The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites

The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane.     

IMC1b is a putative membrane skeleton protein involved in cell shape, mechanical strength, motility, and infectivity of malaria ookinetes

Membrane skeletons are cytoskeletal elements that have important roles in cell development, shape, and structural integrity. Malaria parasites encode a conserved family of putative membrane skeleton proteins related to articulins. One member, IMC1a, is expressed in sporozoites and localizes to the pellicle, a unique membrane complex believed to form a scaffold onto which the ligands and glideosome are arranged to mediate parasite motility and invasion. IMC1b is a closely related structural paralogue of IMC1a, fostering speculation that it could be functionally homologous but in a different invasive life stage. Here we have generated genetically modified parasites that express IMC1b tagged with green fluorescent protein, and we show that it is targeted exclusively to the pellicle of ookinetes. We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity. These findings are consistent with a membrane skeletal role of IMC1b and provide strong experimental support for the view that membrane skeletons form an integral part of the pellicle of apicomplexan zoites and function to provide rigidity to the pellicular membrane complex. The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way. However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.     

The Role of Palmitoylation for Protein Recruitment to the Inner Membrane Complex of the Malaria Parasite

To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.     

A cluster of ring stage-specific genes linked to a locus implicated in cytoadherence in Plasmodium falciparum codes for PEXEL-negative and PEXEL-positive proteins exported into the host cell

Blood stages of Plasmodium falciparum export proteins into their erythrocyte host, thereby inducing extensive host cell modifications that become apparent after the first half of the asexual development cycle (ring stage). This is responsible for a major part of parasite virulence. Export of many parasite proteins depends on a sequence motif termed Plasmodium export element (PEXEL) or vacuolar transport signal (VTS). This motif has allowed the prediction of the Plasmodium exportome. Using published genome sequence, we redetermined the boundaries of a previously studied region linked to P. falciparum virulence, reducing the number of candidate genes in this region to 13. Among these, we identified a cluster of four ring stage-specific genes, one of which is known to encode an exported protein. We demonstrate that all four genes code for proteins exported into the host cell, although only two genes contain an obvious PEXEL/VTS motif. We propose that the systematic analysis of ring stage-specific genes will reveal a cohort of exported proteins not present in the currently predicted exportome. Moreover, this provides further evidence that host cell remodeling is a major task of this developmental stage. Biochemical and photobleaching studies using these proteins reveal new properties of the parasite-induced membrane compartments in the host cell. This has important implications for the biogenesis and connectivity of these structures.     

A systematic analysis of the early transcribed membrane protein family throughout the life cycle of Plasmodium yoelii

The early transcribed membrane proteins (ETRAMPs) are a family of small, highly charged transmembrane proteins unique to malaria parasites. Some members of the ETRAMP family have been localized to the parasitophorous vacuole membrane that separates the intracellular parasite from the host cell and thus presumably have a role in host-parasite interactions. Although it was previously shown that two ETRAMPs are critical for rodent malaria parasite liver-stage development, the importance of most ETRAMPs during the parasite life cycle remains unknown. Here, we comprehensively identify nine new etramps in the genome of the rodent malaria parasite Plasmodium yoelii, and elucidate their conservation in other malaria parasites. etramp expression profiles are diverse throughout the parasite life cycle as measured by RT-PCR. Epitope tagging of two ETRAMPs demonstrates protein expression in blood and liver stages, and reveals differences in both their timing of expression and their subcellular localization. Gene targeting studies of each of the nine uncharacterized etramps show that two are refractory to deletion and thus likely essential for blood-stage replication. Seven etramps are not essential for any life cycle stage. Systematic characterization of the members of the ETRAMP family reveals the diversity in importance of each family member at the interface between host and parasite throughout the developmental cycle of the malaria parasite.     

Identification and initial characterization of three novel cyclin-related proteins of the human malaria parasite Plasmodium falciparum

The molecular mechanisms regulating cell proliferation and development during the life cycle of malaria parasites remain to be elucidated. The peculiarities of the cell cycle organization during Plasmodium falciparum schizogony suggest that the modalities of cell cycle control in this organism may differ from those in other eukaryotes. Indeed, existing data concerning Plasmodium cell cycle regulators such as cyclin-dependent kinases reveal structural and functional properties that are divergent from those of their homologues in other systems. The work presented here lies in the context of the exploitation of the recently available P. falciparum genome sequence toward the characterization of putative cell cycle regulators. We describe the in silico identification of three open reading frames encoding proteins with maximal homology to various members of the cyclin family and demonstrate that the corresponding polypeptides are expressed in the erythrocytic stages of the infection. We present evidence that these proteins possess cyclin activity by demonstrating either their association with histone H1 kinase activity in parasite extracts or their ability to activate PfPK5, a P. falciparum cyclin-dependent kinase homologue, in vitro. Furthermore, we show that RINGO, a protein with no sequence homology to cyclins but that is nevertheless a strong activator of mammalian CDK1/2, is also a strong activator of PfPK5 in vitro. This raises the possibility that "cryptic" cell cycle regulators may be found among the 50% of the open reading frames in the P. falciparum genome that display no homology to any known proteins.     

Malaria IMC1 membrane skeleton proteins operate autonomously and participate in motility independently of cell shape

Plasmodium IMC1 (inner membrane complex 1) proteins comprise components of the subpellicular network, a lattice of intermediate filaments that form a structural part of the pellicle in the zoite stages of malaria parasites. Family members IMC1a and IMC1b are differentially expressed in sporozoites and ookinetes, respectively, but have functionally equivalent roles affecting cell morphology, strength, motility, and infectivity. Because of the coincident effects of previous imc1 gene disruptions on both zoite shape and locomotion, it has been impossible to ascribe a direct involvement in motility to these proteins. We show here that a third family member, IMC1h, has a distinct differential expression pattern and localizes to the pellicle of both ookinetes and sporozoites. Knock-out of IMC1h mimics the loss-of-function phenotypes of IMC1a and IMC1b in their respective life stages, indicating that IMC1 proteins could be operating co-dependently. By generating double null mutant parasites for IMC1h and IMC1b, we tested this hypothesis: double knock-out exacerbated the phenotypes of the single knock-outs in terms of ookinete strength, motility, and infectivity but did not further affect ookinete morphology. These findings provide the first genetic evidence that IMC1 proteins can function independently of each other and contribute to gliding motility independently of cell shape.     

Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum

We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCalpha and PfGCbeta) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCbeta in Escherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCalpha was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct.     

Depletion of Plasmodium berghei plasmoredoxin reveals a non-essential role for life cycle progression of the malaria parasite

Proliferation of the pathogenic Plasmodium asexual blood stages in host erythrocytes requires an exquisite capacity to protect the malaria parasite against oxidative stress. This function is achieved by a complex antioxidant defence system composed of redox-active proteins and low MW antioxidants. Here, we disrupted the P. berghei plasmoredoxin gene that encodes a parasite-specific 22 kDa member of the thioredoxin superfamily. The successful generation of plasmoredoxin knockout mutants in the rodent model malaria parasite and phenotypic analysis during life cycle progression revealed a non-vital role in vivo. Our findings suggest that plasmoredoxin fulfils a specialized and dispensable role for Plasmodium and highlights the need for target validation to inform drug development strategies.     

The Armadillo repeat protein PF16 is essential for flagellar structure and function in Plasmodium male gametes

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite.     

Subcellular location, phosphorylation and assembly into the motor complex of GAP45 during Plasmodium falciparum schizont development

An actomyosin motor complex assembled below the parasite's plasma membrane drives erythrocyte invasion by Plasmodium falciparum merozoites. The complex is comprised of several proteins including myosin (MyoA), myosin tail domain interacting protein (MTIP) and glideosome associated proteins (GAP) 45 and 50, and is anchored on the inner membrane complex (IMC), which underlies the plasmalemma. A ternary complex of MyoA, MTIP and GAP45 is formed that then associates with GAP50. We show that full length GAP45 labelled internally with GFP is assembled into the motor complex and transported to the developing IMC in early schizogony, where it accumulates during intracellular development until merozoite release. We show that GAP45 is phosphorylated by calcium dependent protein kinase 1 (CDPK1), and identify the modified serine residues. Replacing these serine residues with alanine or aspartate has no apparent effect on GAP45 assembly into the motor protein complex or its subcellular location in the parasite. The early assembly of the motor complex suggests that it has functions in addition to its role in erythrocyte invasion.     

Transmission of the malaria parasite requires ferlin for gamete egress from the red blood cell

Ferlins mediate calcium‐dependent vesicular fusion. Although conserved throughout eukaryotic evolution, their function in unicellular organisms including apicomplexan parasites is largely unknown. Here, we define a crucial role for a ferlin‐like protein (FLP) in host‐to‐vector transmission of the rodent malaria parasite Plasmodium berghei. Infection of the mosquito vectors requires the formation of free gametes and their fertilisation in the mosquito midgut. Mature gametes will only emerge upon secretion of factors that stimulate the disruption of the red blood cell membrane and the parasitophorous vacuole membrane. Genetic depletion of FLP in sexual stages leads to a complete life cycle arrest in the mosquito. Although mature gametes form normally, mutants lacking FLP remain trapped in the red blood cell. The egress defect is rescued by detergent‐mediated membrane lysis. In agreement with ferlin vesicular localisation, HA‐tagged FLP labels intracellular speckles, which relocalise to the cell periphery during gamete maturation. Our data define FLP as a novel critical factor for Plasmodium fertilisation and transmission and suggest an evolutionarily conserved example of ferlin‐mediated exocytosis.     

PfSPATR,a Plasmodium falciparum protein containing an altered thrombospondin type I repeat domain is expressed at several stages of the parasite life cycle and is the target of inhibitory antibodies

The annotated sequence of chromosome 2 of Plasmodium falciparum was examined for genes encoding proteins that may be of interest for vaccine development. We describe here the characterization of a protein with an altered thrombospondin Type I repeat domain (PfSPATR) that is expressed in the sporozoite, asexual, and sexual erythrocytic stages of the parasite life cycle. Immunoelectron microscopy indicated that this protein was expressed on the surface of the sporozoites and around the rhoptries in the asexual erythrocytic stage. An Escherichia coli-produced recombinant form of the protein bound to HepG2 cells in a dose-dependent manner and antibodies raised against this protein blocked the invasion of sporozoites into a transformed hepatoma cell line. Sera from Ghanaian adults and from a volunteer who had been immunized with radiation-attenuated P. falciparum sporozoites specifically recognized the expression of this protein on transfected COS-7 cells. These data support the evaluation of this protein as a vaccine candidate.     

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.     

Functional dissection of the apicomplexan glideosome molecular architecture

The glideosome of apicomplexan parasites is an actin- and myosin-based machine located at the pellicle, between the plasma membrane (PM) and inner membrane complex (IMC), that powers parasite motility, migration, and host cell invasion and egress. It is composed of myosin A, its light chain MLC1, and two gliding-associated proteins, GAP50 and GAP45. We identify GAP40, a polytopic protein of the IMC, as an additional glideosome component and show that GAP45 is anchored to the PM and IMC via its N- and C-terminal extremities, respectively. While the C-terminal region of GAP45 recruits MLC1-MyoA to the IMC, the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress. The orthologous Plasmodium falciparum GAP45 can fulfill this dual function, as shown by transgenera complementation, whereas the coccidian GAP45 homolog (designated here as) GAP70 specifically recruits the glideosome to the apical cap of the parasite.     

Gliding Associated Proteins Play Essential Roles during the Formation of the Inner Membrane Complex of Toxoplasma gondii

The inner membrane complex (IMC) of apicomplexan parasites is a specialised structure localised beneath the parasite’s plasma membrane, and is important for parasite stability and intracellular replication. Furthermore, it serves as an anchor for the myosin A motor complex, termed the glideosome. While the role of this protein complex in parasite motility and host cell invasion has been well described, additional roles during the asexual life cycle are unknown. Here, we demonstrate that core elements of the glideosome, the gliding associated proteins GAP40 and GAP50 as well as members of the GAPM family, have critical roles in the biogenesis of the IMC during intracellular replication. Deletion or disruption of these genes resulted in the rapid collapse of developing parasites after initiation of the cell cycle and led to redistribution of other glideosome components.