Transcriptome analysis of antigenic variation in Plasmodium falciparum--var silencing is not dependent on antisense RNA

Abstract

Background: Plasmodium falciparum, the causative agent of the most severe form of malaria, undergoes antigenic variation through successive presentation of a family of antigens on the surface of parasitized erythrocytes. These antigens, known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, are subject to a mutually exclusive expression system, and are encoded by the multigene var family. The mechanism whereby inactive var genes are silenced is poorly understood. To investigate transcriptional features of this mechanism, we conducted a microarray analysis of parasites that were selected to express different var genes by adhesion to chondroitin sulfate A (CSA) or CD36.

Results: In addition to oligonucleotides for all predicted protein-coding genes, oligonucleotide probes specific to each known var gene of the FCR3 background were designed and added to the microarray, as well as tiled sense and antisense probes for a subset of var genes. In parasites selected for adhesion to CSA, one full-length var gene (var2csa) was strongly upregulated, as were sense RNA molecules emanating from the 3' end of a limited subset of other var genes. No global relationship between sense and antisense production of var genes was observed, but notably, some var genes had coincident high levels of both antisense and sense transcript.

Conclusion: Mutually exclusive expression of PfEMP1 proteins results from transcriptional silencing of non-expressed var genes. The distribution of steady-state sense and antisense RNA at var loci are not consistent with a silencing mechanism based on antisense silencing of inactive var genes. Silencing of var loci is also associated with altered regulation of genes distal to var loci.

Figure 1

A dominant var gene is upregulated in CSA binding parasites. Plots of log2 ratio of expression (M) against average log intensity (A) for ring, trophozoite and schizont stages for CSA versus CD36 panned parasites. Only statistically differential data giving a Bonferroni corrected p value (alpha = 0.05) have been displayed. This graph excludes probes corresponding to antisense transcripts and oligos to 3D7 var genes (whose orthologs in FCR3 diverge in sequence). Biological replicates were pooled. The plots reveal a single dominant var transcript (var2csa-marked in orange) that is much more abundant in CSA than in CD36-panned parasites at all life stages. Green dots represent all other oligos corresponding to FCR3 var genes. Several var genes are over-represented in CD36 as compared with CSA-panned parasites. Both log2 ratios of expression and apparent average intensities for var genes decrease through the life cycle.

Figure 2

Consistent sense transcript and interspersed antisense transcript in var2csa gene. Histograms showing apparent absolute abundance of both sense and antisense transcript at the var2csa locus in CD36 (grey) and CSA (white) panned parasites. Different columns show the apparent absolute abundance for oligonucleotides at individual positions along the whole var2csa gene. Left panels show probes corresponding to sense transcript, right panels show probes corresponding to antisense transcripts. Separate histograms show data for ring, trophozoite and schizont stages. Standard deviation is shown. No truncated 5' transcript of the var2csa gene is apparent in CD36 panned parasites, suggesting regulation is not controlled by premature termination of transcription. In ring stages, where var2csa transcript is most abundant in CSA parasites, apparent absolute abundance is also increased for antisense transcripts throughout the gene. Unlike sense transcription, apparent absolute abundance for all antisense transcripts varies greatly between adjacent probes, perhaps indicative of multiple short antisense transcripts initiating throughout the locus. Abundance of sense and antisense transcript in both populations is also shown for a non-var locus, msp2, for which high antisense transcription has previously been measured [34]. Both steady-state sense and antisense levels for the var2csa locus are comparable with those found at the msp2 locus.

Figure 3

No inverse correlation between sense and antisense ratio changes. Scatter plots of log2 ratio of expression (M) (CSA-panned parasites over CD36-panned) for antisense oligonucleotides against sense oligonucleotides for var genes. Data are shown for ring, trophozoites and schizont stages from biological replicate 1. Oligonucleotides corresponding to var2csa are represented by open triangles and the other var genes from the FCR3 strain are displayed as black dots. Oligonucleotides with the highest log2 ratio of expression in CSA- compared with CD36-panned parasites often correspond to those with the highest corresponding ratios for antisense abundance (upper right datapoints). Similarly, several sense transcripts apparently highly upregulated in CD36 correspond to upregulated antisense oligos at the same loci (lower left datapoints). These data are not consistent with a direct transcriptional silencing role for antisense transcription.

Figure 4

A hypothetical model for antisense transcription from var loci. Sense and antisense RNA at several var loci appear to be coordinately regulated. This may result from the altered chromatin state of the encoding genomic DNA, which is differentially modified between silent and active var loci [3]. Silencing factors such as the SIR complex (indicated by blue spheres) bind to inactive var genes, maintaining the chromatin in a condensed state. In the absence of SIR, the active var assumes a relaxed chromatin conformation that makes the surrounding locus competent for transcription. While a stable transcription complex with appropriate assembly of elongation factors generates abundant sense mRNA of full length, transcription from the opposite strand initiates and quickly terminates to produce fragments of antisense. Simultaneous transcription of the same bases from opposite directions is unviable, but in a population, both transcription events may occur at the same time. A chromatin barrier located in the intron [11] may maintain the first exon in a silencing conformation while allowing relaxation of the second exon, leading to partial 3' transcripts from a subset of otherwise silenced var genes.

Figure 5

MESA overexpression in CD36 parasites. (a) Western blot of non-synchronized parasites from FCR3-CD36 and FCR3-CSA parasites. PfHsp70 protein is included as a loading control. A monoclonal antibody (Pf12.8B7.4) against MESA [60] detects approximately 2-4 times more protein in CD36 compared with CSA panned parasites. (b) Immunofluorescence for MESA protein in FCR3-CD36 and FCR3-CSA parasites. The 488-labeled secondary shows that MESA is considerably more abundant in CD36-compared with CSA-panned parasites. The intracellular distribution of MESA is the same in both parasite populations - with most labeling localizing to the periphery of infected erythrocytes.