Figure 8

Mutagenesis in putative hydrophobic cavity of META1 alters secretion in Leishmania. (A) Mutations in putative hydrophobic cavity of META1 alter extracellular SAP activity. Extracellular SAP activity in L. donovani at stationary phase of vector control (Ld GFP), wild-type META1 overexpression line (Ld/Ld-WT), mutant META1 overexpression lines: L58F (Ld/Ld-L58F); L80F (Ld/Ld-L80F) and L58,50F (Ld/Ld-L58,80F) respectively. SAP activity is represented as nmol/min/10⁷ cells. Means of the respective values were compared for statistical significance using a paired Student's t-test. *P < 0.0001 and ** P < 0.0003. (B) Fraction of intracellular v/s total SAP activity. Results are expressed as percentage of intracellular SAP activity of total SAP activity (intracellular and extracellular) measured in nmol/min/10⁷ cells in all the transfectants at stationary phase. Means of the respective values were compared for statistical significance using a paired Student's t-test. *P < 0.004 and ** P < 0.0001. (C) Growth kinetics of L. donovani on META1 overexpression. Growth curves of L. donovani overexpressing either the wild-type META1, Ld/Ld-WT or mutant META1 L58F, L80F and L58,80F were compared to that of Leishmania with control vector, Ld GFP. Cell density (x 10⁶ cells/ml) of each culture was determined at 24 hour time intervals after 48 hours of initial inoculation for up to 7 days. The cell densities plotted are average of at least 3 biological replicates.