Abstract
BackgroundGalactofuranose is an essential cell surface component present in bacteria, fungi and several nematodes such as Caenorhabditis spp., Brugia spp., Onchocerca spp. and Strongyloides spp. This sugar maintains the integrity of parasite surface and is essential for virulence. UDP-Galactopyranose mutase (bmugm) plays a key role in Galf biosynthesis by catalyzing conversion of UDP-Galactopyranose into UDP-galactofuranose and knockout studies of the gene in Leishmania major, Mycobacterium and Aspergillus fumigatus displayed attenuated virulence while RNA interference study in C. elegans exhibited detrimental effects. Presence of UGM in several prokaryotic and eukaryotic microbial pathogens and its absence in higher eukaryotes renders it an attractive drug target. In the present study, RNA interference studies have been carried out to validate bmugm as an antifilarial drug target.MethodsRNA interference studies using two different sequences of siRNAs targeting bmugm were carried out. The in vitro gene silencing of adult B. malayi parasites was undertaken to observe the effects on parasites. Infective larvae were also exposed to siRNAs and their in vivo development in jirds was observed.ResultsThe in vitro gene silencing induced by siRNA1 and 2 individually as well as together knocked down the bmugm gene expression causing impaired viability of the exposed worms along with extremely reduced motility, abridged microfilarial release and adversely effected embryogenesis. The combinatorial in vitro gene silencing revealed marginally better results than both the siRNAs individually. Thus, infective larvae were treated with siRNA combination which showed downregulation of bmugm mRNA expression resulting into sluggish larval movements and/or death. The siRNA-treated actively motile larvae when inoculated intraperitoneally into jirds demonstrated highly reduced transformation of these larvae into adult worms with detrimental effects on embryogenesis. The effects of gene silencing were long-lasting as the adult worms developed from siRNA-treated larvae showed noticeable knockdown in the target gene expression.ConclusionsThe validation studies undertaken here conclude that bmugm is essential for the proper development and survival of the parasite and support its candidature as an antifilarial drug target.
Highlights
Galactofuranose is an essential cell surface component present in bacteria, fungi and several nematodes such as Caenorhabditis spp., Brugia spp., Onchocerca spp. and Strongyloides spp
Visualization of small interfering ribonucleic acids (siRNA) uptake by parasites There was successful uptake of FAM labelled stable off target siRNA, siRNA1, siRNA2 and both siRNA1 + 2 by the parasites on soaking them for 24 h which was visible throughout the body of females (Fig. 1a-d), males (Fig. 2a-d), mf released by siRNA-treated worms (Fig. 3a) and L3 (Fig. 3b) till the end of exposure
Impaired motility and viability of adult B. malayi Silencing of bmugm mediated through siRNA1, siRNA2 and siRNA1 + 2 caused 50–65% inhibition in motility of adult female and 60–70% in male wοrms within 24 h of soaking bringing down their viability between 58 and 70%
Summary
Galactofuranose is an essential cell surface component present in bacteria, fungi and several nematodes such as Caenorhabditis spp., Brugia spp., Onchocerca spp. and Strongyloides spp This sugar maintains the integrity of parasite surface and is essential for virulence. UDP-Galactopyranose mutase (bmugm) plays a key role in Galf biosynthesis by catalyzing conversion of UDP-Galactopyranose into UDP-galactofuranose and knockout studies of the gene in Leishmania major, Mycobacterium and Aspergillus fumigatus displayed attenuated virulence while RNA interference study in C. elegans exhibited detrimental effects. Galactofuranose (Galf ), a five member ring form of galactose, is an essential component of cell wall, cell surface glycolipids and glycoproteins It is widely distributed among several nematodes such as the free-living Caenorhabditis elegans and parasitic Brugia spp., Onchocerca spp. and Strongyloides spp., as well as protozoans and prokaryotes [3]. In the present study, in vitro and in vivo validation of B. malayi UGM as putative antifilarial drug target has been carried out by siRNA mediated gene silencing to understand the biological function of the enzyme in human lymphatic filarial parasite, B. malayi
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