Tobias Spielmann

Functional Analysis of the Leading Malaria Vaccine Candidate AMA-1 Reveals an Essential Role for the Cytoplasmic Domain in the Invasion Process

A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum

Functions have yet to be defined for the majority of genes of Plasmodium falciparum, the agent responsible for the most serious form of human malaria. Here we report changes in P. falciparum gene expression induced by 20 compounds that inhibit growth of the schizont stage of the intraerythrocytic development cycle. In contrast with previous studies, which reported only minimal changes in response to chemically induced perturbations of P. falciparum growth, we find that ∼59% of its coding genes display over three-fold changes in expression in response to at least one of the chemicals we tested. We use this compendium for guilt-by-association prediction of protein function using an interaction network constructed from gene co-expression, sequence homology, domain-domain and yeast two-hybrid data. The subcellular localizations of 31 of 42 proteins linked with merozoite invasion is consistent with their role in this process, a key target for malaria control. Our network may facilitate identification of novel antimalarial drugs and vaccines.

Protein export in malaria parasites: do multiple export motifs add up to multiple export pathways?

Intracellular malaria parasites export numerous proteins into their host cell, a process essential for parasite survival and virulence. Many of these proteins are defined by a short amino acid sequence motif termed PEXEL or VTS that mediates their export, suggesting a collective trafficking route. The existence of several PEXEL-negative exported proteins (PNEPs) indicates that alternative export pathways might also exist. We review recent data on the sequences mediating export of PNEPs and compare this process to PEXEL export taking into account novel findings on the function of this motif. Based on this we propose that, despite the lack of a PEXEL in PNEPs, both groups of proteins might converge in a single export pathway on their way into the host cell.

Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export.

Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite Plasmodium falciparum. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.

Sequence requirements for the export of the Plasmodium falciparum Maurer's clefts protein REX2.

A short motif termed Plasmodium export element (PEXEL) or vacuolar targeting signal (VTS) characterizes Plasmodium proteins exported into the host cell. These proteins mediate host cell modifications essential for parasite survival and virulence. However, several PEXEL‐negative exported proteins indicate that the currently predicted malaria exportome is not complete and it is unknown whether and how these proteins relate to PEXEL‐positive export. Here we show that the N‐terminal 10 amino acids of the PEXEL‐negative exported protein REX2 (ring‐exported protein 2) are necessary for its targeting and that a single‐point mutation in this region abolishes export. Furthermore we show that the REX2 transmembrane domain is also essential for export and that together with the N‐terminal region it is sufficient to promote export of another protein. An N‐terminal region and the transmembrane domain of the unrelated PEXEL‐negative exported protein SBP1 (skeleton‐binding protein 1) can functionally replace the corresponding regions in REX2, suggesting that these sequence features are also present in other PEXEL‐negative exported proteins. Similar to PEXEL proteins we find that REX2 is processed, but in contrast, detect no evidence for N‐terminal acetylation.

Uncovering common principles in protein export of malaria parasites.

For proliferation, the malaria parasite Plasmodium falciparum needs to modify the infected host cell extensively. To achieve this, the parasite exports proteins containing a Plasmodium export element (PEXEL) into the host cell. Phosphatidylinositol-3-phosphate binding and cleavage of the PEXEL are thought to mediate protein export. We show that these requirements can be bypassed, exposing a second level of export control in the N terminus generated after PEXEL cleavage that is sufficient to distinguish exported from nonexported proteins. Furthermore, this region also corresponds to the export domain of a second group of exported proteins lacking PEXELs (PNEPs), indicating shared export properties among different exported parasite proteins. Concordantly, export of both PNEPs and PEXEL proteins depends on unfolding, revealing translocation as a common step in export. However, translocation of transmembrane proteins occurs at the parasite plasma membrane, one step before translocation of soluble proteins, indicating unexpectedly complex translocation events at the parasite periphery.

Selective permeabilization of the host cell membrane of Plasmodium falciparum-infected red blood cells with streptolysin O and equinatoxin II.

Plasmodium falciparum develops within the mature RBCs (red blood cells) of its human host in a PV (parasitophorous vacuole) that separates the host cell cytoplasm from the parasite surface. The pore-forming toxin, SLO (streptolysin O), binds to cholesterol-containing membranes and can be used to selectively permeabilize the host cell membrane while leaving the PV membrane intact. We found that in mixtures of infected and uninfected RBCs, SLO preferentially lyses uninfected RBCs rather than infected RBCs, presumably because of differences in cholesterol content of the limiting membrane. This provides a means of generating pure preparations of viable ring stage infected RBCs. As an alternative permeabilizing agent we have characterized EqtII (equinatoxin II), a eukaryotic pore-forming toxin that binds preferentially to sphingomyelin-containing membranes. EqtII lyses the limiting membrane of infected and uninfected RBCs with similar efficiency but does not disrupt the PV membrane. It generates pores of up to 100 nm, which allow entry of antibodies for immunofluorescence and immunogold labelling. The present study provides novel tools for the analysis of this important human pathogen and highlights differences between Plasmodium-infected and uninfected RBCs.

Targeting of the Ring Exported Protein 1 to the Maurer’s Clefts is Mediated by a Two-Phase Process

Early development of Plasmodium falciparum within the erythrocyte is characterized by the large‐scale export of proteins to the host cell. In many cases, export is mediated by a short sequence called the Plasmodium export element (PEXEL) or vacuolar transport signal; however, a number of previously characterized exported proteins do not contain such an element. In this study, we investigated the mechanisms of export of the PEXEL‐negative ring exported protein 1 (REX1). This protein localizes to the Maurer’s clefts, parasite‐induced structures in the host‐cell cytosol. Transgenic parasites expressing green fluorescent protein–REX1 chimeras revealed that the single hydrophobic stretch plus an additional 10 amino acids mediate the export of REX1. Biochemical characterization of these chimeras indicated that REX1 was exported as a soluble protein. Inclusion of a sequence containing a predicted coiled‐coil motif led to the correct localization of REX1 at the Maurer’s clefts, suggesting that association with the clefts occurs at the final stage of protein export only. These results indicate that PEXEL‐negative exported proteins can be exported in a soluble state and that sequences without any apparent resemblance to a PEXEL motif can mediate export across the parasitophorous vacuole membrane.

Organization of ETRAMPs and EXP‐1 at the parasite–host cell interface of malaria parasites

The parasite–host cell interface is a key compartment of vacuolated intracellular pathogens but little is known about its molecular composition and architecture. We used in vivo cross‐linking to analyse the parasite–host cell interface of asexual stages of the most virulent human malaria parasite Plasmodium falciparum. We show that the integral membrane protein members of the early transcribed membrane protein (ETRAMP) family and exported protein 1 (EXP‐1), which are components of the parasite–host cell interface, form complexes of oligomeric arrays in this compartment. The most notable feature is that each ETRAMP member and EXP‐1 define separate arrays, demonstrating that the protein distribution in this membrane is non‐random. Each of three recombinant ETRAMPs readily oligomerized in bacterial membranes, confirming that these arrays can form independently of other Plasmodium proteins. We propose that the malaria parasite–host cell interface contains patches of integral membrane proteins forming a mosaic of different microdomains in this membrane.

Targeted mutagenesis of the ring-exported protein-1 of Plasmodium falciparum disrupts the architecture of Maurer's cleft organelles

Mature red blood cells have no internal trafficking machinery, so the intraerythrocytic malaria parasite, Plasmodium falciparum, establishes its own transport system to export virulence factors to the red blood cell surface. Maurers clefts are parasite‐derived membranous structures that form an important component of this exported secretory system. A protein with sequence similarity to a Golgi tethering protein, referred to as ring‐exported protein‐1 (REX1), is associated with Maurers clefts. A REX1–GFP chimera is trafficked to the Maurers clefts and preferentially associates with the edges of these structures, as well as with vesicle‐like structures and with stalk‐like extensions that are involved in tethering the Maurers clefts to other membranes. We have generated transfected P. falciparum expressing REX1 truncations or deletion. Electron microscopy reveals that the Maurers clefts of REX1 truncation mutants have stacked cisternae, while the 3D7 parent line has unstacked Maurers clefts. D10 parasites, which have lost the right end of chromosome 9, including the rex1 gene, also display Maurers clefts with stacked cisternae. Expression of full‐length REX1–GFP in D10 parasites restores the 3D7‐type unstacked Maurers cleft phenotype. These studies reveal the importance of the REX1 protein in determining the ultrastructure of the Maurers cleft system.