Susceptibility of cultured erythrocytes to invasion and growth of Plasmodium falciparum . a , Cytospin analysis of cells taken on days 8, 11, 15, and 18. Spots were stained with the May-Gru'a8nwald Giemsa staining technique. Day 8 cells correspond to basophilic erythroblasts, day 11 cells correspond to orthochromatic erythroblasts and are co-cultured on the MS-5 stromal layer, day 15 cells correspond to reticulocytes, and day 18 cells correspond to mature erythrocytes. For day 18 cells, the enucleation percentage is shown as the mean (± standard deviation). b , Expression of maturation markers (glycophorin A, glycophorin C, and Duffy antigen receptor for chemokines [DARC]) on erythrocytes (red blood cells) from normal donors (RBC) and day 18 cultured erythrocytes (cRBC), as measured by flow cytometry. Negative controls for all experiments were isotype control (fluorescein isothiocyanate-conjugated secondary antibody) stained erythrocytes. The percentage of positive staining of erythrocyte gate is indicated. c , Invasion, growth, and reinvasion of P. falciparum parasites into day 18 cultured erythrocytes, as monitored by microscopic analysis of thin smears with Giemsa staining. d , Invasion into cultured erythrocytes and erythrocytes from normal donors, shown as the mean parasite multiplication rate from 2 experiments. Assays were performed in duplicate and triplicate. Error bars represent the standard deviation of the mean. e , Percentage of singly, doubly, and multiply (3, 4, 5, and 6 or more parasites per cell) infected erythrocytes in 2 invasion assays, shown as the mean parasite multiplication rate. Assays were performed in duplicate and triplicate. Error bars represent the standard deviation of the mean. The selectivity index (SI) is shown.

Stable knockdown of glycophorin A (GPA) on the erythrocyte surface resulting in no effect on erythrocyte maturation, but dramatic impact on invasion by Plasmodium falciparum parasites reliant on EBA-175. a , Schematic illustrating the method for reverse genetic analysis of human erythrocytes. Lentiviral particles are produced in 293T cells through co-transfection of small hairpin RNA (shRNA)-expressing plasmids (pLKO), envelope, and packaging plasmids. CD34⁺ hematopoietic stem cells (HSCs) are transduced and puromycin-selected cells are matured into erythrocytes ex vivo, at which point phenotypic analyses are conducted. b , Expression of GPA in cultured erythrocyte untransduced control cells (cRBCs), pLKO scramble- transduced control cells (pLKO), GPA knockdown cells (GPA KD), and isotype control stained erythrocytes (control RBCs). c , Expression of maturation markers (glycophorin C and Duffy antigen receptor for chemokines [DARC]) on transduced cells-pLKO scrambles shRNA-expressing cells (pLKO) and GPA shRNA-expressing cells (GPA KD). Negative controls were isotype control stained erythrocytes. The percentage of positive staining of erythrocyte gate is indicated. d , Erythrocyte morphology on day 18, as assessed by cytospin and May-Grunwald Giemsa staining. e , Expression levels of Band 3- GPA complex antigens (Wright b, Band 3, and CD47) and other surface proteins (Kell) on transduced cells (GPA KD) compared with those on cultured erythrocyte controls (cRBCs). Negative controls were unstained cultured erythrocytes. f , Parasite multiplication rate and invasion efficiency of the 3D7 and W2Mef parasite strains and EBA-175 mutants into GPA knockdown erythrocytes (GPA KD) and invasion efficiency into pLKO-transduced controls (pLKO), shown as the mean invasion percentage for 4 GPA knockdown transduction, performed in triplicate. W2Mef showed a significant difference in invasion percentage between GPA knockdown cells and pLKO control cells (P < .001) by the Student t test (2-tailed; significance level, a = 0.05). Error bars represent the standard deviation of the mean. The selectivity index (SI) is shown. g , Model depicting erythrocyte invasion by both sialic acid-independent (3D7) and sialic acid-dependent (W2Mef) P. falciparum strains. Arrows denote efficient invasion into erythrocytes; the blocked bar denotes inhibited invasion.