Ex-Vivo Cytoadherence Phenotypes of Plasmodium falciparum Strains from Malian Children with Hemoglobins A, S, and C Jeanette T. Beaudry 1,2 , Michael A. Krause 1 , Seidina A. S. Diakite 3 , Michael P. Fay 4 , Gyan Joshi 4 , Mahamadou Diakite 3 , Nicholas J. White 5 , Rick M. Fairhurst 1 * 1 Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 2 Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom, 3 Faculty of Medicine, Pharmacy and Odontostomatology, Malaria Research and Training Center, University of Bamako, Bamako, Mali, 4 Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 5 Mahidol-Oxford Tropical Diseases Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Abstract Sickle hemoglobin (Hb) S and HbC may protect against malaria by reducing the expression of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of parasitized red blood cells (RBCs), thereby weakening their cytoadherence to microvascular endothelial cells (MVECs) and impairing their activation of MVECs to produce pathological responses. Therefore, we hypothesized that parasites causing malaria in HbAS or HbAC heterozygotes have overcome this protective mechanism by expressing PfEMP1 variants which mediate relatively strong binding to MVECs. To test this hypothesis, we performed 31 cytoadherence comparisons between parasites from HbAA and HbAS (or HbAC) Malian children with malaria. Ring-stage parasites from HbAA and HbAS (or HbAC) children were cultivated to trophozoites, purified, and then inoculated in parallel into the same wildtype uninfected RBCs. After one cycle of invasion and maturation to the trophozoite stage expressing PfEMP1, parasite strains were compared for binding to MVECs. In this assay, there were no significant differences in the binding of parasites from HbAS and HbAC children to MVECs compared to those from HbAA children (HbAS, fold-change = 1.46, 95% CI 0.97–2.19, p = 0.07; HbAC, fold-change = 1.19, 95% CI 0.77–1.84, p = 0.43). These data suggest that in-vitro reductions in cytoadherence by HbS and HbC may not be selecting for expression of high-avidity PfEMP1 variants in vivo. Future studies that identify PfEMP1 domains or amino-acid motifs which are selectively expressed in parasites from HbAS children may provide further insights into the mechanism of malaria protection by the sickle-cell trait. Citation: Beaudry JT, Krause MA, Diakite SAS, Fay MP, Joshi G, et al. (2014) Ex-Vivo Cytoadherence Phenotypes of Plasmodium falciparum Strains from Malian Children with Hemoglobins A, S, and C. PLoS ONE 9(3): e92185. doi:10.1371/journal.pone.0092185 Editor: Lars Hviid, University of Copenhagen and Rigshospitalet, Copenhagen, Denmark Received May 13, 2013; Accepted February 20, 2014; Published March 19, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This study was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. 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: rfairhurst@niaid.nih.gov Introduction For millennia, the significant morbidity and mortality of Plasmodium falciparum malaria has selected for red blood cell (RBC) polymorphisms, including sickle hemoglobin (Hb) S, HbC, a-thalassemia, and G6PD deficiency [1–10]. These malaria protective polymorphisms have reached high frequencies in tropical areas despite the spectrum of deleterious consequences associated with their homozygous (or hemizygous) states. HbS (b 6 glu-.val) is a balanced polymorphism in which HbAS heterozy- gotes are protected against both uncomplicated and severe malaria [1]. In contrast, HbC (b 6 glu-.lys) generally affords protection against severe, but not uncomplicated, malaria [1]. Several mechanisms have been proposed to explain this malaria protec- tion, including reduced parasite multiplication rates through reduced invasion, impaired growth, or increased clearance; enhanced innate immunity through inhibition of CD8 + T cells and upregulation of heme-oxygenase 1; accelerated acquisition of immunity; and altered host pathogenic mechanisms (reviewed in [11,12]). One proposed mechanism, which would protect through alterations in host pathogenesis, involves the abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite’s variant surface antigen and cytoadherence ligand, on ‘knobs’ on the surface of parasitized HbAS and HbAC RBCs [13,14]. Abnormal PfEMP1 display is characterized by (i) reduced PfEMP1 levels, (ii) reduced knob densities, (iii) heterogeneous distributions of PfEMP1 and knobs, and (iv) aberrant – wider and more protuberant – knob morphologies [15]. These perturbations are associated with up to 50% reductions in the cytoadherence of parasitized HbAS and HbAC RBCs [13,14]. Cytoadherence, the binding of parasitized RBCs to human microvascular endothelial cells (MVECs), enables mature parasites to sequester in the microvessels of most organs and avoid removal from the bloodstream by the spleen [16]. While enabling parasites to multiply to high densities, cytoadherence also contributes to malaria pathogenesis by activating MVECs, leading to release of inflammatory cytokines, upregulation of adhesion receptors, PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e92185