Adhesion Is Not Linked to clag9 Deletion Figure 4. AFM analysis of erythrocytes infected by P. falciparum FCR3CD36 and D10. A. Representative amplitude images of infected red blood cells. Insets show Giemsa staining of the analysed PRBC. B. Height images were used to measure knob homogeneity, number, diameter and height on infected erythrocytes. Knob homogeneity and number were analyzed using mean 6 standard deviation (5#N cells#15). Knob diameter and height were analyzed using mean 6 standard error of the mean (5#N cells#15). Scale bars: 1 mm. doi:10.1371/journal.pone.0029039.g004 diameter and height of D10 knobs were slightly smaller than that from FCR3CD36 (Fig. 4A and B). These results imply that although the knob morphology is marginally affected by the chromosome 9 deletion it is not causing the loss of cytoadherence of D10 parasites. The CIDR1a domain of the PfEMP1 expressed in D10 binds to CD36 We cloned and sequenced the entire var gene transcribed in D10 (called D10var1) and determined that the open reading frame of this transcript is composed of NTS-DBL1a-CIDR1a-DBL2d- CIDR2b-TM-ATS domains (Fig. 5A). The different domains of PfEMP1 can be classed according to their binding regions allowing the prediction of the corresponding adhesion receptors [5,39]. The M2 minimal domain from PfEMP1 CIDR1a is necessary and sufficient for binding to the CD36 receptor [38]. Thus, to better characterize D10var1, the HA tagged M2 domain was expressed on the surface of COS-7 cells (Fig. 5A). Cells were incubated with recombinant CD36 and M2-CD36 surface complexes we detected using anti-CD36 mAb-coated Dynal beads. COS-7 cells surface- expressing the M2 domain from D10var1 and the positive control MCvarCD36 are shown in Fig. 5B. No significant differences (P = 0.961) in binding were observed. In contrast, COS-7 cells expressing M2 domain from the negative control FCR3var1CSA, showed significantly less coated beads on the surface (P,0.001) (Fig. 5B and C). Transfected COS-7 cells expressing M2 domains were visualized after the binding assay by IFA staining of the HA- domain fused to M2 (green fluorescence). Our results demonstrate that the M2 domain of D10var1 is functional when expressed outside the D10 infected erythrocyte context and indicate that D10 PRBC have lost their capacity for functional expression of parasite-mediated adhesion, maybe due either to a conformational defect, improper insertion into the RBC membrane, or missing post-translational modifications of PfEMP1. PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29039