A metabolite-based resistance mechanism against malaria

Abstract

Jaundice is a common presentation of Plasmodium falciparum malaria, which arises from the accumulation of circulating bilirubin. It is not understood whether it represents an adaptive or maladaptive response to Plasmodium spp. infection. We found that asymptomatic P. falciparum infection in humans was associated with a higher ratio of unconjugated over conjugated bilirubin and parasite burden compared with symptomatic malaria. Genetic suppression of bilirubin synthesis by biliverdin reductase A (BVRA) increased parasite virulence and malaria mortality in mice. Accumulation of unconjugated bilirubin in plasma, through genetic inhibition of hepatic conjugation by UDP glucuronosyltransferase family 1 member A1 (UGT1A1), was protective against malaria in mice. Unconjugated bilirubin inhibited P. falciparum proliferation in red blood cells by a mechanism that suppressed mitochondrial pyrimidine synthesis. Moreover, unconjugated bilirubin inhibited hemozoin crystallization and compromised the parasite's food vacuole. Hence, jaundice appears to represent a metabolic response to Plasmodium spp. infection that limits malaria severity.

Fig. 1.. Unconjugated bilirubin confers resistance to malaria.

(A) Parasite burden (P. falciparum–infected RBCs per μl of blood) and concentrations of (B) total (i.e., conjugated plus unconjugated), (C) conjugated, and (D) unconjugated bilirubin (measured using the Roche Diazo method) in plasma from P. falciparum–infected individuals, stratified according to disease severity as asymptomatic and symptomatic malaria. (E) Plasma concentration of unconjugated bilirubin measured by the UnaG-based assay in same patients as in (A) to (D). (F) Ratio of unconjugated bilirubin measured by the UnaG-based assay (E) to conjugated bilirubin (C). Data in (A) to (F) are shown as box plots; red lines correspond to median values, and error bars correspond to the interquartile range (IQR). (G) Concentration of unconjugated bilirubin in plasma from Blvra^+/+ and Blvra^−/− mice (n = 6 to 9 per genotype), before (day 0) and after Pcc infection (days 4, 7, 15, and 25) as measured by the UnaG-based assay. Data are shown as means ± SD, pooled from two independent experiments with a similar trend. (H) Survival of Pcc-infected Blvra^−/− and control Blvra^+/+ mice. Data are from n = 7 mice per genotype, pooled from two independent experiments with similar trend. (I) Survival of Pcc-infected Blvra^−/− mice receiving bilirubin [30 or 3 mg per kg of body weight (mg/kg) daily and intraperitoneally] or vehicle. Data are from n = 4 to 10 mice per treatment, pooled from two independent experiments with a similar trend. (J) Quantification of Ugt1a1 mRNA [quantitative polymerase chain reaction (qPCR); left] (n = 7 to 9 per genotype) and protein (Western blot; right) (n = 6 per genotype) expression in the liver at day 7 after Pcc infection (Pcc) or in noninfected (NI) C57BL/6J mice. Data are shown as means ± SD, pooled from two independent experiments with a similar trend. (K) Concentration of unconjugated bilirubin in plasma of adult DBA/2 mice transduced 2 to 4 days after birth with AAV8-gRNA-Ugt1a1 repressing hepatic Ugt1a1 or control AAV8-Cas9. Data are from n = 5 mice per group, shown as means ± SD from one experiment representative of three with a similar trend. (L) Survival of the mice from (K) infected with Pcc. Circles correspond to P. falciparum–infected patients in (A) to (F) and to individual mice in (G) to (L). The p values were determined using [(A) to (F)] Mann-Whitney U test; (G) two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test (for genotypes) and ordinary one-way ANOVA with Tukey’s multiple comparison (for days after infection); [(H), (I), and (L)] log-rank (Mantel-Cox) test; [(J), right, and (K)] Student’s t test; and [(J), left] Mann-Whitney U test. NS is not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 2.. Unconjugated bilirubin regulates Plasmodium blood stage development and virulence.

(A) Percentage of infected RBCs and (B) parasite burden (infected RBCs/μl) in Pcc-infected Blvra^−/− and control Blvra^+/+ mice. Data are from n = 7 mice per genotype, pooled from two independent experiments with a similar trend. The same mice as in Fig. 1H are represented here. In (B), the right panel highlights day 8 from the left panel. (C) UMAP projection of single parasite transcriptomes, of circulating infected RBCs isolated by FACS from Blvra^+/+ (n = 1862; left) and Blvra^−/− (n = 3891; right) mice 7 days after Pcc infection. Colors and numbers identify different parasite developmental stages based on scmap projection to a P. falciparum atlas (see data tables S1 to S4). (D) Composition and (E) absolute number of parasite developmental stages. (F) UMAP projection as in (C), with arrows representing the relative change in transcriptional state based on RNA velocity analysis. (G) Survival of Blvra^−/− mice infected with Pcc-infected RBCs isolated from Blvra^−/− or Blvra^+/+ mice (n = 10 or 11 per genotype). (H) Percentage of infected RBCs (left) and parasite burden (infected RBCs/μl, right) for the same mice as in (G). Data are shown as means ± SD, pooled from three independent experiments with a similar trend. Circles in [(B), right] represent individual mice. The p values were determined using [(A), (B), left, and (H)] two-way ANOVA; [(B), right] Mann-Whitney U test; and (G) log-rank (Mantel-Cox) test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 3.. Bilirubin inhibits P. falciparum proliferation and disrupts its mitochondrial integrity and function.

(A) Bilirubin accumulation in Pf3D7-infected RBCs (trophozoites), 8 and 12 hours after exposure to unconjugated bilirubin (41 μM) or vehicle. Data are shown as means ± SD, from one experiment with five technical replicates. (B) Percentage (left) of Pf3D7-infected RBCs, 24 to 72 hours after exposure of ring stages to increasing concentrations of unconjugated bilirubin. Data are shown as means ± SEM, pooled from six independent experiments with a similar trend, with four technical replicates per experiment. On the right are representative Giemsa-stained thin smears of Pf3D7-infected RBCs, 24 hours after exposure to unconjugated bilirubin (41 μM) or vehicle. The black arrowhead highlights parasite nuclear DNA fragmentation. Scale bars are 2 μm. (C) Relative levels of glucose, glucose 6-phosphate, succinate, Asp, fumarate, and AMP in Pf3D7-infected RBCs (trophozoites), as detected by mass spectrometry 12 hours after exposure to unconjugated bilirubin (41 μM) or vehicle. Data are shown as means ± SD, from one experiment with five technical replicates (see data table S5). (D) Schematic representation of differentially expressed genes involved in metabolic processes in Pcc-infected RBCs isolated from Blvra^−/− and Blvra^+/+ mice, as determined by scRNA-seq, in the different clusters identified in Fig. 2, C to F. (E) Representative histograms (left) and quantification of median fluorescence intensity (MFI; right) of mitochondrial volume (MitoTracker Green), 24 hours after exposure of Pf3D7-infected RBCs (rings) to increasing concentrations of unconjugated bilirubin. The gray histogram represents background staining in noninfected RBCs, and the dashed gray line (right) represents the average background signal from all replicates in noninfected RBCs. Data are shown as means ± SD from four replicates in one out of two independent experiments with a similar trend. (F) Relative level of dihydroorotate (DHO; left), orotate (middle), and uridine monophosphate (UMP; right), in Pf3D7-infected RBCs (trophozoites), as detected by mass spectrometry 12 hours after exposure to unconjugated bilirubin (41 μM) or vehicle (see data table S5). DMSO, dimethyl sulfoxide. (G) Percentage (left) of PfD10^TgDHODH-infected RBCs (rings) after exposure to increasing concentrations of unconjugated bilirubin. Data are shown as means ± SEM, pooled from three independent experiments with a similar trend, with four biological replicates per experiment. On the right are representative Giemsa-stained thin smears of Pf3D7-infected RBCs, 24 hours after exposure to unconjugated bilirubin (41 μM) or vehicle. The black arrowhead highlights pyknotic parasites. Scale bars are 2 μm. Dots represent technical replicates in (A), (E), and (F) and individual mice in (C). The p values were determined using [(A) and (E)] ordinary one-way ANOVA with Tukey’s multiple comparison test; [(B) and (G)] two-way ANOVA with Tukey’s multiple comparison test; and [(C) and (F)] Mann-Whitney U test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The concentrations of unconjugated bilirubin in (A) to (C) and (E) to (G) were calculated according to the UnaG-based assay (21, 22) (see fig. S10).

Fig. 4.. Bilirubin inhibits Hz formation and disrupts the P. falciparum food vacuole.

(A) Live confocal microscopy of Pf3D7-infected RBCs (trophozoites), 12 hours after exposure to unconjugated bilirubin (41 μM) or vehicle. Representative images (left) and quantification of Hz cellular intensity distribution (laser reflection mode, cyan; area, right) are shown. RBC membranes are stained with wheat germ agglutinin (RBC; red), and nuclei (N) with Hoechst (DNA; blue). Data are shown as means ± SD from two independent experiments (n = 10 to 15 parasites). Scale bars are 4 μm. (B) Relative inhibition of β-hematin crystallization by bilirubin versus chloroquine. Equimolar amounts of brivanib alaninate (a tyrosine kinase inhibitor) were used as control. The relative inhibition of β-hematin crystallization was inferred from heme accumulation measured at a wavelength of 405 nm (λ₄₀₅ nm) (95) and is represented as the mean of λ/λ₄₀₅ nm ± SD from two replicates in one out of two independent experiments with a similar trend. (C) Hz quantification in blood from Blvra^+/+ and Blvra^−/− mice 7 days after Pcc infection, at daily light (left) and dark (right) cycles. Data for Hz (i.e., nM heme) are shown as means ± SD, pooled from two independent experiments with a similar trend (n = 11 for light cycle and n = 9 or 10 for dark cycle per genotype). (D) Representative images (left) and quantification (right) of the food vacuole. RBC membranes are stained with wheat germ agglutinin (RBC; red), the parasite’s food vacuole (FV) with LysoTracker Green (green), and nuclei (N) with Hoechst (DNA; blue). Data are shown as means ± SD from two independent experiments (n = 10 to 13 parasites), as in (A). Scale bars are 4 μm. AU, arbitrary units; AUC, area under the curve. (E) Representative transmission electron microscopy images of Pf3D7-infected RBCs (trophozoites), 12 hours after exposure to unconjugated bilirubin (41 μM) or vehicle. The images on the right correspond to amplifications of the orange dotted and dashed areas highlighted in the images on the left. EV, endocytic vesicle; FV, food vacuole; Hz, hemozoin; MLB, multilamellar bodies; N, nucleus; RBC, red blood cell. Images are representative of three independent experiments with similar results. Scale bars are 500 nm. (F) Relative levels of amino acids in Pf3D7-infected RBCs (trophozoites), as detected by mass spectrometry, 12 hours after exposure to unconjugated bilirubin (41 μM) or vehicle (see data table S5). Circles represent individual parasites in (A) and (D), individual mice in (C), and technical replicates in (F). The p values were determined using [(A), (C), (D), and (F)] Mann-Whitney U test. NS is not significant; *p < 0.05; **p < 0.01; ****p < 0.0001. The concentrations of unconjugated bilirubin in (A) to (C) and (E) to (G) were calculated according to the UnaG-based assay (21, 22) (see fig. S10).