Plasmodium falciparum Variant Surface Antigen Expression Patterns during Malaria

Abstract

The variant surface antigens expressed on Plasmodium falciparum–infected erythrocytes are potentially important targets of immunity to malaria and are encoded, at least in part, by a family of var genes, about 60 of which are present within every parasite genome. Here we use semi-conserved regions within short var gene sequence “tags” to make direct comparisons of var gene expression in 12 clinical parasite isolates from Kenyan children. A total of 1,746 var clones were sequenced from genomic and cDNA and assigned to one of six sequence groups using specific sequence features. The results show the following. (1) The relative numbers of genomic clones falling in each of the sequence groups was similar between parasite isolates and corresponded well with the numbers of genes found in the genome of a single, fully sequenced parasite isolate. In contrast, the relative numbers of cDNA clones falling in each group varied considerably between isolates. (2) Expression of sequences belonging to a relatively conserved group was negatively associated with the repertoire of variant surface antigen antibodies carried by the infected child at the time of disease, whereas expression of sequences belonging to another group was associated with the parasite “rosetting” phenotype, a well established virulence determinant. Our results suggest that information on the state of the host–parasite relationship in vivo can be provided by measurements of the differential expression of different var groups, and need only be defined by short stretches of sequence data.

Plasmodium falciparum var gene expression is modified by host immunity

“…The immense architectural diversity of var genes, together with their capacity to undergo recombination (15), yields limited positions for PCR amplification and sequence sampling. Therefore we (16) and others (17)(18)(19)(20)(21) have relied on analysis of short, Ϸ350 nucleotide, expressed sequence tags amplified from a region corresponding to a domain that is present in most PfEMP1 variants, DBL␣. To estimate PfEMP1 expression levels, reverse transcriptase PCR products are subcloned into Escherichia coli, bacterial colonies picked and sequenced, and the sequence tags classified and counted.…”

“…To estimate PfEMP1 expression levels, reverse transcriptase PCR products are subcloned into Escherichia coli, bacterial colonies picked and sequenced, and the sequence tags classified and counted. Previous studies have varied in their depth of sequencing from each parasite isolate, the number of patients in different clinical categories, time of RNA sampling within the parasite asexual cycle, method of sequence analysis, and whether the emphasis is placed more on the dominantly expressed sequence(s) in each patient (19,20) or on the overall percentage representation within each patient of different groups of sequence [supporting information (SI) Table S1] (16,22).…”

“…First, although DBL␣ sequence tags contain only a small proportion of a var gene's sequence, specific sequence features present in DBL␣ tags isolated worldwide can be used to classify them (16,22). The vast majority of DBL␣ tags carry either two or four cysteine residues.…”

“…Although they are not exclusive to group A var genes, DBL␣ tags of all group A var genes contain two cysteine residues. A large proportion of DBL␣ tags with 2 cysteine residues (henceforth called cys2 var genes) also carry one of two motifs, MFK and REY, located at two different positions within the sequences but never found together within the same sequence (16).…”

“…In a small pilot study of 12 isolates, children with poorly developed immune responses tended to express cys2 var genes with MFK motifs (16). More recently, Kyriacou et al showed that cys2 var genes were the dominant sequence type expressed by parasites isolated from children with cerebral malaria in Mali (19).…”