Silencing genes by RNA interference in the protozoan parasite Entamoeba histolytica
ABSTRACT Experimental procedures using the RNA interference (RNAi) approach have recently emerged as a powerful tool for gene silencing in eukaryotic microbes for which gene replacement techniques have not yet been developed. Our group has recently explored RNAi to knock down gene-specific expression in the protozoan parasite Entamoeba histolytica, through delivery of small interfering RNA (siRNA) oligonucleotides by the soaking approach. Standardized conditions for the soaking of E. histolytica trophozoites with siRNAs result in highly specific and significant silencing of parasite cognate genes. Real-time PCR analysis indicates that a 16-hour treatment with siRNAs usually results in half-extinction of target messenger RNA. Furthermore, Western blot analysis of trophozoite crude extracts with the use of specific antibodies shows a similar reduction of cognate protein levels after siRNA treatment.
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- "Key to the utilisation of gene-silencing methods is the presence of complete RNA-interference (RNAi) machinery in the parasite. Essential components of this pathway (e.g., DICER) have been well explored in Giardia (Macrae et al., 2006; Prucca et al., 2008), and RNAi has been used for gene perturbation for G. intestinalis (see Gargantini et al., 2011) and E. histolytica (see Solis and Guillen, 2008), suggesting that RNAi pathways are sufficiently complete in both of these organisms. Sequencing of the C. parvum and C. hominis genomes has revealed a lack of identifiable homologues to DICER and Argonaute, suggesting an altered and/or absent role for small interferring RNAs in gene regulation in these organisms (Abrahamsen et al., 2004; Xu et al., 2004), indicating against the use of RNAi technologies for functional exploration of Cryptosporidium . "
ABSTRACT: Parasitic protists are a major cause of diarrhoeal illnesses in humans globally. Collectively, enteric pathogens exceed all other forms of infectious disease, in terms of their estimated global prevalence and socioeconomic impact. They have a disproportionately high impact on children in impoverished communities, leading to acute (diarrhoea, vomiting, dehydration and death) and chronic disease (malabsorption, malnutrition, physical and cognitive stunting and predisposition to chronic, non-communicable disease) consequences. However, historically, investment in research and disease control measures has been disproportionately poor, leading to their current classification as neglected pathogens. A sound understanding of their biology is essential in underpinning detection, treatment and control efforts. One major tool in rapidly improving our knowledge of these parasites is the use of biological systems, including 'omic' technologies. In recent years, these tools have shown significant success when applied to enteric protists. This review summarizes much of this knowledge and highlights the significant remaining knowledge gaps. A major focus of the present review was to provide a perspective on a way forward to address these gaps using advanced biotechnologies.International journal for parasitology 07/2013; DOI:10.1016/j.ijpara.2013.06.005 · 3.40 Impact Factor
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- "The siRNA-A with the scrambled sequence was used at 40 μg per culture following the manufacturer's instructions. Transfection of the trophozoites was performed by soaking as previously reported . In brief, 1 × 105 trophozoites were grown in 6 mL TYI-S33 media in culture flasks. "
ABSTRACT: Encystment is an essential process in the biological cycle of the human parasite Entamoeba histolytica. In the present study, we evaluated the participation of E. histolytica Gln6Pi in the formation of amoeba cyst-like structures by RNA interference assay. Amoeba trophozoites transfected with two Gln6Pi siRNAs reduced the expression of the enzyme in 85%, which was confirmed by western blot using an anti-Gln6Pi antibody. The E. histolytica Gln6Pi knockdown with the mix of both siRNAs resulted in the loss of its capacity to form cyst-like structures (CLSs) and develop a chitin wall under hydrogen peroxide treatment, as evidenced by absence of both resistance to detergent treatment and calcofluor staining. Thus, only 5% of treated trophozoites were converted to CLS, from which only 15% were calcofluor stained. These results represent an advance in the understanding of chitin biosynthesis in E. histolytica and provide insight into the encystment process in this parasite, which could allow for the developing of new control strategies for this parasite.01/2013; 2013:758341. DOI:10.1155/2013/758341
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ABSTRACT: In many eukaryotes, the introduction of double-stranded RNA (dsRNA) into cells triggers the degradation of mRNAs through a post-transcriptional gene-silencing mechanism called RNA interference or RNAi. In the present study, we found that endogenous long-dsRNA was substantially more effective at producing interference than endogenous, or exogenous, short-dsRNA expression in Giardia lamblia . The effects of this interference were not evident in the highly expressed protein tubulin or the stage-specific cyst wall protein 2. However, long-dsRNA caused potent and specific interference in the medium subunits of adaptins, the RNA-dependent RNA polymerase, and the exogenous green fluorescence protein. Our results suggest that the ability of dsRNA antisense to inhibit the expression of these specific types of proteins is indicative of a gene-specific mechanism.Journal of Parasitology 08/2010; 96(4):815-9. DOI:10.1645/GE-2406.1 · 1.26 Impact Factor