Comparative Proteomic Profiling of axenic amastigotes of Leishmania tropica and Leishmania major Isolated in Iranian patients

Introduction and Objectives: Cutaneous leishmaniasis (CL), a neglected tropical disease (NTD), is commonly caused by Leishmania major and Leishmania tropica. Leishmania parasites have two recognizable stages in their life cycle, promastigote and amastigote. In the promastigote stage, the parasite grows in sandfly vector. When the parasite is deployed inside the parasitophorous vacuole of mammalian macrophages differentiated to the amastigote stage. Amastigotes change their protein/gene expression levels to adapt themselves with the environment inside the macrophages, and starts causing the disease. The differential expression of proteins in the amastigote-like of Leishmania tropica was compared with Leishmania major in Iranian Isolates. Materials and Methods: Initially, Leishmania parasites were isolated from the lesions of patients and identified using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). Five isolates of each two species were cultured in N.N.N. medium and then harvested in RPMI-1640 medium at 24°c for achievement the promastigotes phase. In order to convert promastigote to amastigote-like form, the environmental condition was changed. Promastigotes collected in the stationary phase by adding RPMI1640 medium which we previously adjusted the PH to 3.5-4 and then incubated at 35 °C with 5% CO2 for 96 – 120 hours. The media were checked every day and cell suspension containing 107 parasites/ml was collected.Their proteins were extracted. The label-free quantitative proteomic technique (SWATH) approach used to identify significant differentially expressed proteins from amastigote-like form of two types of leishmania. Afterward, Proteins that were expressed at different levels (fold change> 2 and p.value<0.05) were submitted to the STRING database for protein-protein interaction analysis.Results: SWATH is implemented to detect 51 proteins which are differentially expressed. Based on STRING analysis the protein-protein interaction was drawn and main protein (HUB protein) from the point of degree and betweenness centrality respective to the importance were indicated as follows: Inosine-5 -monophosphate dehydrogenase, Putative 60S ribosomal protein L13a, Elongation factor 2, Putative GMP synthase and DEAD/DEAH box helicase-like protein.These proteins were classified into four main categories based on function/biological processes. Out of 51 proteins that had a different expression, 46.1% in the metabolic pathway, 32.7% in translation and protein synthesis, 5.8% in membranous, 1.9% in cell structure and motility and 13.5% of them were uncharacterized proteins.Conclusion: Here, it is shown that Peroxidoxins is involved in the metabolic process, Putative 60S Ribosomal protein L36 is involved in the translation process and ATP-ADP translocase is involved in membraneous protein had the maximum up-regulation in amastigote-like L. tropica whereas the number of differential expression of these proteins were more up-regulated in amastigote-like L. major than amastigote-like L. tropica.