Mutually Exclusive Expression of Virulence Genes by Malaria Parasites Is Regulated Independently of Antigen Production

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Abstract
The primary virulence determinant of Plasmodium falciparum malaria parasite–infected cells is a family of heterogeneous surface receptors collectively referred to as PfEMP1. These proteins are encoded by a large, polymorphic gene family called var. The family contains approximately 60 individual genes, which are subject to strict, mutually exclusive expression, with the single expressed var gene determining the antigenic, cytoadherent, and virulence phenotype of the infected cell. The mutually exclusive expression pattern of var genes is imperative for the parasite's ability to evade the host's immune response and is similar to the process of “allelic exclusion” described for mammalian Ig and odorant receptor genes. In mammalian systems, mutually exclusive expression is ensured by negative feedback inhibition mediated by production of a functional protein. To investigate how expression of the var gene family is regulated, we have created transgenic lines of parasites in which expression of individual var loci can be manipulated. Here we show that no such negative feedback system exists in P. falciparum and that this process is dependent solely on the transcriptional regulatory elements immediately adjacent to each gene. Transgenic parasites that are selected to express a var gene in which the PfEMP1 coding region has been replaced by a drug-selectable marker silence all other var genes in the genome, thus effectively knocking out all PfEMP1 expression and indicating that the modified gene is still recognized as a member of the var gene family. Mutually exclusive expression in P. falciparum is therefore regulated exclusively at the level of transcription, and a functional PfEMP1 protein is not necessary for viability or for proper gene regulation in cultured parasites. Mutually exclusive gene expression refers to the ability of an organism to select one member of a large, multicopy gene family for expression while simultaneously silencing all other members of the family. Human malaria parasites utilize this process in regulating the expression of the major antigenic and virulence-determining proteins encoded by a multicopy gene family called var. In any given parasite, only a single var gene is expressed at a time, while all other members of the family are transcriptionally silenced. The mechanism that regulates this tightly controlled process and coordinates switches in gene expression is largely unknown. Here Dzikowski and colleagues show that this process is regulated entirely at the level of transcription, and that protein production and chromosomal context of the genes are not involved. In addition, they identify the DNA elements required for a var gene promoter to be recognized and co-regulated along with the rest of the family. This knowledge has enabled the authors to create transgenic parasites in which they can manipulate expression of the entire var gene family through selection for expression of specific, modified var genes, thus knocking out expression of the main virulence factor of malaria.