Analysis of var Gene Transcription Pattern Using DBLα Tags

Abstract

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens, which are encoded by a multigene family called var genes, are exported and inserted onto the surface of the infected erythrocytes. PfEMP1 plays a key role in the pathogenesis of severe malaria and are major targets of naturally acquired immunity. Studying the expression pattern of var genes in P. falciparum clinical isolates is crucial for understanding disease mechanism and immunity to malaria. However, var genes are highly variable, which makes it difficult to study their expression in clinical isolates obtained directly from malaria patients. In this chapter, we describe an approach for analysis of var gene expression that targets a region referred to as DBLα tag, which is relatively conserved in all var genes.

Keywords: DBL; Expression; Malaria; PfEMP1; Transcription; var genes.

Fig. 1

Architecture of P. falciparum var genes. (Cartoon adapted from Gardner et al. [1] illustrating the three most common architectures of var genes in the 3D7 genome). Architecture 1 is the most common represented by 38 of the 60 var genes. A semi conserved head structure (separated by the black dotted line) with DBLα-CIDRα combination is seen in 12 out of the 16 architectures seen. The DBLα tag is a small region amplified (red) from the DBLα domain using degenerate AFBR primers (black arrows)

Fig. 2

Depletion of white blood cells. Illustration of depletion of white blood cells from whole blood using Lymphoprep density gradient medium. Figure demonstrates layering of blood before centrifugation and the distinct mononuclear cells, RPMI/plasma and erythrocyte layers after centrifugation

Fig. 3

DBLα tag sequence features. Location of sequence features used in classification of DBLα tags demonstrated using five DBL α sequences derived from clinical P. falciparum isolates. The anchor points are in blue, Positions of Limited Variability (PoLV) are in grey and number of cysteines in green [24]

Fig. 4

Classification of DBLα tags. Venn diagram showing the classification of DBLα tags based on the number of cysteines and the REY/MFK motifs. There is no overlap among the Cys2 sequences containing the REY and MFK motifs. All group 1 sequences are also group A var genes