Expression of var genes using novel integration methods in Plasmodium falciparum

Abstract

Infection with Plasmodium falciparum can result in cerebral and placental malaria. PfEMP1 surface antigens are in large part responsible for this, and are encoded by a ∼60 member var gene family. The var family can be subdivided into four major types (Groups A, B, C, and E) defined by their genetic sequences, genome location and cytoadherence phenotypes. Antibody recognition of these antigen subsets correlates with protection against disease, thus understanding the regulation of these genes and their functional properties may further treatments. Research has been hampered, however, by the silencing in vivo of all but one randomly activated var gene, and by technical limitations imposed by their large size. We applied a novel system of genetic integration into P. falciparum to studies of var gene regulation and in vitro PfEMPI protein expression.;We generated parasite lines containing integrated luciferase reporter cassettes driven by a Group A promoter (termed UpsA) to examine whether silencing of UpsA is mediated by the Group A var intron or a highly conserved downstream non-coding element. UpsA promoter activity was dramatically reduced when either element was present. Significance was confirmed with mathematical modeling of UpsA expression. The UpsA promoter was also capable of nucleating the spread of heterochromatin. Thus Group A promoters are inherently mediators of silencing, but are governed by repressive elements contained within the intron and terminator sequences.;We next applied this technology to in vitro expression of PfEMP1. We inserted attB sites into the IT4var3 locus of the cytoadherent A4 strain. Integration of transgenic cassettes into the ally sites permitted the expression of various miniPfEMP1 proteins that trafficked to parasite-derived structures in the red cell cytoplasm in a manner analagous to endogenous PfEMP1. This work is the first to describe in vitro trafficking of miniPfEMP1 to the surface of infected erythrocytes.;In summary, we have identified repressive cis-elements that reduce expression of a var gene implicated in severe malaria and have developed a miniPfEMP1 expression system. It is our hope that with these new tools, researchers will make great advancements in their pursuit of disease prevention strategies to combat this tenacious pathogen.