Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility

Abstract

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population.

Fig. 1. Hierarchical clustering of PAPs.

Clustering of PAPs from six replicates (R1–R6) of human erythrocyte DRMs is shown as a heatmap. Dendrogram architecture and Pearson’s correlation coefficients corresponding to principal nodes are also reported. Pearson’s correlation values for clusters C1–C9, with coefficients R ≥ 0.6 (P ≤ 0.005), are shown in black. Protein functional assignment in clusters with more than three members is shown as pie charts. Shades of blue relate to the relative abundance values of the identified proteins.

Fig. 2. Subcellular localization of CD55, ART4, AQP1, and Flotillin-1.

a IFA of early trophozoites (ring stages) with anti-ART4, anti-AQP1, and anti-CD55 antibodies. b IFA of a ring-stage parasite with anti-ART4 and anti-N201, used as a parasitophorous vacuole marker. c IFA of trophozoite stage parasites with visible hemozoin, stained with anti-ART4 and anti-Flotillin-1 (Pearson’s correlation coefficient: 0.56). d IFA of invading parasites with anti-ART4 antibody and anti-AQP1 antibody. Anti-RON4 antibody was used as a marker of the moving junction. BF Bright field. Nuclei are stained with DAPI. Scale bar: 10 µm.

Fig. 3. The genetic disruption of ART4 leads to a perturbation in invasion efficiency of P. falciparum.

a Flow plots showing the loss of surface expression of AQP1 and ART4 on their respective ejRBC knockout cells (ΔART4 and ΔAQP1). b Representative images of Giemsa-stained cytospins of differentiated day 8 WT cas9, ΔART4, ΔAQP1, and ΔBSG ejRBCs before (left panel) and after (right panel) invasion by P. falciparum. Arrows point at intracellular P. falciparum parasites. c Graph of invasion efficiencies for WT cas9, ΔART4, ΔAQP1, and ΔBSG ejRBCs (n = 3) during a 18 h invasion assay. Error bars represent standard deviations. d Graph of invasion efficiencies for WT cas9, ΔART4 and ΔBSG ejRBCs (n = 3) during a 4 h invasion assay. NS not significant (p > 0.05). **p ≤ 0.01, ****p ≤ 0.0001 (one-way ANOVA). ΔBSG was used as a negative control in this experimental assay.

Fig. 4. Signals of association between genetic variation at ART4 and AQP1 loci and malaria phenotypes in Mossi children from Burkina Faso.

The figure shows results of association analysis of SNPs at ART4 (a) and AQP1 (b) with malaria outcomes in a case–control sample set from Burkina Faso. In panel (i) each locus is shown in its genomic context (chromosome: start base pair-end base pair) with the arrow indicating transcription direction, empty boxes indicating UTR regions, full boxes indicating exons, and lines indicating introns. In panels (ii) and (iii) the y axis indicates the minus logarithm (base 10) of the p-value for the best model of the likelihood ratio test of association between a given SNP and the malaria phenotype, the x axis indicates SNPs ordered by chromosome position, the horizontal dashed lines indicate significance levels (red: p = 0.01; yellow: p = 0.05), the vertical gray bar indicates the gene region showing significant results with both severe malaria and parasite density, and dots of greater dimension and darker color indicate genotyped SNPs whose results are described in the main text.