clag9 Is Not Essential for PfEMP1 Surface Expression in Non-Cytoadherent Plasmodium falciparum Parasites with a Chromosome 9 Deletion Ade´ la Nacer1,2, Emeric Roux1,2¤a, Se´ bastien Pomel3¤b, Christine Scheidig-Benatar1,2, Hiroshi Sakamoto1,2, Frank Lafont3,4,5,6, Artur Scherf1,2, Denise Mattei1,2* 1 Institut Pasteur, Biology of Parasite-Host Interactions Unit, Department of Parasitology and Mycology, Paris, France, 2 CNRS URA2581, Paris, France, 3 Cellular Microbiology of Infectious Pathogens, Institut Pasteur de Lille, Lille, France, 4 CNRS UMR8204, Lille, France, 5 Inserm U1019, Lille, France, 6 Universite´ Lille Nord de France, Lille, France Abstract 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 non- functional 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 CD36- binding PfEMP1 CIDR1a domain were performed and showed that these parasites express an unusual trypsin-resistant, non- functional 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. In 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. Citation: Nacer A, Roux E, Pomel S, Scheidig-Benatar C, Sakamoto H, et al. (2011) clag9 Is Not Essential for PfEMP1 Surface Expression in Non-Cytoadherent Plasmodium falciparum Parasites with a Chromosome 9 Deletion. PLoS ONE 6(12): e29039. doi:10.1371/journal.pone.0029039 Editor: Thomas J. Templeton, Weill Cornell Medical College, United States of America Received September 20, 2011; Accepted November 18, 2011; Published December 19, 2011 Copyright: 2011 Nacer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by Fondation pour la Recherche Me´dicale grant number DMI20091117309 and Conseil Re´gional Nord Pas-de-Calais. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: denise.mattei@pasteur.fr ¤a Current address: BIO RAD Laboratories, Division LSG, Marnes la Coquette, France ¤b Current address: Groupe Chimiothe´rapie Antiparasitaire, UMR CNRS 8076, Faculte´ de Pharmacie, Universite´ PARIS XI, Chatenay-Malabry, France Introduction An important factor contributing to the virulence of Plasmodium falciparum is the ability of parasitized red blood cells (PRBC) to adhere to receptors such as CD36 or ICAM-1 expressed on the surface of endothelial cells, or chondroitin sulphate A (CSA) expressed on placental syncytiotrophoblasts (see [1] for a review). PRBC sequestration can lead to complete blockage of the microvasculature resulting in severe disease including cerebral malaria that may be fatal [2]. Parasite survival within the newly invaded erythrocyte depends on the synthesis and export of several components, which will be used to build the trafficking pathway and channels necessary for the import and export of essential constituents (see [3]). The variant antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1) is the predominant ligand responsible for adhesion to host endothelial receptors and is essential for parasite survival and establishing chronic infection [2]. Approximately 60 var genes of the parasite’s haploid genome encode for PfEMP1 and their expression occurs in a mutually exclusive manner. The var genes share a two exon structure with exon 1 coding for the extracellular highly variable adhesive region and a conserved cytoplasmic exon 2 that codes for a transmembrane region (TM) and interacts with the red blood cell cytoskeleton [4]. Exon 1 is composed of variable numbers of Duffy binding-like domains (DBL) and Cysteine-Rich Interdomain Regions (CIDR) that specifically bind to the different receptors in adhesion assays when expressed in heterologous expression systems [5–7]. During in vitro culture, var genes are transcribed and translated in ring stages and, despite the absence of an N-terminal signal sequence [8], PfEMP1 is exported through the endoplasmic reticulum pathway, and beyond the parasite’s confines to the PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29039