Vector-based RNA interference of cathepsin B1 in Schistosoma mansoni - PubMed (original) (raw)

Vector-based RNA interference of cathepsin B1 in Schistosoma mansoni

Elissaveta B Tchoubrieva et al. Cell Mol Life Sci. 2010 Nov.

Abstract

In helminth parasites, proteolytic enzymes have been implicated in facilitating host invasion, moulting, feeding, and evasion of the host immune response. These key functions render them potential targets for anti-parasite chemotherapy and immunotherapy. Schistosomes feed on host blood and the digested haemoglobin is their major source of amino acids. Haemoglobin digestion is essential for parasite development, growth, and reproduction. We recently reported the use of pseudotyped Moloney murine leukaemia virus to accomplish transformation of Schistosoma mansoni. Here, we report the design of a viral vector expressing a dsRNA hairpin to silence expression of the schistosome cathepsin B1 (SmCB1) gene. We observed 80% reduction in transcript level 72 h after virus exposure and complete silencing of enzyme activity in transduced worms. This is the first report using this technology in any helminth parasite. It will facilitate the evaluation of potential drug targets and biochemical pathways for novel interventions in schistosomes.

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Figures

Fig. 1

Fig. 1

Peptidases involved in the digestion of host haemoglobin in the gut of S. mansoni. Schistosomes utilize cysteine, aspartic and metallo-proteases to liberate peptides and amino acids from host haemoglobin. Haemolysin activity in the oesophagus lyses red blood cells and haemoglobin is released. It is speculated (red arrows) that an asparaginyl endopeptidase activates pro-cathepsin B1 and other pro-peptidases such as cathepsins L1 and L2, and cathepsin D in vivo. Cathepsin C may further process cathepsin B1 to its mature form

Fig. 2

Fig. 2

Schematic representations of the retroviral vector and the dsRNA hairpin. a The pLNHX vector containing the MMLV long terminal repeats (LTR), the packaging signal (Ψ), and a neomycin selection marker (neor), modified to include the S. mansoni actin 1.1 gene promoter (Act 1.1) driving the transcription of inverted regions of the S. mansoni cathepsin B1 encoding sequence linked by a spacer (loop). b RNA hairpin structure of the transcribed sequence. For clarity, the central part of the molecule has been cut away. The structure was generated using mfold (

http://mfold.burnet.edu.au/

)

Fig. 3

Fig. 3

Gene transcription analysis following transduction of S. mansoni with pLNHX-SmAct-Luc virions. Gene expression is demonstrated by the amplification of transcripts, and specificity of the reaction—by dissociation curves of the primers employed in a qRT-PCR. a Relative luciferase expression is detected at both 24 h (blue symbols) and 72 h (red symbols) after viral transduction. In contrast, no luciferase transcripts were present in the non-transduced parasites (green symbols). Transcription of the S. mansoni tubulin (b) and SmCB1 genes (c) was apparent at all time points and also in the non-exposed parasites

Fig. 4

Fig. 4

Analyses of downstream molecular events following transduction of S. mansoni by virions carrying a dsRNA hairpin loop specific for cathepsin B1 (shCB1RNAi). a Relative transcript levels of cathepsin B1. The results are normalised by the expression levels of the housekeeping gene α-tubulin and represented as a fold difference compared to the wild-type control sample (±SE). Results are averaged from three independent experiments. Significance at p < 0.05 is indicated by an asterisk. b To control for off-target silencing effect, the transcription level of two additional genes SmCD and SmCox1 was also measured at 72 h post transduction with either pLNHX-SmAct-Luc (red bars) or pLNHX-SmAct-CB1RNAi (orange bars) virions and compared to non-transduced worms (grey bars). While transcription of SmCB1 was dramatically reduced after exposure to pLNHX-SmAct-CB1RNAi virions, expression of both SmCD and SmCox1 genes was not affected as a result of exposure to any of the viruses

Fig. 5

Fig. 5

Analysis of SmCB1 proteolytic activity. Fluorescence was measured in a fluorometer with excitation at 355 nm and emission at 460 nm in a 200-μl reaction mix of 1 μg total protein (1:200 dilution) and 20 μM substrate for 200 cycles, with 15 flashes at gain 5 and 37°C. In the control samples, 20 μM of inhibitor was pre-incubated with the extracts prior to addition of the substrate. Data were measured as relative cathepsin B activity in units/sec and expressed as a fold difference compared to the wild-type (not exposed to virions) control. Results are averaged from three independent experiments. Significance at p < 0.0001 is indicated by an asterisk

Fig. 6

Fig. 6

Kaplan–Meier survival curve. A representative set of survival curves (out of three independent experiments) is shown. A 95% confidence interval for fractional survival at any particular time was calculated by both the log-rank (Mantel–Cox) test and the Gehan–Breslow–Wilcoxon test. The survival curves show significance when either the wild-type (p < 0.0001) or the pLNHX-SmAct-Luc control (p = 0.0002) is compared to pLNHX-SmAct-CB1RNAi and no difference is observed when only the two controls are considered (p = 0.07) (GraphPad Prism 5) and [26]

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