (Amersham Biosciences). Background precipitation was removed by treating ³⁵S-labeled in vitro translated products with anti-SmSmad4-linker IgG and Protein A Sepharose beads. The pre-cleared lysates were then used in the above-described reactions. A positive control reaction (lane 3) was included, in which SmSmad2-MH2, or the AAA mutant peptide, were reacted with SmSmad4 in the presence of the active mutant form of type I receptor, SmTβR-I (Q-D). Reactions, which contain either SmSmad2-MH2 or its AAA mutant form with SmSmad4 in the presence of wild-type SmTβRI, represent the negative controls of the assay (lane 4). Immunoprecipitated products were separated by SDS-PAGE and subjected to autofluorography. Lanes are labeled to specify the input components of each reaction. In vitro translated products (20% of input) are shown (lane 1). Percentage values of precipitated reactive radiolabeled product of each reaction are shown at the bottom of each lane.

Figure 8. Semi-Quantitative RT-PCR Analysis of SmGCP mRNA

The bottom panel shows the agarose gel separation of the PCR products of SmGCP (bottom), and the constitutively transcribed control, α-tubulin (top). Panel A: Lanes are numbered and the respective stages are listed at the bottom of the panel. Panel B: Adult worm pairs (42-d-old) were left untreated (lane 1) or treated with human TGF-β1 (1 nM; lane 2) or human BMP2 (5 nM; lane 3). Top of each panel shows a bar graph representation of the relative PCR band intensities (%) of SmGCP compared to that of α-tubulin control. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).

Figure 9. Silencing of TGF-β–Induced Expression of SmGCP by Knocking Down SmTβRII Expression

Semi-quantitative RT-PCR analyses for transcripts of SmGCP as well as various components of schistosomal TGF-β signaling pathways in 35-d-old and 28-d-old old worm pairs, untransformed and transformed with SmTβRII-siRNA, and either left untreated or treated with TGF-β1 (1 nM). The top panel shows the agarose gel separation of the PCR products of SmTβRII (panel B), SmTβRI (panel C), SmGCP (panel D), SmSmad4 (panel E), SmSmad2 (panel F), SmSmad1 (panel G), and the constitutively transcribed control, α-tubulin (panel A). The lanes are labeled to show detailed treatment of each sample. The bar graph representation shows the percentage values of the optical densities in pixels of the PCR bands for each gene compared to the corresponding band of α-tubulin control from the same stage. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).