Fig 1.
Molecular and structural characterization of two SmRho1 isoforms.
(A) The SmRho1 isoforms are proteins of 193 aa. SmRho1.1 and SmRho1.2 contain five GTPase domains named “G boxes” (G1-G5) (in blue) and two functional elements, Switch I and II (boxed in green and yellow, respectively), which can interact with many regulators (GEFs, GAPs and GDIs) and effectors. The insert region (in red) is essential for Rho GTPase functions [37]. The C-terminal part presents a hypervariable domain and a prenylation motif CAAX (C = cysteine residue, A = aliphatic residue, X = terminal aa). (B) Sequence alignment between the S. mansoni Rho1 isoforms, S. mansoni Rho2 (Uniprot G4V9A8) and Rho3 (Uniprot Q8I8A0), S. japonicum Rho1 (Uniprot Q8MUI8), RhoA of S. haematobium (Uniprot A0A094ZFT0), human RhoA (Uniprot P61586) and mouse RhoA (Uniprot Q9QUI0). Sequences were aligned using ClustalW Multiple Alignment (SnapGene). The identical and semi-conserved aa are highlighted in black and gray respectively. Residues not conserved between SmRho1.1 and SmRho1.2 are boxed in orange. SmRho1.1 and SmRho1.2 both share high sequence similarity with HsRhoA (72% et 73% respectively) The secondary structure elements of SmRho1 isoforms and HsRhoA are indicated at the top and below the aligned sequences, respectively with A for α helices, B for β sheets and H for 310 helices. (C) Modeling of the tertiary structure of SmRho1 isoforms and HsRhoA [28]. Shown is a ribbon representation of SmRho1 isoforms and HsRhoA with β-strands (red), α-helices (cyan), and 310-helices (green). (D) Surface representation of proteins structures with Switch I and II (green and yellow, respectively), G boxes (in blue). For SmRho1 isoforms, the CAAX prenylated site is shown in purple and the acetylated lysine in cyan. The aa sequence differences between SmRho1 isoforms and HsRhoA are shown in red.
Fig 2.
SmRho1.1 and SmRho1.2 are orthologous to human RhoA.
Phylogenetic tree of Rho GTPases from vertebrates, platyhelminths (trematodes, cestodes and turbellarians), nematodes and insects, obtained with the ML algorithm. Numbers on internal branches are the bootstrap values. SmRho1 isoforms are circled in red. SmRho1.1 and SmRho1.2 cluster with the human RhoA, B, C clade (in red rectangle). The data generated also suggest that SmRho1.1 and SmRho1.2 are paralogs, which are orthologous to human RhoA, and originate from a recent gene duplication. The schistosome Rho GTPases are indicate with a *.
Fig 3.
Biological processes involving the proteins identified by Mass spectrometry as SmRho1 partners.
(A) Pie chart showing the percentage of involvement of the identified proteins and their biological processes. The processes were defined using the Blast2GO software. Two independent immunoprecipitation experiments were performed and grouped into one graphic display. (B) Venn diagram. Graphic shows common proteins between the two IP experiments (IP1 SmRho1 and IP2 SmRho1). The Venn diagram was established from Venny V2.1 [40].
Fig 4.
SmHDAC8 interacts with SmRho1 in S. mansoni parasites.
Adult worms (A) and schistosomula (B) SmHDAC8 and SmRho1 were immunoprecipitated respectively using an anti-SmHDAC8 and anti-SmRho1 antibodies cross-linked to protein-L agarose beads. The immunoblots were probed with the same antibodies to detect the SmHDAC8 or SmRho1 protein in the input and eluates (labelled as IP SmHDAC8, IP SmRho1 or MOCK). As a negative control, we performed Co-IP (MOCK) with a mouse pre-immune serum alone in each experiment. The whole of IP eluate (60 μL) was loaded on SDS-PAGE. The results presented are from one experiment representative of three carried out.
Fig 5.
The interaction between SmRho1.1 and SmHDAC8 is dependent on the SmRho1.1 C-terminus.
(A) Y2H mating experiments showed that SmHDAC8 interacts specifically with SmRho1.1 protein. AH109 yeasts expressing only Gal4AD (pGADT7) or Gal4AD-fused SmRho1.1 or SmRho1.2 were mated with Y187 yeasts expressing only Gal4DBD (pGBKT7) or Gal4DBDfused SmHDAC8. Diploids were allowed to grow on a minimal SD -Leu/-Trp medium (left panel) and diploids expressing interacting proteins were then selected on SD -Leu/-Trp/-His/Ade medium (right panel). Only yeasts expressing SmHDAC8 and SmRho1.1 grew on the selective medium. The results presented are from one experiment representative of three carried out. (B) SmHDAC8 binds only SmRho1.1 but not SmRho1.2. Co-IP and WB analysis of SmHDAC8 and SmRho1 isoforms expressed in X. laevis oocytes showed an interaction only between SmHDAC8 (Myc-tagged) and SmRho1.1 (HA-tagged). cRNAs encoding HA-tagged SmRho1.1 or SmRho1.2 were co-injected in X. laevis oocytes with cRNA encoding Myc-tagged SmHDAC8. Oocytes were incubated in ND96 medium and lysed. Proteins from soluble extracts were immunoprecipitated by anti-HA or anti-Myc antibodies and analyzed by WB to detect SmHDAC8- Myc (50 kDa) and SmRho1-HA isoforms (22 kDa) with anti-Myc or anti-HA antibodies. Experiments were repeated three times on oocytes from three different females. (C) Schematic structure of SmRho1.1 and SmRho1.2 mutants. Using site-directed mutagenesis, the glutamic acid Glu33 of SmRho1.1 was substituted by a lysine (SmRho1.1 E33K) and the lysine Lys33 of SmRho1.2 by a glutamic acid (SmRho1.2 K33E). SmRho1. 1–143 aa and SmRho1. 1–88 aa proteins are portions of SmRho1.1. produced by site-directed mutagenesis. (D) Co-IP and WB experiments performed in X. laevis oocytes revealed that SmRho1. 1–143 aa and SmRho1. 1–88 aa mutants (HA-tagged) are not able to bind SmHDAC8 (Myc -tagged). cRNAs encoding HA-tagged SmRho1 isoforms, SmRho1.1 mutant or SmRho1.2 mutants were co-injected in X. laevis oocytes with cRNA encoding Myc-tagged SmHDAC8. Oocytes were incubated in ND96 medium and lysed. Proteins from soluble extracts were immunoprecipitated (IP) by anti-HA or anti-Myc antibodies and analyzed by WB to detect SmHDAC8 (50 kDa), SmRho1 isoforms (22 kDa) or SmRho1 mutants (22kDa) with anti-Myc or anti-HA antibodies. SmHDAC8 co-immunoprecipitated only with SmRho1.1 E33K.
Fig 6.
SmDia binds SmRho1.2 but not SmRho1.1.
(A) Y2H mating experiments showed that SmDia-RBD (Rho Binding Domain) interacts specifically with SmRho1.2 protein. AH109 yeasts expressing only Gal4AD (pGADT7) or Gal4AD-fused SmRho1.1 or SmRho1.2 were mated with Y187 yeasts expressing only Gal4DBD (pGBKT7) or Gal4DBD-fused SmDia-RBD. Diploids were allowed to grow on a minimal SD -Leu/-Trp medium and diploids expressing interacting proteins were then selected on medium. Only yeasts expressing SmDia-RBD and SmRho1.2 grew on the SD -Leu/-Trp/His/-Ade selective medium. The results presented are from one experiment representative of three carried out. (B) SmDia-RBD interacts with SmRho1.2. Co-immunoprecipitation and WB analysis of SmDia-RBD and SmRho1 isoforms expressed in X. laevis oocytes showed an interaction only between SmDia-RBD (Myc-tagged) and SmRho1.2 (HA-tagged). cRNAs encoding HA-tagged SmRho1.1 or SmRho1.2 were co-injected in oocytes with cRNA encoding Myc-tagged SmDia-RBD. Oocytes were incubated in ND96 medium and lysed. Proteins from soluble extracts were immunoprecipitated (IP) by anti-HA or anti-Myc antibodies and analyzed by WB to detect SmDia-RBD (6 kDa) and SmRho1 isoforms (22 kDa) with anti-Myc or anti-HA antibodies. Experiments were performed three times on oocytes from three different females.
Fig 7.
Effect of SmHDAC8 inhibition on actin filament of S. mansoni adult worms.
(A) Effect of SmHDAC8 inhibition in male and female adult worms. Freshly perfused adult couples were maintained in culture for 16 hours and incubated with DMSO or with 50 μM of TH65 or 10 μM of TSA, then fixed and stained with phalloidin (green) and DAPI (blue). H2O and DMSO were used as negative controls. As a positive control, schistosomula were treated with a Rho inhibitor I (160 μM). Scale bar represents 20 μm, magnification, x630. Experiments were performed three times on adult worms obtained by three different hepatic portal perfusions of hamsters. (B) Effect of SmHDAC8 transcript knockdown in adult worms. RNA interference was carried out by S. mansoni worms with dsRNA for SmHDAC8, SmRho1 (positive control) or luciferase (negative control) as described in the Methods section. Actin-F was revealed with phalloidin staining and the nuclei were stained with DAPI (blue). Microscopic examination was carried out 5 days after RNAi treatment. Scale bar represents 20 μm, magnification, x630. Experiments were performed three times on adult worms obtained by three different hepatic portal perfusions of hamsters. (C) RT-qPCR results of RNAi treatment with dsRNA for SmHDAC8 (dsSmHDAC8), SmRho1 (dsSmRho1) or luciferase (dsSmLuc) and analyses of relative transcript levels of SmHDAC8, SmRho1.1 or SmRho1.2 in adult worms. SmPSMB7 was used as an internal reference gene (32). The results were analyzed using the 2−ΔΔCT method and experiments were carried out once in duplicate.
Fig 8.
Effect of SmHDAC8 inhibition on actin filaments of S. mansoni schistosomula.
(A) Effect of SmHDAC8 inhibition in schistosomula. Airyscan microscopy images taken of schistosomula treated for 16 hours with a SmHDAC8 selective inhibitor (TH65), at 50 μM and a pan-inhibitor (TSA) at 3 μM. Parasites treated with DMSO or H2O were used as negative controls. As a positive control, schistosomula were treated with a Rho inhibitor I (80 μM). Actin-F was revealed with phalloidin staining. The nuclei were stained with DAPI (blue). Results shown are representative of three independent experiments. Scale bars represent 20 μm, magnification, x630 (top panel). Airyscan images with orthogonal views of treated S. mansoni schistosomula are also shown. Results shown are from one experiment. Scale bar represents 20 μm, magnification, x630 (bottom panel). (B) Effect of SmHDAC8 transcript knockdown in schistosomula. RNA interference was carried out by schistosomula with dsRNA for SmHDAC8, SmRho1 (positive control) or luciferase (negative control) as described in the Methods section. Actin-F was revealed with phalloidin staining and the nuclei were stained with DAPI (Blue). Microscopic examination was carried out 2 days after RNAi treatment. Scale bar represents 20 μm, magnification, x630 (top panel). Airyscan images with orthogonal views of S. mansoni schistosomula are also shown. Results shown are from one experiment. Scale bars represent 20μm, magnification, x630 (bottom panel). (C) RT-qPCR results of RNAi treatment with dsRNA for SmHDAC8 (dsSmHDAC8), SmRho1 dsSmRho1) or luciferase (dsSmLuc) and analyses of relative transcript levels of SmHDAC8, SmRho1.1 or SmRho1.2 in schistosomula. SmPSMB7 was used as an internal reference gene (32). The results were analyzed using the 2−ΔΔCT method and experiments were carried out once in duplicate.
Fig 9.
Model of signaling pathways involving SmHDAC8 and SmRho1 isoforms in cytoskeleton organization based on the human RhoA Signaling Pathway.
A model summarizing the different pathways putatively regulated by the SmRho1 isoforms is shown in Fig 9. In this model, SmRho1.1 and SmRho1.2 would impact on different pathways of cytoskeleton organization via interaction respectively with SmHDAC8 and SmDia. A) Schematic model showing proteins involving in cytoskeletal events in S. mansoni adult worms and schistosomula. We propose the existence of two signaling pathways in the parasite involving the two SmRho1 isoforms. Various extracellular stimuli trigger the activation of RhoGEFs that catalyze the exchange of GDP for GTP to activate SmRho1.1 and SmRho1.2. Activated SmRho1.1 and SmRho1.2 bind to SmHDAC8 and SmDia respectively that transmit Rho mediated signals to downstream target proteins which will regulate actin polymerization. Proteins encircled in grey (RhoGEF and RhoGAP) are absent from the SmRho1 interactome. (B) An overview of SmRho1-interacting proteins.