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, 15 (9), 1585-604

A Key Role for Lipoic Acid Synthesis During Plasmodium Liver Stage Development

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A Key Role for Lipoic Acid Synthesis During Plasmodium Liver Stage Development

Brie Falkard et al. Cell Microbiol.

Abstract

The successful navigation of malaria parasites through their life cycle, which alternates between vertebrate hosts and mosquito vectors, requires a complex interplay of metabolite synthesis and salvage pathways. Using the rodent parasite Plasmodium berghei, we have explored the synthesis and scavenging pathways for lipoic acid, a short-chain fatty acid derivative that regulates the activity of α-ketoacid dehydrogenases including pyruvate dehydrogenase. In Plasmodium, lipoic acid is either synthesized de novo in the apicoplast or is scavenged from the host into the mitochondrion. Our data show that sporozoites lacking the apicoplast lipoic acid protein ligase LipB are markedly attenuated in their infectivity for mice, and in vitro studies document a very late liver stage arrest shortly before the final phase of intra-hepaticparasite maturation. LipB-deficient asexual blood stage parasites show unimpaired rates of growth in normal in vitro or in vivo conditions. However, these parasites showed reduced growth in lipid-restricted conditions induced by treatment with the lipoic acid analogue 8-bromo-octanoate or with the lipid-reducing agent clofibrate. This finding has implications for understanding Plasmodium pathogenesis in malnourished children that bear the brunt of malarial disease. This study also highlights the potential of exploiting lipid metabolism pathways for the design of genetically attenuated sporozoite vaccines.

Conflict of interest statement

All authors hereby confirm that they have no conflict of interest in the publication of this research.

Figures

Fig. 1
Fig. 1. Generation of PbΔLipB knockout parasites in Plasmodium berghei
A. Schematic representation of lipoic acid synthesis and scavenging in the Plasmodium apicoplast and mitochondrion respectively. Enzymes responsible for the synthesis or attachment of lipoic acid to its target proteins are represented in blue. LipB and LipA synthesize lipoic acid from the octanoyl-acyl carrier protein (ACP) precursor generated by the fatty acid biosynthesis type II (FAS-II) pathway. LipB is responsible for the attachment of octanoyl-ACP to the E-2 subunit of the pyruvate dehydrogenase (PDH) complex within the apicoplast. LipA is responsible for creating the thiosulfur bonds. PDH converts pyruvate into acetyl-CoA, which primes the FAS-II pathway. In the mitochondria, LplA1 attaches scavenged lipoic acid to α-ketoglutarate dehydrogenase (KGDH) and branched-chain α-ketoacid dehydrogenase-E2 subunit (BCDH), which both feed into the tricarboxylic acid (TCA) cycle, as well as the H-protein of the glycine cleavage system. Attachment of scavenged lipoic acid can be inhibited by the analog 8-BOA that targets the ligase LplA1. A second lipoate ligase, LplA2, has been localized to both the apicoplast and the mitochondria. B. Schematic representation of the replacement strategy used to delete the PbLipB gene, based on homologous recombination and double crossover events between the pL0001-ΔPbLipB donor plasmid and the PbLipB genomic locus. C. PCR confirmation of the PbLipB gene deletion and its replacement with Tgdhfr-ts. The left panel shows PCR products specific to the PbLipB coding sequence and its 5′ and 3′ UTRs in the parental P. berghei ANKA strain. This showed the expected band sizes of 0.70, 0.76 and 0.98 kb for p1+p2, p3+p4 and p5+p6 respectively. The right panel shows the replacement of PbLipB sequence with the Tgdhfr-ts marker in a PbΔLipB knockout clone. This shows the expected band sizes of 1.0, 0.98 and 0.93 kb for p3+p7, p6+p8 and p9+p10 respectively. D. Reverse transcriptase (RT)-PCR studies showing the loss of PbLipB transcription in PbΔLipB parasites and the expression of this gene in asexual blood stages (ABS) and liver stages (LS) in wild-type (WT) P. berghei ANKA parasites. + and − denote with and without RT. Reactions with the LipB-specific primers p1 + p2 showed the expected 0.7 kb transcription product in WT but not in KO parasites. Control reactions with P. berghei actin I-specific primers yielded the expected products with both KO and WT cDNA preparations (data not shown).
Fig. 2
Fig. 2. PbΔLipB parasites show normal kinetics of blood stage replication but show reduced lipoylation of parasite dehydrogenases
A, B. Blood stage growth kinetics of PbΔLipB and parental wild-type (WT) strains, in mice inoculated with 10,000 or 1,000 asexual blood stage parasites. Five mice were infected with each strain and the experiment performed on three occasions. Mean parasitemias were calculated from each experiment and used to determine the average ± SEM shown here for each day post-inoculation. Mann-Whitney U tests showed no significant differences in growth kinetics between the two strains. C. Apicoplast morphology in WT and PbΔLipB asexual blood parasites. These parasites were collected from infected mice, allowed to mature overnight in vitro, fixed, and stained with ACP-specific antibodies that label the apicoplast as well as DAPI that stains the nucleus. Microscopic examination revealed no significant difference in apicoplast morphology between these strains. D. Western blot analysis of WT and PbΔLipB parasite protein extracts (16 μg per lane) labeled with antibodies specific to lipoic acid. PDH-E2, pyruvate dehydrogenase E2 subunit; BCDH-E2, branched-chain α-ketoacid dehydrogenase E2 subunit; KGDH-E2, α-ketoglutarate dehydrogenase E2 subunit. The membrane was stripped and re-probed with antibodies specific to P. falciparum Hsp70 as a loading control. Data are presented as one representative image from three independent experiments. BCDH-E2 was detected in the PbΔLipB parasites at longer exposures (data not shown). Based on band intensity quantification (using ImageJ; NIH), we estimated that lipoylated PDH-E2, BCDH-E2 and KGDH-E2 were present in the LipB-deficient parasite at 16%, 2% and 98% of WT, normalized for loading based on the intensity of Hsp70 labeling. E. The BCDH-E2 protein localizes to the mitochondria in blood stages. Parasites expressing BCDH-E2-GFP fusion proteins were stained with MitoTracker Red, revealing co-localization with the mitochondrial dye. F. The BCDH-E2 protein is not localized to the apicoplast. BCDH-E2-GFP parasites were fixed and stained with anti-ACP antibodies to identify the location of the apicoplast, which did not localize with BCDH-E2. White bar, 10 μm (for all panels in this Figure).
Fig. 3
Fig. 3. Blood stage PbΔLipB parasites display reduced growth rates following inhibition of lipoic acid scavenging from the host or reduction of host lipid levels with clofibrate
A, B. Effect of 8-BOA on growth rates of LipB KO and WT asexual blood stage parasites. (A) P. berghei WT or (B) PbΔLipB parasites were treated with concentrations of 100, 200 or 400 μM 8-BOA or DMSO control during overnight in vitro incubations, followed by injections of 10,000 parasites into each mouse. Parasitemias were monitored daily, and are presented as means ± SEM of three independent experiments each with 5 mice per group. The 8-BOA treatments showed a dose-dependent effect and a stronger impact on the PbΔLipB parasites than on the WT control. Tests for significance employed the Mann-Whitney U test that compared parasitemias between the groups of 15 mice injected with parasites that were untreated, versus those injected with parasites treated at a given 8-BOA concentration. *P<0.05; **P<0.01; ***P<0.001. C, D. Mice were treated with either 0.5 mg/kg, 5.0 mg/kg of clofibrate or DMSO as vehicle control prior to inoculation with either P. berghei ANKA or PbΔLipB parasites. Untreated controls showed TG and NEFA mean ± SD concentrations of 0.17 ± 0.03 and 0.16 ± 0.06 mM respectively (n=30). Parasitemias were monitored daily for 9 days and are represented as the averages ± SD of three independent experiments each with 5 mice per group. Statistical tests employed the Mann-Whitney U test that compared DMSO-treated controls with each clofibrate concentration. **P<0.01; ***P<0.001.
Fig. 4
Fig. 4. PbΔLipB parasites show no residual lipoylated proteins after 8-BOA treatment
A. HepG2 cells were infected with P. berghei ANKA mCherry-mito sporozoites and treated with either 200 μM 8-BOA or DMSO as vehicle control. At 54 hpi, infected cells were fixed and stained with anti-lipoic acid (shown in green) to identify lipoylated proteins within the parasite and the HepG2 cell host. The mitochondria is shown in red, while the nucleus (stained with DAPI) is in blue. DIC, differential interference contrast. The dashed white line outlines the portion of the parasite magnified as the Inset in the last column (also for panel B). White bar, 10 μm. White arrows in the inset of WT parasites treated with DMSO control illustrate lipoic acid signal in the mitochondrion (evident as an orange signal). B. HepG2 cells infected with PbΔLipB sporozoites were treated with either 200 μM 8-BOA or DMSO as a vehicle control. At 48 hpi, infected cells were stained with MitoTracker Red to label the mitochondria (red). Cells were fixed and stained with anti-lipoic acid to identify lipoylated proteins (shown in green). DAPI dye identified nuclear structures (blue). White bar, 10 μm. The white arrow in the KO parasite treated with DMSO illustrates some lipoic acid signal in the mitochondrion. This signal is largely absent in 8-BOA treated KO parasites. C. The amount of lipoylated protein within the parasite was quantified using Python and the MATLAB Image Analysis Toolbox and the signal intensity determined from the anti-lipoic acid images, after cropping for parasite-specific areas, for WT and PbΔLipB parasites treated with 8-BOA or DMSO vehicle control. Data are presented as the mean ± SEM intensity per μm2 of parasite. This analysis revealed significant differences between WT and KO parasites, including reduced lipoylation in PbΔLipB compared to WT parasites following 8-BOA treatment (*p<0.05; **p<0.01; Mann-Whitney U test; n = 4 to 8 parasites per condition and per strain).
Fig. 5
Fig. 5. PbΔLipB parasites have a less branched apicoplast in comparison to wild-type cells
A. HepG2 cells were infected with WT or PbΔLipB sporozoites and the apicoplast was labeled using ACP-specific antibodies (labeling shown in green) at several time points throughout liver stage development. To visualize the developing parasite, primary antibodies (labeling shown in red) were used as follows: anti-CSP at 6 hpi; anti-Exp-1 at 30 & 48 hpi; anti-MSP-1 at 54 hpi and with mature merozoites (imaged at 62 hpi for WT parasites and 72 hpi for PbΔLipB parasites). White bar, 10 μm. B. Plot of intensity of apicoplast staining for WT vs. KO parasites sampled at 48 and 54 hi. Intensity was measured from the anti-ACP image and determined per μm2 of parasite after cropping the images for the parasite-specific areas (bounded by the parasitophorous vacuole). Results showed a statistically significant two-thirds reduction in apicoplast signal in KO compared to WT parasites (***p<0.001; Mann-Whitney U test, n = 12–18 per group). Additional images of parasites sampled at 48 and 54 hpi are provided in Figs. S4 and S5 respectively.
Fig. 6
Fig. 6. PbΔLipB parasites are defective in late liver stage development
A. HepG2 cells were inoculated with either P. berghei WT or PbΔLipB sporozoites, both constitutively expressing GFP. Sizes of developing parasite forms were measured at 48 hpi from 150 to 200 parasites per well (obtained from two independent experiments performed in duplicate) and were quantified using ImageJ. Data are presented as a box and whiskers plot that illustrates the mean, interquartile range (25–75%) and 95% confidence interval. B. HepG2 cells were infected with equal numbers of WT and KO sporozoites. Parasite numbers were determined by counting fixed and stained developing liver stage parasites, shown for time points 24, 48 and 84 hpi, and counting of live Hoechst-stained detached cells at 65 hpi. Data are shown as the mean ± SEM generated from two independent experiments performed in duplicate. C. Liver stage parasites were imaged for DAPI (blue) and MSP-1 (green) expression at different stages of development. The left column shows WT control parasites imaged at the cytomere stage (sampled at 54 hpi). The three right panels show PbΔLipB liver parasites developing within HepG2 cells at late schizogony, cytomere and merozoite stages (sampled at 54, 62 and 70 hpi). White bar, 10 μm.

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