Genetic manipulation in protozoa such as Leishmania parasites enables us to analyze deeply the consequences of genetic changes and their impact on many aspects of the parasite’s life. Especially, in exploring the immune responses formed during infection. The use of genetically modified Leishmania parasites can solve the mystery of the parasite's contribution in pathogenesis, cellular mechanisms, host-parasite interaction, and the responses of the innate and adaptive immune systems against this pathogen [1]. The possibility of modeling attenuated parasites has been readily available forasmuch the efficiency of the modification processes, the availability of various vectors for genetic manipulations, and due to the abundance of sequences on bioinformatics websites that provide different databases [2]. These mutant parasites which genetically manipulated could serve to investigate the efficacy of new drug targets, and can also be candidates for use in vaccines, depending on the targeted genes.
Through these modifications, self-genes or external genes can be inserted. This could be either by episomal vectors or by genome integrated vectors. In the episomal vectors, the ectopic gene expression is temporary, because this type of expression is under the control of the vector’s promoter and it can be inducible in certain conditions. As for the case of integrated vectors, the inserted genes are directed to merge within a specific region that is located downstream of rRNA promoter site in the genome, to study the effects of the persistent expression during all stages of parasite life and in subsequent cells resulting from its division [3]. This genetic manipulation can be made to target a gene family or multiple different genes at the same time, through the concept of replacement that was generally according to the principle of Homologous Recombination [4]. This methodology has shown great success in elucidating the functions of several Leishmania genes.
The Leishmania expression system (LEXSY) is a protein expression platform developed for Leishmania tarentolae and was designed to combine eukaryotic protein synthesis, folding and modification with higher growth rates comparison with mammalian cells. In Vivo, LEXSY is available in two principal types of expression: constitutive (persistent) or inducible. The constitutive system leads to efficient and consistent production of proteins. It is based on the integration of an expression cassette into the highly repeated chromosomal 18S rRNA (ssu-locus). This site of integration will permit persistent transcription by the endogenous RNA Polymerase I [5, 6].
Genetically, after years of knock-out attempts, only about two hundred genes out of the 9,000 genes in Leishmania have been targeted [7]. But Recently, with the discovery and application of the CRISPR-Cas9 system, the number of targeted genes has increased dramatically. This system is a very efficient and time saver for studying different genes and their functions by knocking out them in previously transfected parasites for expressing Cas9 protein [8]. Genes also can be targeted by knocking down using the antisense RNA (asRNA) approach, which leads to repressing the translation process to some extent. This mechanism was first reported in 1984 in E.coli bacteria [9]. It has been applied as a technology that can control the gene expression processes, and affect the nature of the studied organism such as the Leishmania parasite without making any genomic modifications at the genome level [10]. asRNA sequence is engineered to retard the ribosome binding site (RBS), and to bind the initiation codon of the target mRNA [11]. This specific region is important for initiating translation and thus will prevent the ribosome from binding to the RBS site of the target mRNA. Furthermore, the target mRNA blocked by asRNA may tend to be rapidly degraded in the cell by nuclease enzymes [12]. The efficiency of asRNA varies greatly depending on the target gene, and in general, it is low [13]. Regression and loss of silencing are possible to occur, when culturing and growing for a long time and that’s, in the case when using an episomal vector [14]. To get a successful silencing, the asRNA transcribed by the plasmid vector must be hybridized with the target mRNA, this process will impede the translation of the target protein [15], as well as by increasing the number of transcribed copies of asRNAs in the cell, the higher number of copies achieve the higher silencing efficiency [16].
A set of the crucial proteins in Leishmania are Iron Superoxide dismutase enzymes, these proteins are metalloenzymes that act as antioxidants and protect the cell by converting the superoxide radical into H2O2 [17, 18]. Then peroxidoxine enzyme continues the reaction to get rid of H2O2 by converting it to water and molecular oxygen (O2) [19, 20]. SOD enzymes form a large family of proteins in most organisms, that use metals such as manganese, iron, nickel, copper and zinc as cofactors [21]. In Leishmania, FeSODs were classified into SOD-A which is expressed in the mitochondria as dimeric form, glycosome-specific SODB1 andSODB2 enzymes, and SOD-C recently detected in the mitochondria[18]. The genes encoding SODB1, SODB2, are organized tandemly on the same chromosome, their sequences show a significant similarity of more than 92% in several species of Leishmania parasites (L.major, L.chagasi, L.donovani). In addition, the amino acid sequences of SODB1 and SODB2 are approximately 90% similar, with the main difference between them due to the presence of about 13 codons at the 3' end of the SODB2 gene [22]. When Leishmania parasites infect the host's macrophages, these macrophages go through consequent respiratory oxidation process producing ROS intermediates such as: H2O2, OH. radicals, O2.− and peroxynitrite, as a part of the oxygen-dependent mechanisms. these mechanisms destroy the invading pathogens of phagocytes [23]. It can also activate NO, which is a small organic radical with a strong antimicrobial effect [24, 25]. In humans, the reactive oxygen species (ROS) dominates the elimination weapons of Leishmania parasites. In the early stages of infection after phagocytosis, phagocytes release lethal particles such as O2.− and H2O2 [26–28], so that, FeSODs could play a crucial role in the parasite’s survival.
Although macrophages are the primary host facing the flagellated parasites, neutrophils and dendritic cells can also recognize and phagocytose these parasites [29].
In this study, Leishmania tropica parasites were transfected by two linear cassettes of LEXSY plasmid, the first contained a reporter gene that encodes the green fluorescent protein (GFP), and the second contained a cloned specific designed adverse sequence of SODB1 gene so that it was integrated into the genome in the opposite arrangement, and the antisense RNA molecules could lead to repression of gene expression either by inhibiting the initiation of translation or by stimulating RNase enzymes to degrade the target mRNA [30], then we evaluated the efficiency of the SODB1 down regulating in the transfected strains by western blot analysis followed by infectivity experiment of human peripheral blood macrophage cells (PBMC).