title: Design of a variant surface antigen-supplemented microarray chip for whole transcriptome analysis of multiple Plasmodium falciparum cytoadherent strains, and identification of strain-transcendent rif and stevor genes.
abstract: BACKGROUND
  The cytoadherence of Plasmodium falciparum is thought to be mediated by variant surface antigens (VSA), encoded by var, rif, stevor and pfmc-2tm genes. The last three families have rarely been studied in the context of cytoadherence. As most VSA genes are unique, the variability among sequences has impeded the functional study of VSA across different P. falciparum strains. However, many P. falciparum genomes have recently been sequenced, allowing the development of specific microarray probes for each VSA gene.
  
  
  METHODS
  All VSA sequences from the HB3, Dd2 and IT/FCR3 genomes were extracted using HMMer software. Oligonucleotide probes were designed with OligoRankPick and added to the 3D7-based microarray chip. As a proof of concept, IT/R29 parasites were selected for and against rosette formation and the transcriptomes of isogenic rosetting and non-rosetting parasites were compared by microarray.
  
  
  RESULTS
  From each parasite strain 50-56 var genes, 125-132 rif genes, 26-33 stevor genes and 3-8 pfmc-2tm genes were identified. Bioinformatic analysis of the new VSA sequences showed that 13 rif genes and five stevor genes were well-conserved across at least three strains (83-100% amino acid identity). The ability of the VSA-supplemented microarray chip to detect cytoadherence-related genes was assessed using P. falciparum clone IT/R29, in which rosetting is known to be mediated by PfEMP1 encoded by ITvar9. Whole transcriptome analysis showed that the most highly up-regulated gene in rosetting parasites was ITvar9 (19 to 429-fold up-regulated over six time points). Only one rif gene (IT4rifA_042) was up-regulated by more than four fold (five fold at 12 hours post-invasion), and no stevor or pfmc-2tm genes were up-regulated by more than two fold. 377 non-VSA genes were differentially expressed by three fold or more in rosetting parasites, although none was as markedly or consistently up-regulated as ITvar9.
  
  
  CONCLUSIONS
  Probes for the VSA of newly sequenced P. falciparum strains can be added to the 3D7-based microarray chip, allowing the analysis of the entire transcriptome of multiple strains. For the rosetting clone IT/R29, the striking transcriptional upregulation of ITvar9 was confirmed, and the data did not support the involvement of other VSA families in rosette formation.
authors: Centre for Immunity, Infection and Evolution, Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, West Mains Rd, Edinburgh, EH9 3JT, UK.
authors: Antoine
authors: A
authors: Claessens
authors: Ashfaq
authors: A
authors: Ghumra
authors: Archna P
authors: AP
authors: Gupta
authors: Sachel
authors: S
authors: Mok
authors: Zbynek
authors: Z
authors: Bozdech
authors: J Alexandra
authors: JA
authors: Rowe
mesh_terms: D000953:Antigens, Protozoan / Q000235:genetics*; D000954:Antigens, Surface / Q000235:genetics*; D020869:Gene Expression Profiling; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010963:Plasmodium falciparum / Q000235:genetics*; D020741:rho GTP-Binding Proteins / Q000235:genetics*
chemical_list: D000953:Antigens, Protozoan; D000954:Antigens, Surface; C474556:STEVOR antigen, Plasmodium falciparum; D020741:rho GTP-Binding Proteins