RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro

Abstract

Background: Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).

Methodology/principal findings: Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200-320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.

Conclusions/significance: In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.

Figure 1. Survival of Fasciola hepatica newly excysted juveniles (NEJs) in Fasciola saline (FS) vs RPMI 1640.

NEJ viability in FS is improved both by regular media changes (−change = without media changes; +change = with media changes), or by maintenance in larger volumes (50 µl vs 1000 µl). Experiments were replicated 3 times, employing 15 worms per treatment. Symbols represent mean±SEM.

Figure 2. Time-dependent transcript knockdown under constant exposure to long double-stranded (ds)RNA.

A, knockdown of virulence gene target transcripts after 72 h exposure to long dsRNA (CTRL, negative control; FABP, fatty acid binding protein; LAP, leucine aminopeptidase; μGST, μ-class glutathione transferase; ωGST, ω-class glutathione transferase; CatB, cathepsin B; CatL, cathepsin L; σGST, σ class glutathione transferase); B–D, time-dependent knockdown of cathepsin B (B), cathepsin L (C), σ-class glutathione transferase (D). Square brackets e.g. [CatB] represent qPCR amplicon. Target ΔΔCt (Y-axes) represents ratio of abundance of target transcript to a reference gene (glyceraldehyde phosphate dehydrogenase, GAPDH), in treated samples, relative to the abundance of those transcripts in untreated samples. Statistical significances are indicated relative to effects of negative control dsRNA (dsCTRL, complementary to neomycin phosphotransferase). dsCTRL treatments were performed in parallel with all experimental treatments. Experiments were repeated ≥4 times, employing 20–30 flukes per replicate. *, P<0.05; **, P<0.01; ***, P<0.001. Symbols represent mean±SEM.

Figure 3. Transcript knockdown following burst exposure to long double-stranded (ds)RNA.

Exposure conditions are indicated in panel A, where newly-excysted juveniles were incubated in 50 ng/µl dsRNA (black bar), 2.5 ng/µl dsRNA (grey bar) or zero/sub-effective dsRNA (clear bar), for time periods as indicated in scale. Media were replaced at 2–3 day intervals for the duration of the experiments. Transcript knockdown was assayed in all cases by quantitative PCR, 72 h after start of soak. Target ΔΔCt (Y-axes) represents ratio of abundance of target transcript (B, cathepsin B; C, cathepsin L; D, sigma-class glutathione transferase) to a reference gene (glyceraldehyde phosphate dehydrogenase, GAPDH) in treated samples, relative to the abundance of those transcripts in untreated samples. E, Silencing of σGST ortholog in Fasciola gigantica, using long dsRNA exposure under Protocol IV conditions. Data expressed as mean±SEM. Statistical significances are indicated relative to effects of negative control dsRNA (dsCTRL, complementary to neomycin phosphotransferase). dsCTRL treatments were performed in parallel with all experimental treatments. Experiments were repeated ≥3 times, employing 20–30 flukes per replicate. *, P<0.05; **, P<0.01; ***, P<0.001. Symbols represent mean±SEM.

Figure 4. Effects of RNA interference on target protein abundance during maintenance in vitro.

Densitometry analysis performed of bands generated by immunoblot analyses of newly-excysted juvenile (NEJ) crude protein extracts, at 9 and 21 days post exposure to long double-stranded (ds)RNA: A, σGST; B, cathepsin L, C, cathepsin B. Each panel includes typical examples of target bands imaged from immunoblots using: (A) sigma glutathione transferase antiserum (25 KDa), and actin antiserum (47 KDa) loading control; (B) cathepsin L antiserum (preproenzyme, 37 KDa; partially-processed intermediate, 30 KDa; mature enzyme, 24.5 KDa), and actin antiserum (47 KDa) loading control; (C) anti-cathepsin B antiserum (preproenzyme, 36 KDa), and anti-FheσGST (sigma glutathione transferase, 25 KDa) loading control. Bar graphs illustrate densitometric analyses of western blot protein bands, where target band density is normalised to loading control band density, and expressed relative to untreated sample (where untreated control = 100%). Statistical significances are indicated relative to effects of negative control dsRNA (dsCTRL, complementary to neomycin phosphotransferase). Untreated and dsCTRL-treated samples were performed in parallel with all experimental treatments; these columns represent means of both time points. Experiments were repeated ≥3 times, employing 50–60 flukes per replicate. *, P<0.05; **, P<0.01; ***, P<0.001. Symbols represent mean±SEM.

Figure 5. Persistence of transcript knockdown during maintenance in vitro.

Following exposure to target long double-stranded (ds)RNA (A, cathepsin B; B, cathepsin L; C, sigma-class glutathione transferase), newly-excysted juveniles (NEJs) were maintained in vitro for up to 25 days. NEJs were collected and assayed by quantitative PCR at 3, 9 and 25 days post dsRNA exposure. Target ΔΔCt (Y-axes) represents ratio of abundance of target transcript to a reference gene (glyceraldehyde phosphate dehydrogenase, GAPDH), in treated samples, relative to the abundance of those transcripts in untreated samples. Statistical significances are indicated relative to effects of negative control dsRNA (dsCTRL, complementary to neomycin phosphotransferase). dsCTRL treatments were performed in parallel with all experimental treatments. Experiments were repeated ≥3 times, employing 20–30 flukes per replicate. *, P<0.05; **, P<0.01; ***, P<0.001. Symbols represent mean±SEM.

Figure 6. Concentration-dependent effects of long double-stranded (ds)RNA and short interfering (si)RNA triggers on transcript knockdown.

Target long dsRNAs (A, cathepsin B; B, cathepsin L; C, sigma-class glutathione transferase), and σGST siRNAs (D) serially diluted in RPMI 1640, were applied to newly-excysted juveniles at the concentrations indicated. Transcript knockdown was assayed in all cases by quantitative PCR, 72 h after start of soak. Target ΔΔCt (Y-axes) represents ratio of abundance of target transcript to a reference gene (glyceraldehyde phosphate dehydrogenase, GAPDH), in treated samples, relative to the abundance of those transcripts in untreated samples. Statistical significances are indicated relative to effects of negative control dsRNA (dsCTRL, complementary to neomycin phosphotransferase), or siRNA (siCTRL, commercially supplied scrambled sequence). Control treatments were performed in parallel with all experimental treatments. dsCTRL and siCTRL bars illustrate the impacts of the highest trigger concentrations tested (50 ng/ul). Experiments were repeated ≥3 times, employing 20–30 flukes per replicate. *, P<0.05; **, P<0.01; ***, P<0.001. Symbols represent mean±SEM.

Figure 7. Combinatorial silencing and specificity of cathepsin B, cathepsin L and sigma glutathione transferase RNAi on target transcripts.

(A) Newly-excysted juveniles exposed to a cocktail of long double-stranded (ds)RNA against all three targets (individual dsRNAs at 16.7 ng/µl concentration, final total dsRNA concentration, 50 ng/µl). (B) RNAi specificity, assayed via non-specific transcriptional impacts of three target long dsRNAs. Target ΔΔCt (Y-axes) represents ratio of abundance of target transcript to a reference gene (glyceraldehyde phosphate dehydrogenase, GAPDH), in treated samples, relative to the abundance of those transcripts in untreated samples. Statistical significances are indicated relative to effects of negative control dsRNA (dsCTRL, complementary to neomycin phosphotransferase). dsCTRL treatments were performed in parallel with all experimental treatments. Experiments were repeated 4 times, employing 20–30 flukes per replicate. *, P<0.05; **, P<0.01; ***, P<0.001. Symbols represent mean±SEM.