A Plasmodium falciparum host-targeting motif functions in export during blood stage infection of the rodent malarial parasite Plasmodium berghei

Plasmodium falciparum (P. falciparum) secretes hundreds of proteins--including major virulence proteins--into the host erythrocyte. In order to reach the host cytoplasm, most P. falciparum proteins contain an N terminal host-targeting (HT) motif composed of 11 amino acids. In silico analyses have suggested that the HT motif is conserved throughout the Plasmodium species but experimental evidence only exists for P. falciparum. Here, we show that in the rodent malaria parasite Plasmodium berghei (P. berghei) a reporter-like green fluorescent protein expressed by the parasite can be exported to the erythrocyte cytoplasm in a HT-specific manner. This provides the first experimental proof that the HT motif can function as a signal for protein delivery to the erythrocyte across Plasmodium species. Further, it suggests that P. berghei may serve as a model for validation of P. falciparum secretome proteins. We also show that tubovesicular membranes extend from the vacuolar parasite into the erythrocyte cytoplasm and speculate that these structures may facilitate protein export to the erythrocyte.     

Sorting of malarial antigens into vesicular compartments within the host cell cytoplasm as demonstrated by immunoelectron microscopy

A double and triple immunogold labeling technique has been applied to demonstrate that several malarial antigens of the erythrocytic stages of Plasmodium falciparum are exported from the parasite into distinct compartments within the host cell cytoplasm. Multiple species of vesicles, each with specifically packaged contents, are consistent with a sorting function of vesicular structures in the Plasmodium infected erythrocyte. During schizogony, two parasite antigens, an S-antigen and a parasitophorous vacuole membrane antigen, QF 116, become packaged into such vesicles and are transported into the erythrocyte cytoplasm. At this stage of parasite development, host cell material is taken in through the parasitophorous vacuole membrane into the vacuolar space surrounding the parasite.     

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BACKGROUND: The development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised. METHODS: P. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database. RESULTS: Following four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library. CONCLUSION: The construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.     

The exported Plasmodium berghei protein IBIS1 delineates membranous structures in infected red blood cells

The importance of pathogen-induced host cell remodelling has been well established for red blood cell infection by the human malaria parasite Plasmodium falciparum. Exported parasite-encoded proteins, which often possess a signature motif, termed Plasmodium export element (PEXEL) or host-targeting (HT) signal, are critical for the extensive red blood cell modifications. To what extent remodelling of erythrocyte membranes also occurs in non-primate hosts and whether it is in fact a hallmark of all mammalian Plasmodium parasites remains elusive. Here we characterize a novel Plasmodium berghei PEXEL/HT-containing protein, which we term IBIS1. Temporal expression and spatial localization determined by fluorescent tagging revealed the presence of IBIS1 at the parasite/host interface during both liver and blood stages of infection. Targeted deletion of the IBIS1 protein revealed a mild impairment of intra-erythrocytic growth indicating a role for these structures in the rapid expansion of the parasite population in the blood in vivo. In red blood cells, the protein localizes to dynamic, punctate structures external to the parasite. Biochemical and microscopic data revealed that these intra-erythrocytic P. berghei-induced structures (IBIS) are membranous indicating that P. berghei, like P. falciparum, creates an intracellular membranous network in infected red blood cells.     

Deciphering the export pathway of malaria surface proteins

The intra-erythrocytic stages of Plasmodium falciparum assemble a unique protein trafficking system that targets parasite proteins to the red cell cytoplasm and cell surface. It is through this trafficking pathway that the primary virulence determinants of P. falciparum infections are targeted to the erythrocyte surface to mediate adhesion to host endothelial cells. A recent study has shown that SBP-1, a parasite protein associated with Maurer's clefts in the infected red cell cytosol, is essential for transport of the virulence factor PfEMP-1. This discovery sheds new light on the little-understood mechanisms that regulate protein trafficking in infected cells.     

Illuminating Plasmodium falciparum-infected red blood cells

The malaria parasite undergoes a remarkable series of morphological transformations, which underpin its life in both human and mosquito hosts. The advent of molecular transfection technology coupled with the ability to introduce fluorescent reporter proteins that faithfully track and expose the activities of parasite proteins has revolutionized our view of parasite cell biology. The greatest insights have been realized in the erythrocyte stages of Plasmodium falciparum. P. falciparum invades and remodels the human erythrocyte: it feeds on haemoglobin, grows and divides, and subverts the physiology of its hapless host. Fluorescent proteins have been employed to track and dissect each of these processes and have revealed details and exposed new paradigms.     

Effect of calpain inhibitors on the invasion of human erythrocytes by the parasite Plasmodium falciparum

17 different proteinase inhibitors were screened for their effect on the erythrocyte invasion by the malaria parasite Plasmodium falciparum. The effect was tested when the inhibitors were present in the culture medium and when they were trapped into erythrocyte ghosts. A very strong inhibition of invasion was observed in the presence of calpain inhibitors, with IC50 in the order of 10(-7) M. Chymostatin, leupeptin, pepstatin A and bestatin also caused inhibition of the invasion, but with IC50 in the order of 10(-5) M. The results suggest that participation of various proteinases in the process and point to the possibility of a calpain-mediated proteolytic event. This study may explain previous observations on the role of calcium in the invasion of the human erythrocyte by Plasmodium falciparum.     

Erythrocyte exit: Out, damned merozoite! Out I say!

A new study has combined video microscopy with fluorescent labeling of host and parasite membranes to follow Plasmodium falciparum merozoites as they exit their host erythrocyte. The result has yielded some arresting images, which make compelling viewing irrespective of whether or not you have an interest in cell motility in general or P. falciparum erythrocyte exit in particular. Moreover, this work injects important new insights into the long-running debate about the biological mechanisms that underpin merozoite release.     

Sphingolipid synthesis and membrane formation by Plasmodium

Plasmodium falciparum is a protozoan parasite that causes the most virulent o f human malarias. The asexual blood-stage organism invades and multiplies in a vacuole in the mature erythrocyte. During intravacuolar growth, it induces the formation of a novel network o f tubovesicular membranes, the TVM, that is not present in uninfected red blood cells. Recent data suggest that sphingomyelin biosynthesis by the parasite is an essential requirement for the assembly o f the TVM. Furthermore, sphingolipid synthesis as well as the formation and function o f the TVM may provide new targets for chemotherapy against malaria parasites.     

Home improvements: malaria and the red blood cell

In real-estate agent's terms, the red blood cell is a renovator's dream. The mature human erythrocyte has no internal organelles, no protein synthesis machinery and no infrastructure for protein trafficking. The malaria parasite invades this empty shell and effectively converts the erythrocyte back into a fully functional eukaryotic cell. In this article, Michael Foley and Leann Tilley examine the Plasmodium falciparum proteins that interact with the membrane skeleton at different stages of the infection and speculate on the roles of these proteins in the remodelling process.     

Vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes

During the development of the asexual stage of the malaria parasite, Plasmodium falciparum, the composition, structure and function of the host cell membrane is dramatically altered, including the ability to adhere to vascular endothelium. Crucial to these changes is the transport of parasite proteins, which become associated with or inserted into the erythrocyte membrane. Protein and membrane targeting beyond the parasite plasma membrane must require unique pathways, given the parasites intracellular location within a parasitophorous vacuolar membrane and the lack of organelles and biosynthetic machinery in the host cell necessary to support a secretory system. It is not clear how these proteins cross the parasitophorous vacuolar membrane or how they traverse the erythrocyte cytosol to reach their final destinations. The identification of: (1) a P. falciparum homologue of the protein Sar1p, which is an essential component of the COPII-based secretory system in mammalian cells and yeast and (2) electron-dense, possibly coated, secretory vesicles bearing P. falciparum erythrocyte membrane protein 1 and P. falciparum erythrocyte membrane protein 3 in the host cell cytosol of P. falciparum infected erythrocytes recently provided the first direct evidence of a vesicle-mediated pathway for the trafficking of some parasite proteins to the erythrocyte membrane. The major advance in uncovering the parasite-induced secretory pathway was made by incubating infected erythrocytes with aluminium tetrafluoride, an activator of guanidine triphosphate-binding proteins, which resulted in the accumulation of the vesicles into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminium fluoride treatment, making their capture by electron microscopy possible. It appears that malaria parasites export proteins into the host cell cytosol to support a vesicle-mediated protein trafficking pathway.     

Targeting malaria parasite proteins to the erythrocyte

The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.     

Protein transport across the parasitophorous vacuole of Plasmodium falciparum: into the great wide open

The human malaria parasite Plasmodium falciparum resides and multiplies within a membrane-bound vacuole in the cytosol of its host cell, the mature human erythrocyte. To enable the parasite to complete its intraerythrocytic life cycle, a large number of parasite proteins are synthesized and transported from the parasite to the infected cell. To gain access to the erythrocyte, parasite proteins must first cross the membrane of the parasitophorous vacuole (PVM), a process that is not well understood at the mechanistic level. Here, we review past and current literature on this topic, and make tentative predictions about the nature of the transport machinery required for transport of proteins across the PVM, and the molecular factors involved.     

Plasmodium falciparum erythrocyte membrane protein-1 specifically suppresses early production of host interferon-gamma

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a variable antigen expressed by P. falciparum, the malarial parasite. PfEMP-1, present on the surface of infected host erythrocytes, mediates erythrocyte binding to vascular endothelium, enabling the parasite to avoid splenic clearance. In addition, PfEMP-1 is proposed to regulate host immune responses via interactions with the CD36 receptor on antigen-presenting cells. We investigated the immunoregulatory function of PfEMP-1 by comparing host cell responses to erythrocytes infected with either wild-type parasites or transgenic parasites lacking PfEMP-1. We showed that PfEMP-1 suppresses the production of the cytokine interferon-gamma by human peripheral blood mononuclear cells early after exposure to P. falciparum. Suppression of this rapid proinflammatory response was CD36 independent and specific to interferon-gamma production by gammadelta-T, NK, and alphabeta-T cells. These data demonstrate a parasite strategy for downregulating the proinflammatory interferon-gamma response and further establish transgenic parasites lacking PfEMP-1 as powerful tools for elucidating PfEMP-1 functions.     

Functional analysis of Plasmodium falciparum merozoite antigens: implications for erythrocyte invasion and vaccine development

Malaria is a major human health problem and is responsible for over 2 million deaths per year. It is caused by a number of species of the genus Plasmodium, and Plasmodium falciparum is the causative agent of the most lethal form. Consequently, the development of a vaccine against this parasite is a priority. There are a number of stages of the parasite life cycle that are being targeted for the development of vaccines. Important candidate antigens include proteins on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to manipulate the genome of Plasmodium species has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. This has provided new information on the role of merozoite antigens in erythrocyte invasion and also allows new approaches to address their potential as vaccine candidates.     

Invasion by P. falciparum merozoites suggests a hierarchy of molecular interactions

Central to the pathology of malaria disease are the repeated cycles of parasite invasion and destruction of human erythrocytes. In Plasmodium falciparum, the most virulent species causing malaria, erythrocyte invasion involves several specific receptor-ligand interactions that direct the pathway used to invade the host cell, with parasites varying in their dependency on these different pathways. Gene disruption of a key invasion ligand in the 3D7 parasite strain, the P. falciparum reticulocyte binding-like homolog 2b (PfRh2b), resulted in the parasite invading via a novel pathway. Here, we show results that suggest the molecular basis for this novel pathway is not due to a molecular switch but is instead mediated by the redeployment of machinery already present in the parent parasite but masked by the dominant role of PfRh2b. This would suggest that interactions directing invasion are organized hierarchically, where silencing of dominant invasion ligands reveal underlying alternative pathways. This provides wild parasites with the ability to adapt to immune-mediated selection or polymorphism in erythrocyte receptors and has implications for the use of invasion-related molecules in candidate vaccines.     

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.     

Taking Charge: Feeding Malaria via Anion Channels

The human malaria parasite Plasmodium falciparum increases red blood cell membrane permeability during infection to allow for import of nutrients and other solutes. Nguitragool et?al. (2011) have now identified parasite-encoded CLAG3 proteins as key components of the import channel located on the erythrocyte membrane.     

Functional analysis of proteins involved in Plasmodium falciparum merozoite invasion of red blood cells

Plasmodium falciparum causes the most lethal form of malaria in humans and is responsible for over two million deaths per year. The development of a vaccine against this parasite is an urgent priority and potential protein targets include those on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to transfect P. falciparum has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. In this review, we describe the use of this technology to examine the role of merozoite antigens in erythrocyte invasion and to address their potential as vaccine candidates.