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Strict tropism for CD71+/CD234+ human reticulocytes limits the zoonotic potential of Plasmodium cynomolgi

Varakorn Kosaisavee, Rossarin Suwanarusk, Adeline C. Y. Chua, Dennis E. Kyle, Benoit Malleret, Rou Zhang, Mallika Imwong, Rawiwan Imerbsin, Ratawan Ubalee, Hugo Sámano-Sánchez, Bryan K. S. Yeung, Jessica J. Y. Ong, Eric Lombardini, François Nosten, Kevin S. W. Tan, Pablo Bifani, Georges Snounou, Laurent Rénia and Bruce Russell

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Article Figures & Data

Figures

  • Figure 1.

    P cynomolgi RBC tropism and specificity for DARC. (A-B) Invasion efficiencies of P cynomolgi into macaque or human RBCs, reticulocytes, and normocytes (CD71). Two representative photomicrographs of P cynomolgi invasions in monkey normocytes and human reticulocytes (scale bar = 20 μm). The invasion data were derived from 8 independent experiments, and significance was determined by one-way analysis of variance (ANOVA) and post hoc analysis. (C) Inhibition of P cynomolgi invasion by enzymatic treatment of RBCs. (D) Inhibition of macaque or human RBC invasion by anti-Fy6 (2C3 antibody) or anti-FyB. (E) In-depth analysis of inhibition of macaque RBC invasion by anti-Fy6 (2C3 antibody), anti-FyB, or the chemokine IL-8. (F) The extracellular domain 1 (ECD1) of DARC showing the region targeted by IL-8 and the antibodies and used in this study. Residues in red represent the 2C3 antibody recognition region, with the key binding residues marked by an asterisk; residues in green represent the position of the Fy group (FyB in macaque, G D); residues in yellow indicate the IL-8 binding sites.26 (G) Alignment of partial sequences of DARC from 5 of the monkey donors (D1-5) used in this study. The color notations correspond to the ECD1 diagram of DARC. *P < .05; ***P < .001. n.s., nonsignificant.

  • Figure 2.

    Erythrocytic development of P cynomolgi in monkey and human hosts. Giemsa-stained thin films of P cynomolgi in (A) monkey (M mulatta) normocytes and (B) human reticulocytes matured ex vivo over a period of 42 hours. The parasite was staged into the following categories: tiny ring (0-6 hours after invasion), small ring (6-12 hours after invasion), large rings (12-18 hours after invasion), early trophozoites (18-24 hours after invasion), late trophozoites (24-30 hours after invasion), early schizonts (30-36 hours after invasion), and mature schizonts (36-42 hours after invasion). Scale bar = 10 μm.

  • Figure 3.

    Comparative morphological and rheological characteristics of P cynomolgi and P vivax infected RBCs. (A) The morphologic similarity of P cynomolgi early trophozoites (18-24 hours after invasion) in M mulatta normocytes and human reticulocytes compared with that of P vivax in human reticulocytes. Giemsa-stained thin film smear is the most commonly used method for species diagnosis in areas where P vivax and P cynomolgi are co-endemic. Scale bar = 10 μm. (B) The effect of P cynomolgi invasion and development on the deformability of monkey and human RBCs. The plot shows the membrane mean shear modulus (SM) (a higher SM indicates a reduced membrane deformability) for different RBC types (monkey and human) and stages of P vivax erythrocytic development (geometric mean ± 95% confidence intervals; n > 15 cells from 3 independent trials). The red lightning icons indicate the tropism of P cynomolgi when invading human or monkey RBCs. Pictures of respective cell types before and during membrane SM measurement by micropipette aspiration are shown under the graph. Mean (geometric) SMs were compared by using ANOVA (Bonferroni correction) and multiple comparison test (Tukey). Uninfected normocytes were significantly more deformable than uninfected reticulocytes (P < .001). However, both ring and trophozoite P vivax stages become progressively more deformable (P < .05) until schizont stage (the very mature schizonts segmenters were especially rigid). Scale bar = 5 μm. (C) Comparative phenotypic characterization of caveolae in P cynomolgi- and P vivax–infected RBCs in monkeys and humans as revealed by atomic force microscopy. (D) The mean density of caveolae per μm2 (n > 10 cells scanned for each condition over 3 separate ex vivo maturation experiments) on early and late trophozoite-infected RBCs. (E) The mean diameter of caveolae of P vivax and P cynomolgi in human reticulocytes. No significant difference in caveola density or diameter was observed.