The Evaluation of Gene Expression Analysis of Isolates Containing Leishmania RNA Virus
Project no. 23784 · Status: Kapanmış · 2022
Summary
<p>Leishmaniasis is a neglected tropical disease caused by the parasite Leishmania, which has complex pathology ranging from skin ulcers to fatal organ damage. They are transmitted by the bite of infected sandflies and infect host macrophages.</p><p>Some Leishmania strains have been found to harbour the Leishmania RNA virus (LRV). Leishmania RNA virus is a small parasitic virus containing a capsid protein and a dsRNA genome encoding an RNA-dependent RNA polymerase. It is found only in the cytoplasm of the parasite. Leishmania RNA virus, a double-stranded RNA virus, is divided into two groups in the New and Old World, LRV1 and LRV2, respectively. Many studies show that LRV, a Leishmania endosymbiont, is one of the factors affecting disease severity, metastasis and resistance to treatment in New World species. There is no study on the interaction mechanism of LRV2, which was recently detected by molecular methods in Leishmania major (L.major) and Leishmania tropica (L. tropica)species in Turkey, with the host cell and the possible effect of the virus on the disease process. This project is structured around four main objectives. First, to determine the transcriptome profile of LRV2 -/+ L. tropica strains detected by us for the first time. Second, to evaluate the transcriptome results by bioinformatic analyses. Third, to identify differentially expressed genes (DEGs). And finally, to validate the expression of these DEGs. </p><p>In our study, the following methodology was implemented for virus-negative and positive L. tropica strains isolated from Turkey: (i) analysis of transcriptome data; (ii) evaluation of the obtained data through bioinformatics analyses to identify distinctive gene targets and differentially expressed genes (DEGs); (iii) comparison of gene expression levels of 3 selected DEGs (both upregulated and downregulated) using qRT-PCR in a control group consisting of 30 Leishmania strains isolated from Turkey with known virus-positive/negative status, and visualization of some of the significant gene regions at the protein level using Western blotting.</p><p>Gene expression analysis between LRV2 -/+ L. tropica strains revealed 5023 down-regulated and 1751 up-regulated genes. Significantly significant results were obtained in LRV2+ L. tropica for genes involved in basic vital activities of Leishmania, such as intracellular parasite survival, transcription, translation, signal transduction, biosynthetic process, parasite proliferation and mechanisms of action on host cells. Among the up-and down-regulated genes examined were fatty acid elongase 2, vamp8 snare protein, histone 4, gp63, ppgi, signalling mucin Msb2, cytosolic triparedoxin peroxidase, DNA repair protein alkB, etc. Many of them were found to be related to defence, signalling, reproduction and survival mechanisms.</p><p>Our study, the first successful RNA-Seq and functional annotation analysis of LRV2 - and + L. tropica, provides an essential overview of the relationship between LRV2 and Leishmania parasites. It sheds light on their impact on disease, the potential to specifically target the virus as a therapeutic strategy, and the identification of potential virus components as targets. Understanding the role and contribution of LRV2 in disease pathogenesis deepens our understanding of virally infected parasites and opens new possibilities for evaluating LRV2 as a potential therapeutic target</p>