Quantitative proteomic analysis to determine differentially expressed proteins in axenic amastigotes of Leishmania tropica and Leishmania major.

Cutaneous leishmaniasis is commonly caused by Leishmania major and Leishmania tropica. In the present study, the differential expression of proteins was identified in the amastigote-like forms of L. tropica and L. major in Iranian isolates. Initially, the samples were cultured and identified using PCR-RFLP technique. The Leishmania isolates were then grown in host-free (axenic) culture and prepared to amastigote-like forms, followed by the extraction of their proteins. To identify significant differentially expressed proteins (DEPs) of two types of Leishmania, the label-free quantitative proteomic technique was used based on sequential window acquisition of all theoretical fragment ion spectra mass spectrometry. A total of 51 up/down-DEPs (fold change >2 and p-value <.05) were identified between the axenic amastigote forms of L. major and L. tropica. Of these, 34 and 17 proteins were up-regulated in L. major and L. tropica, respectively. Several enriched GO terms were identified via biological process analyses for DEPs; furthermore, the metabolic process and translation were disclosed as top category in the up-regulated proteins of both L. major and L. tropica species. Also, the KEGG analysis revealed carbon metabolism and metabolic pathways term as the top pathways in the proteins up-regulated in L. major and L. tropica, respectively. Taken together, the numerous novel DEPs identified between the studied species could help fully understand the molecular mechanisms of pathogenesis and provide potential drug targets and vaccine candidates.

Comparative Two-dimensional Gel Electrophoresis Maps for Amastigote-like Proteomes of Iranian Leishmania Tropica and Leishmania Major Isolates.

BACKGROUND: Leishmania major and Leishmania tropica are the main causative agents of cutaneous leishmaniasis. Proteomics as a novel approaches could be used to evaluate protein expression levels in different stages of Leishmania species. We compare the protein contents of amastigote-like forms in L. tropica and L. major using two-dimensional gel electrophoresis (2-DE) and bioinformatics methods. MATERIALS AND METHODS: Leishmania parasites were isolated from the lesions of Iranian patients and identified using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). Five isolates of each two species were cultured in specific media to obtain amastigote-like forms to be prepared for proteomics study. Total protein contents were separated using 2-DE. The gels were stained by silver nitrate and scan was imaged. The protein spots with different expression changes in each gel were analyzed using Progenesis SameSpots software. RESULTS: A total of 354 protein spots were detected in both amastigote-like forms. Comparative analysis of protein spots with different expressions in the two amastigote-like form species showed 173 highly expressed spots of which 74 L. tropica and 99 L. major proteins were spotted with fold≥2. Also, 16 and 20 new protein spots were uniquely found in L. tropica and L. major, respectively. Clustering of different detected proteins using correlation analysis divided the proteins into two clusters based on their expression level. Furthermore, clustering results were confirmed by principal component analysis. CONCLUSION: Using proteomics methods specially 2-DE and statistical analysis demonstrated significant changes in protein expression levels in amastigote-like forms of L. tropica and L. major isolates.