Adhesion Is Not Linked to clag9 Deletion Figure 7. Immunofluorescence and binding assays of clag9 E5KO. A. Labelling of CLAG9 in 3D7 and E5KO. Note there is labelling of E5KO rhoptries with mAb7H8/50 but not with anti-CLAG9 specific sera due to gene disruption. Scale bars = 5 mm. B. Trafficking of PfEMP1 and KAHRP by E5KO; labelling was identical to 3D7. Scale bars = 5 mm. C. Chemiluminescent western blot analysis of normal red blood cells (NRBC), E5KO and 3D7 parasite extracts reacted with anti-CLAG9 antibodies. D. Binding of clag9 E5KO on C32 melanoma cells. E. Binding assay of 3D7 and E5KO PRBC on purified CD36 receptor. Error bars indicate the standard error of the mean from 4 experiments. doi:10.1371/journal.pone.0029039.g007 suggesting that this may be a common adhesion phenotype linked to chromosome 9 deletion. Trypsin and chymotrypsin digestion experiments of surface iodinated PRBC proteins suggest that PfEMP1 molecules expressed at the surface of D10 display an unusual conformation that protect them from the predicted protease cleavage. It may also be the reason for the loss of the expected binding to CD36. This idea is supported by the fact that recombinant CIDR1aof D10var1 binds efficiently to CD36 when expressed by another eukaryotic cell. PfEMP1 may need ‘helper’ proteins acting as a support or chaperones to assume the right shape to function as a ligand. Alternatively, post-translational modifications and/or processing of the protein during export may be needed to obtain functional adhesion molecules. Further studies are required to explore this hypothesis. Are there candidate genes deleted on chromosome 9 of D10 that could contribute to the described adherence phenotype of D10? Several studies have implicated clag9 in parasite adhesion to CD36 and CSA [15,16]. We revisited this question by irreversibly disrupting the clag9 gene by allelic replacement. The clag9 knockout in 3D7 resulted initially in low CD36 binding parasites, which can be explained by the time taken to obtain cloned knockout lines. However, after one panning step on CD36, these clag9 mutants gained an adhesion capacity similar to the reference parasite 3D7. Thus, our data demonstrate that 3D7, in contrast to previous studies, does not depend on clag9 for the PfEMP1 adhesion process. At this stage, it is difficult to explain the different set of clag9 knockout data. It cannot be ruled out that other gene deletions known to occur during in vitro culture of 3D7, or associated with the gene deletion [13], may have been picked up at the cloning step after the knockout selection. This may mask the observed phenotype of clag9 in the study reported by the Kemp laboratory [16]. clag genes form a small gene family (5 members) and two members (clag3.1 and 3.2) have recently been shown to integrate into the erythrocyte membrane and induce a new permeation pathway [51]. It is tempting to speculate that CLAG9 may accomplish a similar function, a role that is not essential during in vitro culture. Since CLAG9 apparently integrates into the erythrocyte membrane, it may interact with a particular member of the adhesion molecule family as has recently been suggested for var2CSA [15]. Further receptor binding studies of our clag9 knockout are needed to fully explore this possibility. PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e29039