Figure 1.
Identification of Lipoylated Proteins in P. falciparum
P. falciparum protein extracts (15 μg) were separated on an SDS-PAGE (4%–12%, Invitrogen) and blotted onto nitrocellulose as described in the Materials and Methods section. The blots were analysed using four different antibodies: (lane 1) rabbit anti-H-protein at 1:2,000 recognising a 25 kDa band; (lane 2) rabbit anti-BCDH-E2 at 1:100 recognising a 50 kDa band; (lane 3) rat anti-KGDH-E2 at 1:5,000 recognising a 47 kDa band; (lane 4) rabbit anti-lipoic acid at 1:500 recognising four major protein bands corresponding to PDH-E2 (75 kDa, Allary et al. [14]), BCDH-E2 (50 kDa), KGDH-E2 (47 kDa) and H-protein (25 kDa).
Figure 2.
Southern Blot of P. falciparum LipB Mutants
Genomic DNA was isolated from wild-type (P. falciparum 3D7 and D10) as well as four independent LipB mutant clones (KO1–1, KO1–2; KO2–1 and KO2–2). The DNA was digested with NdeI and separated on a 0.8% agarose gel before blotting to nylon and probing with the LipB coding region. The band shown in lanes 1 and 2 corresponds to the endogenous LipB gene locus (2.8 kb). The 2.8 kb band disappears and is replaced by two bands diagnostic for the disruption of the endogenous LipB locus (3.7 kb and 4.9 kb) in all four cloned parasite lines (lanes 3–6).
Figure 3.
Parasite growth was assessed over six days according to Sanders et al. [21]. (A) 3D7 wild-type and KO1–1 and KO1–2; (B) D10 wild-type and KO2–1 and KO2–2. The experiment was started with highly synchronised ring-staged cultures which were monitored daily and diluted 5-fold every 48 h. For each determination of percentage parasitemia, the number of infected erythrocytes per 1,000 erythrocytes was recorded. Means and standard errors of three separate experiments are shown.
Figure 4.
Progression of Wild-Type and LipBKO Mutants through Intraerythrocytic Cell Cycle
Progression through the intraerythrocytic developmental cycle was assessed by analysing Giemsa stained thin-smears of highly synchronised cultures that were taken every 8 h for a period of 48 h. The experiments were started with ring-stage parasites (rings) and the development through one intraerythrocytic life cycle was followed by determining the occurrence of rings, trophozoites, and schizonts by light microscopy. The images represent typical appearance of these parasite stages. The numbers show the percentage of rings, trophozoites, and schizonts counted at the respective time point. The progression of the LipB mutants (3D7-based, panel A and D10-based, panel B) through the intraerythrocytic cell cycle is accelerated by about 4 to 8 h in comparison with wild-type 3D7 and D10. Scale bars: 5 μm.
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).
Table 1.
Lipoic Acid Levels in Wild-Type and LipBKO P. falciparum
Figure 6.
Effect of Triclosan, tert-Butylhydroperoxide and N-Methylphenazonium Methosulfate on LipB Mutants
The effect of triclosan (an inhibitor of FabI) (A) and two pro-oxidants (tert-butyl hydroperoxide (B) and N-methylphenazonium methosulfate (C) on parasite growth were analysed as described in Materials and Methods. The IC50 values of wild-type and LipBKO mutants do not differ markedly suggesting that LA depletion has no effect on fatty acid biosynthesis and response to pro-oxidants. All three figures show means ± standard errors of three independent experiments.
Figure 7.
LplA2 Functional Complementation of LipB and LplA/LipB Deficient E. coli
The functionality of LplA2 was analysed by complementing two bacterial lines with three different expression constructs as described in Materials and Methods. (A) Growth of KER 184 in the presence and absence of LplA2 expression constructs as described in Materials and Methods. Key: 1, pASK-IBA3-LplA2fl; 2, pASK-IBA3-LplA2-S1; 3, pASK-IBA3-LplA2-S2; 4, pASK-IBA3-LplA1; 5, pASK-IBA3; 6, untransformed (B) Growth of TM 136 in the presence of LplA1 and LplA2 expression constructs. Key: 1, pASK-IBA3-LplA2fl; 2, pASK-IBA3-LplA2-S1; 3, pASK-IBA3-LplA2-S2; 4, pASK-IBA3-LplA1.
Figure 8.
The localisation of LplA2 cannot be predicted and therefore it was analysed by localising a C-terminally GFP-tagged protein in P. falciparum erythrocytic stages. Expression of the GFP-tagged protein in P. falciparum 3D7 was analysed by fluorescence light microscopy. The parasites were costained with MitotrackerTM Ros (Molecular Probes) to assess whether LplA2-GFP colocalises with the parasite's mitochondrion. In panel 1 LplA2 is clearly colocalising with the mitochondrion. However, as is obvious from panel 2 LplA2-GFP localised also to an organelle close to the mitochondrion but clearly distinct from it. In panel 3 LplA2-GFP colocalises with the mitochondrion but also is present in a second organelle closely associated with the mitochondrion. Scale bars: 5 μm.
Figure 9.
Localisation of LplA2 by Immunofluorescence Studies
Immunofluorescent analyses were performed on P. falciparum 3D7 using anti-LplA2 and anti-aLipDH (aE3) antibodies (specifically staining the apicoplast of the parasites) as outlined in the Materials and Methods section. The data suggest a similar distribution of LplA2 as that shown by expressing a C-terminally tagged LplA2-GFP construct in the parasites. Panel 1 shows staining of an organelle distinct from the apicoplast, probably being the mitochondrion. Panel 2 shows costaining of both antibodies, suggesting LplA2 is located in the apicoplast, and panel 3 shows staining of both the apicoplast and potentially the mitochondrion. Scale bars: 5 μm.