Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum

Abstract

Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.

Keywords: Kennedy pathway; Plasmodium falciparum; environmental sensing; lysophosphatidylcholine; malaria; phospholipid metabolism; sexual differentiation; transmission.

Graphical abstract

Figure 1

P. falciparum Sexual Commitment Is Subject to Host-Factor Availability (A) EVs induce gametocyte formation at high concentrations. Parasites were challenged with EVs isolated from high-parasitemia cultures at the original (1×) and 10× concentrations. Bars show fold change of sexual differentiation normalized to the untreated (no EVs) condition. Absolute differentiation rates were generally low (average of 0.41% in “no EVs” and 1.12% in “10xEVs” condition). n = 3, standard deviations are shown; ns, not significant; ^∗ p < 0.05, Student’s t test. Parasites of strain Pf2004 were used (transfected with reporter plasmid 164tdTom [Pf2004/164tdTom]). (B) Conditioned medium induces sexual commitment independently of parasite-derived vesicles. +SerM, serum-supplemented medium; CM, conditioned medium. Bars show sexual differentiation rates (left axis). Effect on parasite growth is indicated (right axis). n = 3; standard deviations are shown; ns, not significant, Student’s t test. (C) Parasites induce sexual differentiation in response to serum depletion in −SerM conditions. n = 3, standard deviations are shown, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, Student’s t test. CM, conditioned medium; “/Serum” indicates supplementation with 10% serum. (D) Serum depletion induces sexual commitment across different P. falciparum strains. n = 3, standard deviations are shown, ^∗∗ p<0.01, Student’s t test. Parasites of strain HB3 (transfected with reporter plasmid 748tdTom [HB3/748tdTom]) and wild-type parasites of strain NF54 were used. (E) Sexual differentiation is irreversibly determined after 38 ± 2 hpi. Except for the +SerM control, all cultures were exposed to −SerM at 28 ± 2 hpi. −SerM was exchanged by +SerM at indicated time points. n = 3, standard deviations are shown, ^∗∗ p < 0.01; ns, not significant; Student’s t test.

Figure 2

Identification of LysoPC as a Regulator of Transmission-Stage Formation in P. falciparum (A) Polarity-based separation of serum components yields LysoPC-enriched fractions with sexual differentiation-inhibiting activity (highlighted in red). See Figure S1 for details on activity and composition of fractions. (B) Sexual commitment inhibiting activity of LysoPC-containing fractions (left panel) and LC-MS chromatograms and mass spectra of most active fractions (right panel) are shown. An extracted ion chromatogram for the [M+H]⁺ ion of LysoPC (16:0) is highlighted in red. Bars in the left panel quantify sexual differentiation normalized to a −SerM-exposed control population. n = 3, data from a representative experiment is shown. Standard deviations of technical triplicates are indicated. (C) LysoPC inhibits sexual differentiation of Pf2004/164tdTom parasites (half maximal inhibitory concentration [IC50]). Effect of 20 μM LysoPC on HB3/748tdTom parasites is shown in grey. Sexual differentiation is normalized to −SerM-cultured control populations. n = 3, standard errors are shown. (D) LysoPC is internalized by asexual P. falciparum parasites. Uptake was analyzed by live microscopy using TopFluor-labeled LysoPC (green), ER tracker (red) and DNA dye Hoechst (blue). Incorporation is apparent after completion of ring stage development (upper left panel) and the label accumulates at the ER of trophozoites within 75 s after addition of TopFluor LysoPC (lower left panel). LysoPC uptake in the parasite is quantified relative to accumulation at the erythrocyte surface (right panel). Mean fluorescence intensities (MFIs) and standard deviations are shown. Scale bar, 4 μm. 100 cells were analyzed per stage in triplicate experiments. (E) LysoPC is depleted in parasite-exposed CM. Bars quantify area under the curve. n = 3, standard errors are shown. (F) In contrast to different LysoPC species, non-hydrolyzable analogs of LysoPC fail to prevent −SerM-induced sexual differentiation. All molecules were tested at 20 μM. n = 3, standard deviations are shown, ^∗∗∗ p < 0.001, Student’s t test. See Figure S1C for chemical structures. (G) LysoPC-depleted culture conditions (−SerM) result in reduced number of daughter merozoites per schizont (left axis) and reduced multiplication rate (right axis) compared to +SerM and −SerM/LysoPC conditions. n = 100. Interquartile ranges are shown. See also Figures S1 and S2.

Figure S1

Related to Figure 2 (A) Stepwise fractionation of serum (fractions A1–A8) identifies LysoPC as the active component. See Figure 2A for schematic of experimental approach. Bars quantify sexual differentiation normalized to a −SerM- or CM-exposed control population. Active fractions were tested in biological triplicates or quadruplicates as described (Brancucci et al., 2015). Data from a representative experiment is shown and standard deviations of technical triplicates are indicated. CM, 80% conditioned medium. (B) LC-MS chromatograms and mass spectra of B fractions (see above) are shown; LysoPC species are highlighted in color. (C) Chemical structures of tested analogs.

Figure S2

Related to Figures 2 and 3 (A) Live cell microscopy comparing uptake of TopFluor-labeled LysoPC between P. falciparum gametocytes and asexual parasites. LysoPC uptake in the parasite is quantified relative to accumulation at the erythrocyte surface (left panel). 100 cells were analyzed per stage. Representative pictures are shown (right panel). Standard deviations are shown, (^∗∗∗p < 0.001, Student’s t test). (B) Increase of exogenous supply of Kennedy pathway metabolites does not affect P. falciparum parasite sexual differentiation. DG, diglycerides; PA, phosphatidic acids; PC, phosphatidylcholines; PE, phosphatidylethanolamine. n = 3, standard deviations are shown. (C) Levels of parasite phosphatidic acids (PAs), diglycerides (DGs), phosphatidylcholines (PCs), LysoPC(16:0) and choline are significantly increased in the presence of a high concentration of choline. Bars show chromatographic areas under the curve. Colors indicate the contribution of labeled and unlabeled molecules to the total peak areas. n = 3, standard errors of the means are shown. (D) Glucose levels have no autonomous effect on parasite sexual differentiation. While glucose can prevent sexual differentiation in absence of LysoPC (see Figure 3F), limiting levels of this sugar do not induce gametocyte formation under LysoPC-rich conditions. n = 3, standard deviations are shown.

Figure 3

LysoPC Is Metabolized and Drives PC Biosynthesis via the Kennedy Pathway (A) Addition of heavy-isotope-labeled LysoPC increases levels of palmitate and choline in parasites. Bars show chromatographic areas under the curve. Colors indicate the contribution of labeled and unlabeled molecules to the total peak areas. n = 3, standard errors of the means are shown. ¹³C palmitate LysoPC or ²H choline LysoPC was used. (B) Levels of parasite phosphatidic acids (PAs), diglycerides (DGs), and phosphatidylcholines (PCs) are significantly increased in presence of LysoPC and elevated lipids contain LysoPC-derived building blocks. Bars show chromatographic areas under the curve. Colors indicate the contribution of labeled and unlabeled molecules to the total peak areas. n = 3, standard errors of the means are shown. (C) The Kennedy pathway requires CDP-choline and DGs deriving from LysoPC and glycolysis products, respectively, for PC synthesis. LysoPA, lysophosphatidic acid; DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; PA, phosphatidic acid; P-choline, phosphocholine; CDP-choline, cytidine diphosphate choline; DG, diglyceride. (D) Live cell microscopy shows inefficient incorporation of fluorescent Kennedy metabolites PC (TopFluor-labeled) and PA (nitrobenzoxadiazole [NBD]-labeled) compared to LysoPC (TopFluor-labeled). Scale bar, 4 μm. Representative pictures are shown. (E) Choline inhibits sexual differentiation at super-physiological concentrations (IC50 of 207 μM; 95% CI 169–253 μM); human serum contains ∼10 μM choline (Psychogios et al., 2011). Activity of choline was assayed in presence of 11.1 mM glucose in −SerM medium. n = 3, standard errors are shown. (F) In absence of exogenous LysoPC, glucose is required in addition to excess choline to inhibit sexual differentiation, reiterating the importance of Kennedy-mediated PC synthesis in this process. n = 3, standard deviations are shown, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, Student’s t test. (G) In presence of glucose, exogenously added choline mirrors the effect of LysoPC and elevates levels of parasite PA, DG, and PC. n = 3, standard errors of the means are shown. See also Figure S2 and Dataset S1.

Figure 4

LysoPC Depletion Induces Activity of More Than 300 Genes in P. falciparum (A) Transcriptional responses of two parasite strains (Pf2004/164tdTom and NF54; combined) to absence of LysoPC are shown (left panel). Significantly up- and downregulated genes are highlighted in red and blue, respectively. Data show differential transcription between parasites cultured in −SerM and −SerM/20 μM LysoPC. No significant differential expression could be detected between parasites cultured in +SerM and −SerM/20 μM LysoPC (right panel). (B) Clustering of differentially expressed genes reveals that LysoPC depletion elicits a temporal transcriptional cascade in P. falciparum. Differential induction of genes (−SerM vs. −SerM/LysoPC conditions) per time point is shown in a heat map (left panel) and stacked line graph (middle panel). For each time point, the highest-enriched gene ontology (GO) term (p < 0.05) and a subset of significantly induced genes are shown (right panel). Factors involved in epigenetic regulation and/or differentiation are marked in green. (C) CK inhibitor BR23 (Serrán-Aguilera et al., 2016) reverses activity of LysoPC and excess choline, corroborating importance of the Kennedy pathway in regulating sexual differentiation. n = 3, standard deviations are shown, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, Student’s t test. (D) PMT expression is increased in sexually committed schizonts. Enzyme levels were quantified in a time-course experiment. While PMT is generally induced under −SerM conditions, sexually committed cells show a more prominent induction. MFI, mean fluorescence intensity; a minimum of 100 infected red blood cells (iRBCs) were analyzed by confocal microscopy. Measurements were repeated in triplicate experiments. Stadard errors of the means are shown. See also Figures S3 and S4 and Dataset S2.

Figure S3

Related to Figure 4 (A) Schematic depiction of parasite culturing setup used for transcriptional analysis (upper left panel). Pf2004/164tdTom and NF54 parasite cultures were split at 30±2 hpi and parasites were subsequently grown under conditions that either induce (−SerM) or do not induce (−SerM/LysoPC; +SerM) sexual differentiation. Time points of RNA harvest are indicated. Effect of culture conditions on parasite sexual differentiation and multiplication is shown (lower left panels, standard deviations are indicated). Global effect of culture conditions on differential gene expression (Pf2004/164tdTom and NF54, combined) is shown in scatterplots on the right. FPKM, fragments per kilobase of exon per million mapped reads. (B) Effect of LysoPC on gene expression of selected genes. Normalized read counts are given for different conditions (−SerM and −SerM/LysoPC) and time points. Differential gene expression is indicated by shaded areas (red, Pf2004/164tdTom; blue, NF54). FPKM, fragments per kilobase of exon per million mapped reads.

Figure S4

Related to Figure 4 (A) Schematic maps of the endogenous ap2-g locus (PF3D7_1222600) in wild-type parasites (top), the pH_gC-ap2g-3’ and pD_ap2g-gfp transfection vectors (center), and the edited ap2-g-gfp locus after CRISPR/Cas9-based marker-free fusion of the gfp sequence to the 3’end of the ap2-g coding sequence in NF54/AP2-G^GFP parasites (bottom). Numbers refer to nucleotide positions on chromosome 12 (http://plasmodb.org/). The position of the sgt_ap2g3’ sgRNA target sequence 60bp downstream of the ap2-g coding sequence is indicated. The pH_gC-ap2g-3’ Cas9/sgRNA suicide plasmid contains expression cassettes for SpCas9, the sgRNA and the hdhfr resistance marker. In the pD_ap2g-gfp donor plasmid two homology regions (5’HR and 3’HR) flanking the gfp coding sequence facilitate repair of the Cas9-induced double-strand break and marker-free tagging of the ap2-g gene by double crossover homologous recombination. Yellow asterisks indicate the position of translation termination codons. Positions of PCR primer binding sites used to confirm successful gene editing are indicated by horizontal black arrows. (B) PCR on gDNA isolated from NF54/AP2-G^GFP and 3D7 wild-type control parasites. Primers apF and apR bind to wild-type sequences outside the ap2-g 5’HR and ap2-g 3’HR homology regions and amplify a 2218bp or 1502bp fragment from the edited ap2-g-gfp or wild-type ap2-g locus, respectively. The ap2F/gfpR and gfpF/ap2R primer combinations are specific for the edited ap2-g-gfp locus in NF54/AP2-G^GFP parasites and amplify 1170 and 1222bp fragments, respectively.

Figure 5

LysoPC Is Metabolized, but Does Not Control Differentiation, in Rodent Malaria Parasites (A) Phylogenetic analysis reveals loss of key LysoPC-responsive genes in the rodent malaria lineage. Lineage-specific losses are marked by hatched area. (B) LysoPC does not affect sexual differentiation of rodent parasites. Gametocyte production of P. berghei parasites in response to different ex vivo culture conditions (+SerM, −SerM, or −SerM/LysoPC) is shown. Effect on sexual differentiation was measured after parasites were delivered into a recipient mouse. n = 5, standard deviations are shown, (ns, not significant; Student’s t test). (C and D) LysoPC depletion reduces number of progeny in P. berghei. Growth response to different ex vivo culture conditions (+SerM, −SerM, or −SerM/LysoPC) is shown. (C) Average merozoite counts per infected erythrocyte were quantified after subtracting gametocyte-infected cells (single nucleated cells in +SerM condition at 30 hpi were used to define gametocyte proportion in samples). Interquartile ranges are shown. (D) Merozoite number was quantified per infected erythrocyte and assigned to one of five categories (indicated). Shown are relative proportions of each category. Merozoites of 30–65 infected erythrocytes were counted per time point.

Figure 6

Physiological LysoPC Levels Are Conducive to Sexual Commitment (A) LysoPC levels drop during bacterial and parasitic infection in humans. The effect of different infections on serum LysoPC concentration is shown (Drobnik et al., 2003, Lakshmanan et al., 2012, Lamour et al., 2015, Ollero et al., 2011, Orikiiriza et al., 2017). CF, cystic fibrosis. (B) LysoPC levels drop during rodent malaria infection. LysoPC species were quantified in mice infected with P. berghei (parasitemia of 10%–14%) and in healthy control mice. (C) LysoPC levels in P. berghei-infected mice correlate negatively with parasite burden. Shown is the correlation between LysoPC concentration (different species are indicated) and parasitemia in peripheral blood of five mice. Correlation coefficients are indicated. Concentrations of LysoPC species are normalized to maximum concentration found across all mice (see Figure S5 for absolute quantities). Standard errors of the mean are shown. (D) Elevated BSA levels increase the IC50 of LysoPC for sexual commitment. Higher LysoPC concentrations are required to prevent parasite sexual commitment in presence of 3.9% BSA compared to 0.39% BSA. n = 3. Standard errors are shown. (E) LysoPC levels are lower in bone marrow compared to serum. Shown are LysoPC levels in serum and cell-free bone marrow extracts from healthy mice. n = 5. Standard errors of the means are shown. (F) Sexual differentiation rates of Pf2004/164tdTom parasites cultured in erythrocytes of different maturity. Parasites were allowed to invade into reticulocyte-enriched erythrocyte populations, and sexual differentiation was monitored for inducing (−SerM) and for non-inducing (−SerM/LysoPC and +SerM) conditions. Each data point represents a biological replicate (mean of technical triplicates). Sexual differentiation rates were normalized to a reticulocyte-negative control population cultured under inducing −SerM conditions. See also Figure S5.

Figure S5

Related to Figure 6 LysoPC drops in response to parasite infection. Serum LysoPC levels were quantified in 5 mice infected with P. berghei and in 2 non-infected controls. LysoPC concentrations decrease in a parasitemia-dependent manner. Bars quantify area under the curve. Shown are standard errors of the means from 3 technical replicates.