Figure 5
META1 expression in Leishmania donovani. (A) Quantitative RT-PCR analysis of META1 transcript in virulent and attenuated lines. The relative expression of META1 gene in various stages is presented as fold difference over expression in V3. The average of 3 biological replicates is presented. Bar represents standard deviation. V3 and A3: virulent and attenuated log phase promastigotes respectively; V7 and A7: virulent and attenuated stationary phase promastigotes respectively; VA and AA: virulent and attenuated amastigotes respectively. (B) META1 protein expression in virulent and attenuated lines. Total cell lysates from various stages of L. donovani were obtained at the indicated growth stages. The samples were assessed by western blotting with META1 (upper panel) and BiP (lower panel) antibodies. Leishmania BiP is a chaperone protein in the endoplasmic reticulum and was used as a loading control. (C) Time-course kinetics of META1 expression in promastigotes and amastigotes. Total cell lysates at various time intervals after switch to amastigote culture conditions from promastigotes of L. donovani were obtained at the indicated time points. Lysates were probed with META1 (upper panel) and BiP (lower panel) antibodies. Left panel represents virulent and right panel represents attenuated L. donovani respectively. D3: log phase promastigotes; A24 and A48: 24 hours and 48 hours after shift to amastigote culture conditions respectively; A48/P24, A48/P48 and A48/P72: 48 hours amastigotes shifted to promastigote conditions for 24 hours, 48 hours and 72 hours respectively. (D) Heat inducible expression of META1. Total cell lysates after individual or concomitant shift to pH 5.5 or 37°C for 48 hours of L. donovani promastigotes at pH 7.4 and 26°C were obtained for both virulent (lanes 1-4) and attenuated (lanes 5-8) L. donovani. Lysates were probed with META1 (upper panel) and BiP (lower panel) antibodies. D3, pH7.4, 26°C: log phase promastigotes; D3, pH7.4, 37°C and D3, pH5.5, 26°C: log phase promastigotes at pH 7.4 shifted to either 37°C or pH 5.5 at 26°C for 48 hours respectively; VA and AA: virulent and attenuated amastigotes respectively.