Abstract
BackgroundPlasmodium falciparum parasites export more than 400 proteins into the cytosol of their host erythrocytes. These exported proteins catalyse the formation of knobs on the erythrocyte plasma membrane and an overall increase in erythrocyte rigidity, presumably by modulating the endogenous erythrocyte cytoskeleton. In uninfected erythrocytes, Band 4.1 (4.1R) plays a key role in regulating erythrocyte shape by interacting with multiple proteins through the three lobes of its cloverleaf-shaped N-terminal domain. In P. falciparum-infected erythrocytes, the C-lobe of 4.1R interacts with the P. falciparum protein mature parasite-infected erythrocyte surface antigen (MESA), but it is not currently known whether other P. falciparum proteins bind to other lobes of the 4.1R N-terminal domain.MethodsIn order to identify novel 4.1R interacting proteins, a yeast two-hybrid screen was performed with a fragment of 4.1R containing both the N- and α-lobes. Positive interactions were confirmed and investigated using site-directed mutagenesis, and antibodies were raised against the interacting partner to characterise it’s expression and distribution in P. falciparum infected erythrocytes.ResultsYeast two-hybrid screening identified a positive interaction between the 4.1R N- and α-lobes and PF3D7_0402000. PF3D7_0402000 is a member of a large family of exported proteins that share a domain of unknown function, the PHIST domain. Domain mapping and site-directed mutagenesis established that it is the PHIST domain of PF3D7_0402000 that interacts with 4.1R. Native PF3D7_0402000 is localized at the parasitophorous vacuole membrane (PVM), and colocalizes with a subpopulation of 4.1R.DiscussionThe function of the majority of P. falciparum exported proteins, including most members of the PHIST family, is unknown, and in only a handful of cases has a direct interaction between P. falciparum-exported proteins and components of the erythrocyte cytoskeleton been established. The interaction between 4.1R and PF3D7_0402000, and localization of PF3D7_0402000 with a sub-population of 4.1R at the PVM could indicate a role in modulating PVM structure. Further investigation into the mechanisms for 4.1R recruitment is needed.ConclusionPF3D7_0402000 was identified as a new binding partner for the major erythrocyte cytoskeletal protein, 4.1R. This interaction is consistent with a growing body of literature that suggests the PHIST family members function by interacting directly with erythrocyte proteins.
Highlights
Plasmodium falciparum parasites export more than 400 proteins into the cytosol of their host erythrocytes
As the parasite matures the erythrocyte becomes more rigid, loses its biconcave shape for a more spherocytic one, and small electron dense protrusions, known as knobs, form on the surface [1]. These modifications enable the infected erythrocyte to adhere to and block the microvasculature and microcapillaries, which in turn contributes to specific complications such as cerebral and placental malaria [2,3]. Plasmodium falciparum achieves these massive changes in erythrocyte structure by exporting proteins into the erythrocyte cytosol, some of which interact with components of the host cytoskeleton and plasma membrane [3]
A few of these exported proteins have been studied in detail, such as knobassociated histidine rich protein (KAHRP), an essential structural component of knobs that secure the major cytoadherence ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the erythrocyte cytoskeleton through interactions with spectrin and actin [4,5,6], and mature parasite infected erythrocyte surface antigen (MESA), which binds to the erythrocyte cytoskeleton associated protein Band 4.1, known as 4.1R [7,8]
Summary
Plasmodium falciparum parasites export more than 400 proteins into the cytosol of their host erythrocytes. As the parasite matures the erythrocyte becomes more rigid, loses its biconcave shape for a more spherocytic one, and small electron dense protrusions, known as knobs, form on the surface [1] These modifications enable the infected erythrocyte to adhere to and block the microvasculature and microcapillaries, which in turn contributes to specific complications such as cerebral and placental malaria [2,3]. Plasmodium falciparum achieves these massive changes in erythrocyte structure by exporting proteins into the erythrocyte cytosol, some of which interact with components of the host cytoskeleton and plasma membrane [3]. One PHISTb family member, ring infected surface antigen (RESA), is known to interact directly with the erythrocyte cytoskeleton by binding to spectrin tetramers, resulting in increased resistance to shear stress and thermal damage [13], while another binds to the C-terminal tail of PfEMP1 [14] but in the majority of cases no binding partner has been identified
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