Partial inhibition of glyceraldehyde-3-phosphate dehydrogenase translation by gentamicin in the attenuated Leishmania major: structure study

Context An attenuated line of Leishmania major was generated by continuous in vitro culture of promastigotes under gentamicin pressure. The growth rate of promastigotes exposed to high concentrations of gentamicin was significantly reduced compared with those cultured at lower concentrations. The present study addressed a key challenge in developing an attenuated L. major understanding how parasite molecules change in response to gentamicin exposure. Methods Western blot membranes were individually incubated with pooled sera from vaccinated or naturally infected subjects as primary antibodies. For proteomic analysis, lysate of the attenuated L. major H-line was separated by SDS–PAGE, and protein band at approximately 30 kDa were excised. The band was excised and subjected to comparative proteomic analysis using liquid chromatography-mass spectrometry (LC-MS/MS). Quantitative real-time PCR (qRT-PCR) was performed to assess glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcription levels. Results Western blot analysis revealed a markedly reduced protein expression at band ~30 KDa (approximately 4.5-fold). LC–MS/MS analysis identified GAPDH as the most significantly modulated protein. Downregulation of GAPDH in the attenuated line parasite relative to the wild type was further confirmed at the transcriptional level by qRT-PCR. Conclusions These findings suggest that passage of L. major wild type in a low concentration of gentamicin (20 µg/ml) partially inhibits GAPDH translation, leading to the selection of avirulent cells. In contrast, exposure to a higher gentamicin concentration (200 µg/ml) suppresses promastigote growth and may fully inhibit GAPDH translocation. WHO | Iranian Registry of Clinical Trials (IRCT): (IRCT2015101544604N1).