Using LEXSY plasmids for sodb1 gene silencing in Leishmania tropica parasites

Abstract

Abstract Background: Genetic modifications and manipulations have topped the list of recent research, as they target the causes, not the results, and are currently considered one of the most powerful methodologies used to study the biology of the Leishmania parasites. These methods have expanded since the publication of the first study in which the genetic replacement of one of the Leishmania genes was done, which provided an opportunity to analyze and study the biology of the parasite genomically. In this study, the SODB1 gene was targeted by antisense RNA. This gene encodes one of the important enzymes in the infectivity of Leishmania tropica parasites within macrophages, which is the superoxide dismutase enzyme (SODB1).Methods: An inverted sequence of part of SODB1 ORF and 3’UTR were cloned in LEXSY plasmid, and after obtaining the silencing constructions, the Leishmania tropica parasites were transfected by electroporation. Western Blot analysis of SODB1 expression and the infectivity of these mutant parasites in human macrophages was studied and evaluated in comparison with a wild-type negative control and another control containing the GFP gene that codes for Green Fluorescent Protein (GFP).Results: The results showed that using LEXSY plasmids for SODB1 gene silencing was efficient and the knocking-down was clear forasmuch the decrease in both infectivity and parasite load in human macrophages in vitro. On the other hand, western blot analysis revealed a lower expression level of SODB1 in the mutant parasites than wild-type. The results were subjected to statistical analysis and the decreases in the infectivity and parasites load of the mutant parasites were very significant in comparison with the wild-type.Conclusion: Our study confirms the efficiency of the produced silencing system, and the importance of the SODB1 enzyme in the amastigote parasites’ ability to grow and survive within the host's macrophages despite the presence of many other SODs enzymes. This is the first study that affirms the success of using the LEXSY gene expression system for gene silencing according to the antisense RNA approach. Thus, this work will allow using of the produced silencing system to target other important genes in Leishmania, and continue studying the knocked-down strain in vivo.