Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference

Abstract

Background: Cathepsin-like enzymes have been identified as potential targets for drug or vaccine development in many parasites, as their functions appear to be essential in a variety of important biological processes within the host, such as molting, cuticle remodeling, embryogenesis, feeding and immune evasion. Functional analysis of Caenorhabditis elegans cathepsin L (Ce-cpl-1) and cathepsin Z (Ce-cpz-1) has established that both genes are required for early embryogenesis, with Ce-cpl-1 having a role in regulating in part the processing of yolk proteins. Ce-cpz-1 also has an important role during molting.

Methods and findings: RNA interference assays have allowed us to verify whether the functions of the orthologous filarial genes in Brugia malayi adult female worms are similar. Treatment of B. malayi adult female worms with Bm-cpl-1, Bm-cpl-5, which belong to group Ia of the filarial cpl gene family, or Bm-cpz-1 dsRNA resulted in decreased numbers of secreted microfilariae in vitro. In addition, analysis of the intrauterine progeny of the Bm-cpl-5 or Bm-cpl Pro dsRNA- and siRNA-treated worms revealed a clear disruption in the process of embryogenesis resulting in structural abnormalities in embryos and a varied differential development of embryonic stages.

Conclusions: Our studies suggest that these filarial cathepsin-like cysteine proteases are likely to be functional orthologs of the C. elegans genes. This functional conservation may thus allow for a more thorough investigation of their distinct functions and their development as potential drug targets.

Figure 1. dsRNA-mediated silencing of the B. malayi cathepsin-like genes, Bm-cpl-1, Bm-cpl-5, and Bm-cpz leads to a reduction in microfilaria release from B. malayi in vitro.

Following 24 h culture in normal culture medium, two groups of 3 female B. malayi worms were soaked for 18 h in 2 mg/ml gene-specific dsRNA (Bm-cpl-1, Bm-cpl-5, Bm-cpz, Ov-cpz-Int2) or medium alone (control). Following dsRNA treatment, individual worms were transferred to dsRNA-free medium and cultured for an additional 48 h. Released microfilariae were collected and counted daily. Results are expressed as Mf release before RNAi treatment (A), 24 h (B) and 48 h (C) after treatment. Each graph represents one experiment which is representative of at least 3 separate experiments. P values denote a significant difference between dsRNA-treated worms and either untreated medium controls or negative control (Ov-cpz-Int2) (Mann-Whitney U-test).

Figure 2. Demonstration of uptake of Cy3-labeled dsRNAs or siRNA by B. malayi.

Adult female B. malayi (2 groups of 4 worms) were soaked in normal culture medium containing Cy3-dsRNA (0.01 mg/ml) for 24–72 h. Uptake was examined for Bm-cpl-5 dsRNA (∼800 bp) (A), Bm-cpl-5 siRNA (B), Bm-cpl Pro dsRNA (∼400 bp) (C) and Bm-cpl Pro siRNA (D). Fluorescence was visualized using a Zeiss Axiovert fluorescence microscope using the rhodamine filter set, using emission 590 nm.

Figure 3. dsRNA treatment of adult female B. malayi with Bm-cpl-5 and the pro-region of Bm-cpl (Bm-cpl Pro) leads to an inhibition of B. malayi microfilaria secretion in vitro.

8 female worms (2 groups of 4 worms) were treated for 3 d with 1.5 mg/ml gene-specific dsRNA or 5 µM siRNA, corresponding to Bm-cpl-5 or Bm-cpl-Pro. Worms were then cultured for a further 2 d in culture medium alone. Mf secretion was recorded daily and release expressed as a reduction in release in comparison to the dsRNA-free medium control group on day 1 (open bars), day 3 (hashed bars), and day 5 (closed bars). Data represents one representative experiment from at least 3 separate experiments.

Figure 4. Embryogenesis effects following treatment of adult female B. malayi with dsRNA or siRNA.

Intrauterine progeny from individual female worms (groups of four adult worms) were examined 2 d after dsRNA treatment and expressed as the relative proportions of progeny at different stages of development. Data represents one representative experiment from at least 3 separate experiments.

Figure 5. Treatment of adult female B. malayi with Bm-cpl-1, Bm-cpl-5, and Bm-cpz dsRNAs leads to phenotypic changes in developing embryos.

Intrauterine progeny from individual female worms were examined 2 d after treatment with medium control (A), Ov-cpz-Int2 control dsRNA (B), Bm-cpl-5 dsRNA (C) and Bm-cpl Pro dsRNA (D).

Figure 6. Soaking B. malayi adult female worms in dsRNA results in Bm-cpl gene-specific inhibition of expression.

At the end of the experiment (2 d after treatment), groups of four adult female worms were removed from culture and analyzed for differences in Bm-cpl gene-specific transcript levels by qRT-PCR. The relative amounts of Bm-cpl amplicon were determined by using the comparative C_T method and normalizing against the endogenous control gene (Bm-tub-1). The median value of the control group was set to 100% and the reduction in expression in the treated groups was calculated as a percentage of the control. ## denotes a significant difference between dsRNA or siRNA-treated worms and medium control, while *** denotes a significant difference between dsRNA or siRNA-treated worms and negative control (dsRNA of Ov-cpz-Int2) (Mann-Whitney U-test).