Fig 1.
Homology trees and multiple sequence alignment of cathepsin L protease sequences.
(A) Phylogenetic tree constructed with amino acid sequences of Cathepsin L proteases from two schistosome species and two host species. The scale of 0.1 is shown below the tree. The frequency for each sequence is shown in bracket before the names. (B) Multiple sequence alignment of SjCLs. Dark Blue, violet, light blue and white indicated 100%, ≥ 75%, ≥ 50% and 0% identity, respectively. Type I-29 protease inhibitor is underlined in black. ERFNIN and GNFD motifs present in the propeptides are overlined with amino acid residues. Peptidase_C1 domain is underlined in red. The catalytic triad residues (C, H and N) are overlined with amino acid residues highlighted in red. Six cysteines forming three putative disulfide bonds that are present the catalytic domain are marked by red asterisk. Putative glycosylation sites are highlighted with yellow boxes. CL: cathepsin L, SjCL: Schistosoma japonicum CL, SmCL: Schistosoma mansoni CL, hCL: Homo sapiens CL, mCL: Mus musculus CL.
Fig 2.
Detection of recombinant protein of SjCLs by SDS-PAGE and western blot.
Recombinant proteins of SjCL1-5 were resolved in 12% SDS-PAGE and stained with Coomassie brilliant blue (A), and analyzed by western blot using a mouse monoclonal antibody against the His-tag (B), or a mixture of serum samples (in equal volume) from 6 S. japonicum-infected BALB/c mice (C) and 5 S. japonicum-infected rabbits (D) collected at 42 dpi, respectively. Lane M: Marker, Lane 1: SjCL1, Lane 2: SjCL2, Lane 3: SjCL3, Lane 4: SjCL4, Lane 5: SjCL5.
Fig 3.
Evaluation of the immunoprotective effects of SjCLs in a murine model.
BALB/c mice (n = 10 per group) were immunized with recombinant SjCL1, SjCL2, SjCL3, SjCL4, SjCL5, respectively, or with PBS as control. Following the final immunization, the mice were challenged with cercariae. The blank group (n = 10) consists of mice immunized with PBS but without cercariae challenge. The adult worm burden (A) and liver egg burden (B) were assessed at 42 dpi. for the SjCL1-, SjCL2-, SjCL3-, SjCL4-, SjCL5-immunized groups and PBS control group. The weight of the bodies (C), livers (D) and spleens (E) were measured for all the groups at 42 dpi. Data are presented as mean ± SD. Comparisons were performed between diffident groups with the PBS group, *, p < 0.05; **, p < 0.01; ***, p < 0.0001. The results are representative of two independent experiments.
Fig 4.
Expression of SjCL1 in parasites at different developmental stages.
(A) Relative mRNA expression of SjCL1 in the five developmental stages of S. japonicum was detected by qRT-PCR. Data are presented as mean + standard deviation. ***, ###, and ΔΔΔ indicate the comparisons with SjCL1 expression levels in eggs, hepatic schistosomula, and adult males, respectively with p < 0.001. (B) Expression of SjCL1 and Actin in the five developmental stages of S. japonicum were evaluated by western blot. (C) Cryosections of hepatic schistosomula, male adults (longitudinal sections) and female adults (cross sections) were incubated with rabbit anti-SjCL1 polyclonal antibodies, and male adults was incubated with normal rabbit IgG as control, followed by Alexa Fluor 555 donkey anti-rabbit IgG (green fluorescence). The samples were subsequently counterstained with DAPI (in blue). Representative images from three independent experiments are presented, with a minimum of three worms analyzed for each developmental stage in every experiment. E: eggs, C: cercariae, S: hepatic schistosomula, M: adult males, F: adult females. T: tegument, G: gastrodermis, IL: intestinal lumen.
Fig 5.
SjCL1 is necessary for normal parasite growth in vitro.
(A) Schistosome parasites obtained from infected mice were cultured in vitro for 8 h. The supernatant was collected and analyzed by western blot using anti-SjCL1 polyclonal antibodies. M: markers; Lane 1, RPMI 1640; lane 2, hepatic schistosomula; lane 3, adult males; lane 4, adult females. (B) Representative hepatic schistosomula cultured in vitro with anti-SjCL1 polyclonal antibodies or a rabbit IgG control for 10 days. Scale bar indicates 1.0 mm. (C) Length analysis for the worms cultured under the diffident conditions. Data are presented as mean + SD. *, p < 0.05; **, p < 0.01; ***, p < 0.0001. The results are representative of two independent experiments.