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

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.

study samples have been deposited in the European Genome-Phenome Archive (EGA; study accession EGAS00001001311); whole-genome sequence read data have been deposited in the EGA (study accession EGAS00001003648); access to MalariaGEN datasets on EGA is by application to an independent data access committee.
For co-localization analyses, two biological replicates were taken into account and at least 30 cells were analyzed in each replicate, leading to average Pearson's correlation coefficients of 0.97 for AQP1-ART4, 0.96 for CD55-ART4 and 0.96 for N201-ART4 with standard deviations <0.01. For the functional analysis, 3 biological replicates were performed, each carried out in triplicate. A minimum of 30 parasites were counted in each experiment, leading to P values probabilities (one-way ANOVA) of P # 0.01 for the wt/ART4 ko comparison and non significance for the wt/AQP1 ko. For genetic association study, sample size was not pre-determined based on statistical calculation, but was based on the availability of collected clinical data/biological specimens and on pre-established quality control filters. The same case-control sample set has been used in previously published studies that could demonstrate significant associations of a range of effect sizes (doi: 10.1038/35104556; doi: 10.1038/ sj.gene.6364456; doi: 10.1038/s41467-019-13480-z).
No data were excluded from the analyses, except for proteomic data. For proteomic analysis, proteins identified with 1 unique peptide were excluded from data analysis (functional annotation, comparative analysis). To improve robustness of the cluster analysis (Pearson's correlation (R) $0.6 with a Probability value (P) # 0.005), proteins identified in less than 3 out of 6 replicates were excluded.
For proteomic data analysis, effectiveness and reproducibility of Detergent Resistant Membrane (DRM) separation was assessed by probing sucrose gradient fractions with an antibody against Flotillin (a DRM marker). To assess reproducibility of the Protein Abundance Profiles (PAPs), the Pearson's correlation was calculated. We verify that more than 70% of PAP pairs are conserved (R$0.6, P<0.005). The presence of a protein subset with less conserved PAPs was explained by the presence of DRMassociated proteins residing in membrane contexts partially susceptible to detergent extraction. Moreover, PAP reproducibility was confirmed in a different blood sample by an alternative method (Western blot).
The wild type and CRISPR knock out erythroid cell lines were differentiated three independent times. From these independent differentiation experiments, invasion assays were set up in triplicate. Cytospins taken at 0hrs and 20hrs post invasion were counted by light microscopy. The ART4 phenotype replicated across these three experimental repeats.
No randomization was performed.
For the functional analysis, slides were counted blind. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Genome browser session The target species for all commercial antibodies used was human. All commercial antibodies were validated by manufacturer for target species. In all cases human was the primary target species. Anti-RON4 and anti-N201 were validated in previous works (doi:10.1074/mcp.M113.029272 and doi:10.1128/EC.00040-06).
Immortalized erythroid progenitor cell line (EJ cells) generated from peripheral blood mononuclear cells ( https:// doi.org/10.1002/ajh.25543). This cell line was generated in house within the Duraisingh lab.
EJ cells were characterized as erythroid cells through light microscopy examination of cytospins post differentiation and surface expression of known erythroid markers as observed by flow cytometry. CRISPR knock outs with the EJ background were verified by flow cytometry and TIDE software.
Not tested.

N/A.
Subjects are children aged 0-180 months, of both sexes, and belonging to Mossi ethnic group (self-reported ethnicity of both parents) from Burkina Faso.
The sample of severe malaria cases was recruited at the 158-bed paediatric ward of the Ouagadougou University Hospital. In line with WHO guidelines, severe malaria was defined by the presence of P. falciparum in the thick blood film associated with at least one of the following conditions: prostration (incapacity of the child to sit without help in the absence of coma), unrousable coma (score between 0 and 2 on the Glasgow modified coma scale), repeated generalised convulsions (more than two episodes in the preceding 24h), severe anaemia (haemoglobin <5g per 100ml), hypoglycaemia (<40mg per 100ml), pulmonary oedema/respiratory distress, spontaneous bleeding and renal failure (plasma creatinine>3mg per 100ml). The sample of healthy control children was recruited during malaria cross-sectional surveys performed in the Ouagadougou area.
The study received approval from the ethical committees of the Ministry of Health of Burkina Faso and the University of Oxford.