Adhesion Is Not Linked to clag9 Deletion Figure 5. Expression and binding of D10var1 CIDR1a minimal domain. A. Diagram of D10var1. The different domains are shown, including the location of the PfEMP1 CIDR1a minimal domain (M2) used for expression by COS-7 cells. The region spanning the intron between exons 1 and 2 of D10var1 was sequenced from cDNA. B. Beads bound to COS-7 cells expressing M2 on the surface. C. Quantification of binding on COS-7 cells. The CIDR1a minimal domain of the D10var1 PfEMP1 binds equally well to COS-7 cells as that of the Malayan Camp (MCvarCD36, positive control). In contrast, there is no significant binding of the FCR3var1CSA M2 domain. pDisplay control represents COS-7 cells transfected with pDisplay not containing the insert. doi:10.1371/journal.pone.0029039.g005 CLAG9 is not essential for binding to CD36 The site of the chromosome breakpoint leading to the deletion of the subtelomeric region was determined by PCR on the telomere repeats and the genes suspected to be located there. Based on the 3D7 genome sequence D10 has deleted around 120 kbp of the right arm of chromosome 9 [50] representing 25 genes, including clag9 (Fig. 6A), encoding a rhoptry protein [37]. CLAG9 was initially implicated in parasite adhesion to CD36 and C32 cells [16] and recently in the adhesion to placental CSA [15]. To determine its potential role in the cytoadherence phenotype, we inactivated the clag9 gene from P. falciparum 3D7. Targeted disruption of clag9 was performed by replacing exons 2 through 6 with the hDHRF selectable marker (Fig. 6B). Gene knockout and integration of the hDHFR cassette were confirmed by PCR analysis (Fig. 6C) and Southern blotting using probes for exons 6 and hDHFR (Fig. 6D–F). CLAG9 was not detected in two knockout clones (E5KO and F4KO), either by immunofluores- cence (Fig. 7A and B) or western blotting (Fig. 7C). In contrast to earlier reports implicating CLAG9 in cytoadhesion, our KO parasites bound to both C32 cells (Fig. 7D) and CD36 (Fig. 7E) at levels comparable to our control 3D7 parasites. These results show that the clag9 gene product is not directly implicated in cytoadhesion to CD36 and, therefore, in the non-cytoadherent phenotype of the D10 strain. Discussion P. falciparum export several proteins beyond the parasite membrane to the erythrocyte surface assuring the parasite’s survival within the human host. A large number of these exported molecules have been described that are essential for, or contribute to, the trafficking of the major virulence factor PfEMP1 to the erythrocyte surface. Here we describe a novel adhesion mutant phenotype. In D10 PfEMP1 is trafficked to the surface of PRBC but is unable to bind to host adhesion receptors. Even repeated panning experiments on C32 cells or recombinant CD36 bound to plastic dishes did not yield any increase in binding, showing that no other var gene is able to restore the adhesion phenotype. Bulk- cultured parasites typically contain minor populations expressing a number of different var gene members (including CD36-binders). Thus, our data indicate that in D10 non-functional PfEMP1 surface expression is implicated in the irreversible loss of cytoadherence. We also have characterized another non-cytoad- herent strain, T9-96 [46] with similar results (data not shown), PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29039