Kirk William Deitsch

The exceptional virulence of the human malaria parasite, Plasmodium falciparum , is attributed to the adhesive properties of infected red blood cells and the parasite’s ability to avoid antibody recognition through antigenic variation. Both properties are derived from the hypervariable surface protein PfEMP1, which is encoded by members of the multi-copy var gene family. Waves of parasitemia during an infection are thought to correspond to var transcriptional switching, enabling parasites to avoid elimination by antibodies targeting previously expressed forms of PfEMP1. The mechanisms underlying and regulating var transcriptional switching remain incompletely understood. Here, we show how transient activation of the var2csa locus mediates var switching, while the expression of non-coding RNAs from this locus contributes to repression of var2csa transcription and affects var switching frequencies. Furthermore, we find that an upstream open reading frame in the 5’-untranslated region of the var2csa transcript destabilizes the var2csa mRNA through the induction of the nonsense-mediated RNA decay pathway. This process promotes transcriptional activation of an alternative var gene. Our findings provide molecular insights into the coordinated transcriptional switching of the var gene family, which contributes to chronic infection.

The exceptional virulence of the human malaria parasite, Plasmodium falciparum , is in large part attributed to the adhesive properties of infected red blood cells and the parasite’s ability to avoid antibody recognition through antigenic variation. Both properties are derived from a hypervariable protein placed on the infected cell surface called PfEMP1, different versions of which are encoded by members of the multi-copy var gene family. Over the course of an infection, rising and falling waves of parasitemia correspond to transcriptional switching between var genes, with parasites expressing alternative PfEMP1 isoforms replacing those eliminated by antibodies targeting the previously expressed PfEMP1. var gene expression is controlled epigenetically, however what triggers transcriptional switching and determines which genes are activated is largely unknown. Previous work implicated a unique, universally conserved gene called var2csa as a central component in transcriptional switching, however the molecular basis for its role was obscure. Here we describe how noncoding RNAs, an upstream open reading frame and the nonsense mediated mRNA decay pathway interact to enable var2csa to function as a transiently activated “switching hub” that unifies the var gene family into a single coordinated network, thereby perpetuating chronic infections.

Plasmodium falciparum evades antibody recognition through transcriptional switching between members of the var gene family, which encodes the major virulence factor and surface antigen on infected red blood cells. Previous work with clonal P. falciparum populations revealed var gene expression profiles inconsistent with uniform single var gene expression. However, the mechanisms underpinning this and how it might contribute to chronic infections were unclear. Here, using single-cell transcriptomics employing enrichment probes and a portable microwell system, we analysed var gene expression in clonal 3D7 and IT4 parasite lines. We show that in addition to mono-allelic var gene expression, individual parasites can simultaneously express multiple var genes or enter a state in which little or no var gene expression is detectable. Reduced var gene expression resulted in greatly decreased antibody recognition of infected cells. This transcriptional flexibility provides parasites with greater adaptive capacity and could explain the antigenically ‘invisible’ parasites observed in chronic asymptomatic infections.

SUMMARY: The human malaria parasite Plasmodium falciparum is known for its ability to maintain lengthy infections that can extend for over a year. This property is derived from the parasite’s capacity to continuously alter the antigens expressed on the surface of the infected red blood cell, thereby avoiding antibody recognition and immune destruction. The primary target of the immune system is an antigen called PfEMP1 that serves as a cell surface receptor and enables infected cells to adhere to the vascular endothelium and thus avoid filtration by the spleen. The parasite’s genome encodes approximately 60 antigenically distinct forms of PfEMP1, each encoded by individual members of the multicopy var gene family. This provides the parasite with a repertoire of antigenic types that it systematically cycles through over the course of an infection, thereby maintaining an infection until the repertoire is exhausted. While this model of antigenic variation based on var gene switching explains the dynamics of acute infections in individuals with limited anti-malarial immunity, it fails to explain reports of chronic, asymptomatic infections that can last over a decade. Recent field studies have led to a re-evaluation of previous conclusions regarding the prevalence of chronic infections, and the application of new technologies has provided insights into the molecular mechanisms that enable chronic infections and how these processes evolved.

Chronic, asymptomatic malaria infections contribute substantially to disease transmission and likely represent the most significant impediment preventing malaria elimination and eradication. Plasmodium falciparum parasites evade antibody recognition through transcriptional switching between members of the var gene family, which encodes the major virulence factor and surface antigen on infected red blood cells. This process can extend infections for up to a year; however, infections have been documented to last for over a decade, constituting an unseen reservoir of parasites that undermine eradication and control efforts. How parasites remain immunologically “invisible” for such lengthy periods is entirely unknown. Here we show that in addition to the accepted paradigm of mono-allelic var gene expression, individual parasites can simultaneously express multiple var genes or enter a state in which little or no var gene expression is detectable. This unappreciated flexibility provides parasites with greater adaptive capacity than previously understood and challenges the dogma of mutually exclusive var gene expression. It also provides an explanation for the antigenically “invisible” parasites observed in chronic asymptomatic infections.

For Plasmodium falciparum, the most widespread and virulent malaria parasite that infects humans, persistence depends on continuous asexual replication in red blood cells, while transmission to their mosquito vector requires asexual blood-stage parasites to differentiate into non-replicating gametocytes. This decision is controlled by stochastic derepression of a heterochromatin-silenced locus encoding AP2-G, the master transcription factor of sexual differentiation. The frequency of ap2-g derepression was shown to be responsive to extracellular phospholipid precursors but the mechanism linking these metabolites to epigenetic regulation of ap2-g was unknown. Through a combination of molecular genetics, metabolomics and chromatin profiling, we show that this response is mediated by metabolic competition for the methyl donor S-adenosylmethionine between histone methyltransferases and phosphoethanolamine methyltransferase, a critical enzyme in the parasite’s pathway for de novo phosphatidylcholine synthesis. When phosphatidylcholine precursors are scarce, increased consumption of SAM for de novo phosphatidylcholine synthesis impairs maintenance of the histone methylation responsible for silencing ap2-g, increasing the frequency of derepression and sexual differentiation. This provides a key mechanistic link that explains how LysoPC and choline availability can alter the chromatin status of the ap2-g locus controlling sexual differentiation.