Apicoplast Lipoic Acid Protein Ligase B Is Not Essential for Plasmodium falciparum
Figure 5
Effect of LipB Disruption on Lipoic Acid Levels and PDH-E2 Lipoylation
(A) WT 3D7 and LipBKO mutants were analysed for their LA content. The data shown were determined in two independent experiments and each sample was measured in triplicate. Wild-type LA levels are around 40 nmoles/108 cells, whereas LipBKO lines had drastically reduced LA levels between 0.9 nmoles/108 cells and 2.5 nmoles/108 cells.
(B) WT 3D7 and LipBKO2–2 mutant were analysed for their fatty acid content. Fatty acids released by acid and base treatment were converted to methyl esters and analysed by GC-MS (see Materials and Methods for details). The figure shows an enlargement of the total ion current (TIC) chromatogram between 25.0 min and 30.0 min in which the fatty acids, including the internal standard (C17:0) at 28.5 min, C14:0 at 25.2 min, C16:1 at 27.3 min, C16:0 at 27.5 min, C18:2, C18:1, and C18:0, as well as the short chain octanoic acid derived LA (oxidised) at 26.4 min, and LA (reduced) at 27.2 min are marked. The levels of myristic acid (C14:0) clearly increase in the LipBKO line in comparison to wild-type parasites.
(C) Analysis of lipoylation pattern of WT 3D7 and LipBKO mutants. Parasite extracts (15 μg) were separated on 4%–12% SDS-PAGE, blotted to nitrocellulose, and subsequently were probed with a rabbit anti-LA antibody as described in Materials and Methods. Lane 1, 3D7 wild-type; lane 2, LipBKO2–1; lane 3, LipBKO2–2. The bands correspond to PDH-E2 (75 kDa), BCDH-E2 (50 kDa), and KGDH-E2 (47 kDa). PDH-E2 lipoylation is greatly reduced in the mutant lines in comparison with wild-type, showing that the decrease in LA levels is primarily due to loss of the cofactor from the apicoplast PDH-E2-subunit. The blot was reprobed with an antibody directed against the 22 kDa 2-Cys peroxiredoxin PfTrx-Px1 as a loading control (Trx-Px1).