erythrocyte surface via the Maurer’s clefts by interacting with the structural component Knob-Associated Histidine-Rich Protein (KAHRP) [9]. In general, a single var gene is expressed in a parasite, but expression can switch to another member in the absence of an immune pressure, leading to antigenic and phenotypic variation at the PRBC surface [10]. This is believed to drive escape from the host’s immune response and is implicated in pathogenesis. A recent large-scale knockout study highlighted the complexity of the interaction between parasite proteins secreted into the RBC cytoplasm and cytoadhesion [11]. In this work and other studies several proteins were identified that contribute either in loading PfEMP1 into Maurer’s clefts [11] or transfer from the clefts to the erythrocyte surface [11–13]. Although this fascinating mechanism is being intensely studied, many cellular processes involved in trafficking of parasite proteins into the host cell remain elusive [3,14]. In this work we identified P. falciparum laboratory lines that have irreversibly lost their adhesive properties but express non- functional and trypsin-resistant PfEMP1 molecules on the surface of PRBC. Furthermore, to determine if loss of cytoadherence may have resulted from the absence of the cytoadherence-linked asexual gene (clag9) due to the truncation of chromosome 9 in D10, we performed targeted disruption of clag9 and show, that in contrast to previous studies [15,16], this gene is not essential for the cytoadhesion of PfEMP1. Methods Parasites and cell cultures Plasmodium falciparum D10, a cloned line derived from FC27 [17], FCR3 [18], Malayan Camp (MC) [19] and 3D7 [20] were cultured as described [21]. All parasite strains were systematically selected for the presence of knobs by gelatine flotation (Plasmion, Fresenius Kabi, France) [22]. Parasite cultures were synchronized by sorbitol lysis [23] and verified for the absence of mycoplasma that could interfere with cytoadhesion [24]. Amelanotic C32 melanoma cells [25] and COS-7 cells [6] were grown in RPMI 1640 without glutamine (Invitrogen), supplemented with 10% foetal bovine serum (heat-inactivated), 0.4 mM L-glutamine, 20 mM HEPES and penicillin/streptomycin. Nucleic acids extraction and analysis Total RNA from 3D7, D10 and FCR3 was isolated by the addition of TRIzol (Invitrogen) to the harvested ring-, trophozoite- and schizont-PRBC [26], processed as described [27], transferred onto Hybond N+ (Amersham) and hybridized with 32P-labelled DBL1a or ATS (Acidic Terminal Segment) probes. Cultured parasites were permeabilized with 0.15% saponin [28] before extraction of genomic DNA (GenElute Mammalian Genomic DNA miniprep kit, Sigma). The full PfEMP1 gene sequence was obtained by PCR amplification and determined by gene walking (Supplementary Table 1), and is available from Genbank (accession number JF342594). For Southern blotting genomic DNA was digested with EcoRI and SpeI (BioLabs), and processed as described [29]. Northern and Southern blots were washed with sodium saline citrate (SSC), sodium dodecyl sulfate (SDS) at 60uC and exposed with BioMax MS films and Transcreen HE (Kodak). clag9 Knockout P. falciparum 3D7 genomic DNA was used to PCR amplify clag9 exons 1 (bp 1 to 761) and 8 (bp 3652 to 4418) (Supplementary Table 1), the fragments were digested with SacII/SpeI and EcoRI/NcoI, and cloned into the vector pCC1 [30]. P. falciparum Adhesion Is Not Linked to clag9 Deletion was transfected with this construct leading to the disruption of clag9 by allelic exchange and insertion of the hDHFR selectable marker. Antibodies Pooled human immune sera [31], anti-Pf332 mAb51-22 and rabbit purified IgG [32], anti-KAHRP mAb89 [33], anti-CD36 mAbFA6-152 [34], and mAb7H8/50 [35] have been described. Anti-Gluthathione S-transferase (GST) mAb clone GST-2 (Sig- ma), anti-hemagglutinin A mAb16B12 (Abcam), fluorochrome- conjugated secondary antibodies (Molecular Probes), and guinea pig anti-ATS serum (Charles Rivers Laboratories, France) were also used. Indirect Immunofluorescence Assay (IFA) Immunofluorescence assays were done as previously described [32]. Surface immunofluoresence assays of D10 and FCR3CD36 were performed on trypsin-treated PRBC using pooled human immune sera (1:5) followed by anti-human Alexa Fluor 488 (1:100). Nuclei were stained with 0.2 mg/ml 49,6-diamidino-2- phenylindole (DAPI; Molecular Probes) and 10 mg/ml propidium iodide (Sigma) to confirm cell viability. Parasites were visualized by confocal fluorescence microscopy (Nikon Eclipse TE2000-E), the images were captured with a Nikon D-Eclipse C1 camera and Nikon EZ-C1 software version 3.80. Iodination and Immunoprecipitation Plasmion-enriched PRBC were labelled with 125I before incubation with different concentrations of trypsin (0, 10, or 100 mg/ml) or chymotrypsin (100 mg/ml) as described [36]. The Triton-X100 insoluble-SDS soluble (TX100ins-SDSsol) fractions were immunoprecipitated with a pool of human immune sera [31]. Western blot Western blots were performed as described [32]. TX100ins- SDSsol extracts from trypsin-treated mature parasites were reacted with anti-ATS serum (1:400) followed by alkaline phosphatase- conjugated anti-guinea pig secondary antibodies (1:2500) and developed with NBT/BCIP (Promega). The clag9 knockout clones KOE5 and KOF4 were similarly analysed using rabbit anti- CLAG9 antibodies (1:1000) [37] and revealed with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Thermo Scientif- ic). CIDR1a binding analysis The M2 CIDR1a domain (minimal CD36 binding site) [38] from the following var genes: D10var1 (this work), FCR3var1CSA [36], and MCvarCD36 [38] were cloned into the pDisplay vector (Invitrogen) (Supplementary Table 1) and expressed on the surface of COS-7 cells [39]. CD36 binding assays were performed as described [36,39] using magnetic protein A-conjugated Dynal beads (Invitrogen) and mAbFA6-152. The efficiency of COS-7 transfection was verified by detection of the pDisplay HA tag by IFA with mAb16B12 followed by anti-mouse Alexa Fluor 488. Cytoadherence assay Assays were performed as described [36] with 10 ml of purified receptors immobilized on plastic Petri dishes: 10 mg/ml CD36 and ICAM-1 (R&D systems), 5 mg/ml gC1qR [40], 1 mg/ml CSA and CSC (Sigma). Control spots were coated with 1% BSA (fraction V, Sigma). FCR3 panned on CD36 (FCR3CD36) was used as positive control. PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29039