Transcription of the var genes from a freshly-obtained field isolate of Plasmodium falciparum shows more variable switching patterns than long laboratory-adapted isolates

Abstract

Background: Antigenic variation in Plasmodium falciparum involves switching among multicopy var gene family and is responsible for immune evasion and the maintenance of chronic infections. Current understanding of var gene expression and switching patterns comes from experiments conducted on long laboratory-adapted strains, with little known about their wild counterparts.

Methods: Genome sequencing was used to obtain 50 var genes from a parasite isolated from the China-Myanmar border. Four clones with different dominant var genes were cultured in vitro in replicates for 50 generations. Transcription of the individual var gene was detected by real-time PCR and then the switching process was analysed.

Results: The expression of multicopy var genes is mutually exclusive in clones of a wild P. falciparum isolate. The activation of distinct primary dominant var genes leads to different and favoured switching patterns in the four clones. The on/off rates of individual var genes are variable and the choice of subsequent dominant var genes are random, which results in the different switching patterns among replicates of each clonal wild P. falciparum isolate with near identical initial transcription profiles.

Conclusions: This study suggests that the switching patterns of var genes are abundant, which consist of both conserved and random parts.

Figure 1

Subclassification of DBL domains of FCYN0906-5H. a. Distance tree of DBL sequences of FCYN0906-5H generated using the p-distance/NJ method. DBL domains are divided into six major groups and the DBLα branch separated from the other groups. b. Phylogenetic trees of all DBLα amino acid sequences between the laboratory clone (3D7) and the wild isolate(FCYN0906-5H). The var genes of 3D7 are labelled as circles and those of FCYN0906-5H are labelled as triangles. Var gene groups are marked by different colours. Although upsB and upsC sub-groups overlapped, the upsA sub-group could be distinguished from others. Meanwhile, var4 and var84 had no upstream sequences but were assumed to be a sub-type of upsA as they were also within the upsA sub-group.

Figure 2

Schematic diagram of the repertoire of var genes of the wild Plasmodium isolate, FCYN0906-5H. The gene names, Ups sequence type and domain architecture are listed. Where known, the var genes have been grouped according to 5′ flanking sequence (Ups type). The extracellular domains of PfEMP1 have been classified by sequence criteria. * var4 and var84 had no upstream sequences but had DBLα sequences of group A. # indicates var genes that had no upstream sequences but belong to either group B or group C according to the DBLα amino acid sequences.

Figure 3

Histograms showing the mutually exclusive expression of var genes in the four clones. The four clones expressed different dominant var genes which were present at proportions ranging from 44 to 69%. There were also minor transcripts with var21 as the second dominant var gene across all four clones.

Figure 4

Diagram depicting the variable var gene switching patterns of the four clones and the replicates. The second dominant var gene was identified when its transcription level reached 40% of that of the dominant var gene. Clone 4C: var167 switches to var21 in 4C-A, 4C-D, var143 in 4C-C, and var98 in 4C-B. However, at the last time point, the dominant var gene in 4C-A, 4C-B, both switch to var45. Clone 4H: var15 is maintained for over 50 generations in 4H-A, 4H-B, however, its expression falls in 4H-C, 4H-D, switches to var21 in 4H-C. Clone5H: var51 switches off quickly and immediately switches to var21 in 5H-A, 5H-B, but in 5H-C, 5H-D, var51 slows down the off rate and switches to var45, var212. Clone 6G: var47 switches to var136 in 6G-A and var21 in 6G-B. The change in the transcriptional trend is similar in 6G-C and 6G-D; var47 switches to var163 gradually. var51 and var47 were predicted to be upsC and upsB group individually, var167 and var15 should belong to either upsB or upsC but not upsA group from the phylogenetic trees of all var-DBLα-contig amino acid sequences of the wild isolate.