Suppression of mRNAs encoding CD63 family tetraspanins from the carcinogenic liver fluke Opisthorchis viverrini results in distinct tegument phenotypes

Abstract

The liver fluke Opisthorchis viverrini infects 10 million people in Southeast Asia and causes cholangiocarcinoma (CCA). Fluke secreted and tegumental proteins contribute to the generation of a tumorigenic environment and are targets for drug and vaccine-based control measures. Herein, we identified two tetraspanins belonging to the CD63 family (Ov-TSP-2 and Ov-TSP-3) that are abundantly expressed in the tegument proteome of O. viverrini. Ov-tsp-2 and tsp-3 transcripts were detected in all developmental stages of O. viverrini. Protein fragments corresponding to the large extracellular loop (LEL) of each TSP were produced in recombinant form and antibodies were raised in rabbits. Ov-TSP-2 and TSP-3 were detected in whole worm extracts and excretory/secretory products of O. viverrini and reacted with sera from infected hamsters and humans. Antibodies confirmed localization of Ov-TSP-2 and TSP-3 to the adult fluke tegument. Using RNA interference, Ov-tsp-2 and tsp-3 mRNA expression was significantly suppressed for up to 21 days in vitro. Ultrastructural observation of tsp-2 and tsp-3 dsRNA-treated flukes resulted in phenotypes with increased tegument thickness, increased vacuolation (tsp-2) and reduced electron density (tsp-3). These studies confirm the importance of CD63 family tegument tetraspanins in parasitic flukes and support efforts to target these proteins for vaccine development.

Figure 1

Predicted membrane spanning architecture of Ov-TSP-2 and TSP-3. Schematic illustration of the structure of Ov-TSP-2 (A) and Ov-TSP-3 (B) proteins visualized by Protter. The four membrane spanning domains are highlighted: small extracellular loop (SEL), large extracellular loop (LEL) with three conserved motifs (CCG, -PXSC and GC motifs), a small inner loop (IL) and short cytoplasmic tail are featured. The sequences labeled in colour indicate identity of amino acids between Ov-TSP-2 and Ov-TSP-3.

Figure 2

Neighbor joining phylogenetic tree of Opisthorchis viverrini tetraspanins and homologs belonging to the CD63 and CD9/81 lineages. Unrooted phylogenetic tree based on complete open reading frames. Ov-TSP-2 and TSP-3 grouped within the same clade as other CD63-like plathyhelminth sequences. The tree was calculated using 1,000 bootstrap samplings and values are shown on each branch.

Figure 3

Recombinant Ov-TSP-2 and TSP-3 were expressed as thioredoxin fusion proteins in E. coli and were immunologically detected by sera from liver fluke infected hamsters and human subjects. The expressed and purified recombinant Ov-TSP-2 (A) and Ov-TSP-3 (B) proteins were separated using SDS-PAGE. SDS-PAGE of E. coli lysate (Lysate), and, purified protein using Ni-NTA affinity (Purif.) are shown. Western blot analysis of recombinant Ov-TSP-2 (rOv-TSP-2) and Ov-TSP-3 proteins (rOv-TSP-3), immunoblot of recombinant proteins probed with pre-immunization hamster serum (NSR); and immunoblot of recombinant proteins probed with rabbit anti-rOv-TSP-2 or rOv-TSP-3 (α-TSP-2 and α-TSP-3). Ov-TSP-2 or Ov-TSP-3 in crude worm protein extracts (worm) and in excretory/secretory products (ES) of O. viverrini were probed with antibodies against rOv-TSP-2 or rOv-TSP-3. Sera from liver fluke infected humans and hamsters recognize rOv-TSP-2 and rOv-TSP-3 (infect human and infect hams, respectively). [In panels A and B, the two lanes at the left of each labeled Lysate and Purif. were cropped (with Adobe Photoshop) from images of the entire gels; specifically lanes Lys and F3 for both panels A and B. Supplementary Fig. S2 presents the full length, intact gels.]

Figure 4

Immunohistochemical detection of Ov-TSP-2 and Ov-TSP-3 in Opisthorchis viverrini from the bile ducts of infected hamsters. Panels A and B: Liver sections of infected hamsters probed with pre-immunization sera showing no immunoreactivity. Panels C and D: Liver sections of infected hamsters probed with rabbit anti Ov-TSP-2 (C) and Ov-TSP-3 (D) sera showed positive staining in the tegument (T) and eggs (E) within the uteri of the flukes and in the biliary epithelium (B) of hamsters adjacent to the flukes but not in the gastrodermis (G) of the parasite.

Figure 5

Expression levels of Ov-tsp-2 and Ov-tsp-3 in different developmental stages of Opisthorchis viverrini. Expression levels of Ov-tsp-2 (A) and Ov-tsp-3 (B) mRNAs relative to Ov-actin were analysed by qRT-PCR. Protein expression levels in the same developmental stages were evaluated by Western blot probed with rabbit anti-Ov-TSP-2 (C) or rabbit anti-Ov-TSP-3 (D); Western blot band intensity was quantified using densitometry. The experiments were performed in biological duplicate and represented as the mean ± SD. Juvenile (Juv), O. viverrini adult worm (Adult), Metacercariae (Meta). Supplementary Fig. S3 provides the full length ethidium bromide stained agarose gels of RT-PCR products, and Supplementary Fig. S4 provides the full length western blot membrane strips.

Figure 6

Suppression of Ov-tsp-2 and Ov-tsp-3 mRNA expression in adult O. viverrini flukes by RNA interference. mRNA expression levels of Ov-tsp-2 (A) and Ov-tsp-3 (B) relative to Ov-actin (mean ± S.E.) in adult worms electroporated with 50 µg dsRNA of Ov-tsp-2 or Ov-tsp-3 or luciferase control followed by soaking in culture media containing 2 µg/ml dsRNA for 21 days. mRNA expression was measured by qRT-PCR. The protein expression levels of Ov-TSP-2 (C) and Ov-TSP-3 (D) in worms after electroporation with dsRNAs were analysed by Western blot and quantified using imageJ. The experiments were performed in biological duplicate and represented as the mean ± SD. Supplementary Fig. S5 provides the full length ethidium bromide stained agarose gels of RT-PCR products, and Supplementary Fig. S6 provides the full length western blot membrane strips.

Figure 7

Ultrastructure of the tegument of Opisthorchis viverrini adult worms treated with Ov-tsp-2 and tsp-3 dsRNAs. O. viverrini adult worms were electroporated with RPMI medium (A) or the mock control firefly luciferase dsRNA (B), Ov-tsp-2 dsRNA (C), and Ov-tsp-3 dsRNA (D), further cultured in RPMI medium containing 2 µg dsRNA for 3 days, and analysed using transmission electron microscopy (magnification 40,000x). The tegument (T) of worms treated with Ov-tsp-2 dsRNA was thicker than controls (P < 0.0001) (Fig. 7C,E) and displayed extensive vacuolation (P < 0.0001) (Fig. 7C,F; red arrows).The tegument of Ov-tsp-3 dsRNA-treated worms was also thicker (P < 0.0001) (Fig. 7D,E) and less electron dense when compared to controls (Fig. 7D,F). The lines indicate the width of fluke tegument that was used for measurement of tegument thickness.