clag9 Is Not Essential for PfEMP1 Surface Expression in Non-Cytoadherent Plasmodium falciparum Parasites with a Chromosome 9 Deletion

Background: The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a nonfunctional PfEMP1. Methodology/Principal Findings: Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36binding PfEMP1 CIDR1a domain were performed and showed that these parasites express an unusual trypsin-resistant, nonfunctional PfEMP1 at the erythrocyte surface. However, the CIDR1a domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9 .I n contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36. Conclusions/Significance: Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications.