Abstract
The microaerophilic protozoan Giardia lamblia is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA), which can silence expression of a target gene through the RNA interference (RNAi) pathway. Small silencing RNAs were detected and quantified in transformants expressing dsRNA by a stem-loop RT-qPCR approach. The results showed that, in transformants expressing dsRNA of 100–200 base pairs, the level of NADHox mRNA was reduced by around 30%, concomitant with a decrease in enzyme activity and a reduction in the number of trophozoites with respect to the wild type strain, indicating that NADHox is indeed an important enzyme for Giardia viability. These results suggest that it is possible to induce the G. lamblia RNAi machinery for attenuating the expression of genes encoding proteins of interest. We propose that our silencing strategy can be used to identify new potential drug targets, knocking down genes encoding different structural proteins and enzymes from a wide variety of metabolic pathways.
Highlights
Giardia lamblia is a unicellular flagellate protozoan causing giardiasis, which is catalogued as a global public health problem
Considering that the NADH oxidase (NADHox) gene plays a fundamental role in the redox metabolism of Giardia, we decided to study the effect of silencing this gene through an RNA interference (RNAi) approach
For the silencing of the NADHox gene, four plasmids were constructed based on the pTubGdh-RNAi vector and four more based on the pTubGdh-eGFP-RNAi vector, containing DNA
Summary
Giardia lamblia is a unicellular flagellate protozoan causing giardiasis, which is catalogued as a global public health problem. Children and immuno-compromised patients are the main affected groups. Compounds derived from benzimidazoles and nitroimidazoles have been used in pharmacotherapy against giardiasis [3]; side effects in the host and the existence of resistant strains to these drugs have been reported [3,4]. To overcome these problems, several groups have attempted different strategies to find new antigiardiasic compounds [3,5,6]
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