Introduction: Recent research has focused on genetic modifications and manipulations, which address the root causes rather than just the outcomes. These methods are now recognized as powerful tools for studying Leishmania parasites’ biology. In this study, the sodb1 gene was targeted using antisense RNA. This gene encodes an essential enzyme, superoxide dismutase (SODB1), which plays a crucial role in the infectivity of Leishmania tropica parasites within macrophages.
Methods: The inverted sequence of a part of the sodb1 ORF and 3’UTR was cloned into the LEXSY plasmid. After obtaining the knock-down constructions, Leishmania tropica parasites were transfected using electroporation. Western blot analysis was performed to study and evaluate SODB1 expression and the infectivity of these mutant parasites in human macrophages, comparing them with a wild-type negative control and another control containing the EGFP gene, which codes for the enhanced Green Fluorescent Protein (EGFP).
Results: The use of LEXSY plasmids for sodb1 gene knock-down was efficient, resulting in a clear decrease in both infectivity and parasite load in human macrophages in vitro. Western blot analysis revealed a lower expression level of SODB1 in the mutant parasites compared to the wild- type. Statistical analysis confirmed that the decreases in infectivity and parasite load of the mutant parasites were highly significant compared to the wild-type. Statistical analysis confirmed that the decreases in infectivity and parasite load of the mutant parasites were highly significant compared to the wild-type.
Conclusion: Our study confirms the efficiency of the produced knock-down system and the importance of the SODB1 enzyme in the amastigote parasites’ ability to grow and survive within the host's macrophages. This is the first study that affirms the success of using the LEXSY gene expression system for gene knock-down according to the antisense RNA approach. This work will allow for the use of the produced knock-down system to target other important genes in Leishmania and continue studying the knocked-down strain in vivo.