Plasmodium falciparum Plasmodium helical interspersed subtelomeric proteins contribute to cytoadherence and anchor P. falciparum erythrocyte membrane protein 1 to the host cell cytoskeleton

Adherence of Plasmodium falciparum‐infected erythrocytes to host endothelium is conferred through the parasite‐derived virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), the major contributor to malaria severity. PfEMP1 located at knob structures on the erythrocyte surface is anchored to the cytoskeleton, and the Plasmodium helical interspersed subtelomeric (PHIST) gene family plays a role in many host cell modifications including binding the intracellular domain of PfEMP1. Here, we show that conditional reduction of the PHIST protein PFE1605w strongly reduces adhesion of infected erythrocytes to the endothelial receptor CD36. Adhesion to other endothelial receptors was less affected or even unaltered by PFE1605w depletion, suggesting that PHIST proteins might be optimized for subsets of PfEMP1 variants. PFE1605w does not play a role in PfEMP1 transport, but it directly interacts with both the intracellular segment of PfEMP1 and with cytoskeletal components. This is the first report of a PHIST protein interacting with key molecules of the cytoadherence complex and the host cytoskeleton, and this functional role seems to play an essential role in the pathology of P. falciparum.     

Protein phosphatase 1, a Plasmodium falciparum essential enzyme, is exported to the host cell and implicated in the release of infectious merozoites

The malarial parasite Plasmodium falciparum transposes a Golgi-like compartment, referred to as Maurer's clefts, into the cytoplasm of its host cell, the erythrocyte, and delivering parasite molecules to the host cell surface. We report here a novel role of the Maurer's clefts implicating a parasite protein phosphatase 1 (PP1) and related to the phosphorylation status of P. falciparum skeleton-binding protein 1 (PfSBP1), a trans-membrane protein of the clefts interacting with the host cell membrane via its carboxy-terminal domain. Based on co-immunoprecipitation and inhibition studies, we show that the parasite PP1 type phosphatase modulates the phosphorylation status of the amino-terminal domain of PfSBP1 in the lumen of Maurer's clefts. Importantly, the addition of a PP1 inhibitor, calyculin A, to late schizonts results in the hyperphosphorylation of PfSBP1 and prevents parasite release from the host cell. We propose that the hyperphosphorylation of PfSBP1 interferes with the release of merozoites, the invasive blood stage of the parasite, by increasing the red cell membrane stability. Moreover, the parasite PP1 phosphatase is the first enzyme essential for the parasite development detected in the Maurer's clefts.     

A revised mechanism for how Plasmodium falciparum recruits and exports proteins into its erythrocytic host cell

Plasmodium falciparum exports ~10% of its proteome into its host erythrocyte to modify the host cell’s physiology. The Plasmodium export element (PEXEL) motif contained within the N-terminus of most exported proteins directs the trafficking of those proteins into the erythrocyte. To reach the host cell, the PEXEL motif of exported proteins is processed by the endoplasmic reticulum (ER) resident aspartyl protease plasmepsin V. Then, following secretion into the parasite-encasing parasitophorous vacuole, the mature exported protein must be unfolded and translocated across the parasitophorous vacuole membrane by the Plasmodium translocon of exported proteins (PTEX). PTEX is a protein-conducting channel consisting of the pore-forming protein EXP2, the protein unfoldase HSP101, and structural component PTEX150. The mechanism of how exported proteins are specifically trafficked from the parasite’s ER following PEXEL cleavage to PTEX complexes on the parasitophorous vacuole membrane is currently not understood. Here, we present evidence that EXP2 and PTEX150 form a stable subcomplex that facilitates HSP101 docking. We also demonstrate that HSP101 localises both within the parasitophorous vacuole and within the parasite’s ER throughout the ring and trophozoite stage of the parasite, coinciding with the timeframe of protein export. Interestingly, we found that HSP101 can form specific interactions with model PEXEL proteins in the parasite’s ER, irrespective of their PEXEL processing status. Collectively, our data suggest that HSP101 recognises and chaperones PEXEL proteins from the ER to the parasitophorous vacuole and given HSP101’s specificity for the EXP2-PTEX150 subcomplex, this provides a mechanism for how exported proteins are specifically targeted to PTEX for translocation into the erythrocyte.     

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.     

A conserved region in the EBL proteins is implicated in microneme targeting of the malaria parasite Plasmodium falciparum

The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBA-GFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.     

A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum.

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).     

Proteomic analysis identifies novel proteins of the Maurer's clefts, a secretory compartment delivering Plasmodium falciparum proteins to the surface of its host cell

A novel method was validated for the efficient distinction between malaria parasite-derived and host cell proteins in mass spectrometry analyses. This method was applied to a ghost fraction from Plasmodium falciparum-infected erythrocytes containing the red blood cell plasma membrane, the erythrocyte submembrane skeleton, and the Maurer's clefts, a Golgi-like apparatus linked to and addressing parasite proteins to the host cell surface. This method allowed the identification of 78 parasite proteins. Among these we identified seven novel proteins of the Maurer's clefts based on immunofluorescence studies and proteinase K digestion assays. The products of six contiguous genes located on chromosome 5 were identified, and the location within the Maurer's clefts was established for two of them. This suggests a clustering of genes encoding Maurer's cleft proteins. Our study sheds new light on the biological function of the Maurer's clefts, which are central to the pathogenesis and to the intraerythrocytic development of P. falciparum.     

Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum

Gametocyte maturation in Plasmodium falciparum is a critical step in the transmission of malaria. While the majority of parasites proliferate asexually in red blood cells, a small fraction of parasites undergo sexual conversion and mature over 2 weeks to become competent for transmission to a mosquito vector. Immature gametocytes sequester in deep tissues while mature stages must be able to circulate, pass the spleen and present themselves to the mosquito vector in order to complete transmission. Sequestration of asexual red blood cell stage parasites has been investigated in great detail. These studies have demonstrated that induction of cytoadherence properties through specific receptor-ligand interactions coincides with a significant increase in host cell stiffness. In contrast, the adherence and biophysical properties of gametocyte-infected red blood cells have not been studied systematically. Utilizing a transgenic line for 3D live imaging, in vitro capillary assays and 3D finite element whole cell modelling, we studied the role of cellular deformability in determining the circulatory characteristics of gametocytes. Our analysis shows that the red blood cell deformability of immature gametocytes displays an overall decrease followed by rapid restoration in mature gametocytes. Intriguingly, simulations suggest that along with deformability variations, the morphological changes of the parasite may play an important role in tissue distribution in vivo. Taken together, we present a model, which suggests that mature but not immature gametocytes circulate in the peripheral blood for uptake in the mosquito blood meal and transmission to another human host thus ensuring long-term survival of the parasite.     

Plasmodium falciparum and the permeation pathway of the host red blood cell

Invasion of red blood cells by malaria parasites leads to a huge increase in solute traffic across the membrane of a normally tight cell. Recent electrophysiological investigations strongly support earlier evidence from transport and pharmacological studies that the permeability pathway, which the parasite induces in the host cell membrane, is an anion-selective channel. This article analyzes the evidence and controversies concerning the nature of this channel, surveys the main open questions and suggests directions for future research in this area.     

Molecular factors responsible for host cell recognition and invasion in Plasmodium falciparum.

In Plasmodium falciparum, the rhoptries involved in the invasion process are a pair of flask-shaped organelles located at the apical tip of invading stages. They, along with the more numerous micronemes and dense granules, constitute the apical complex in Plasmodium and other members of the phylum Apicomplexa. Several proteins of varying molecular weight have been identified in P. falciparum rhoptries. These include the 225-, 140/130/110-, 80/60/40-, RAP-1 80-, AMA-1 80-, QF3 80-, and 55-kDa proteins. Some of these proteins are lost during schizont rupture and release of merozoites. Others such as the 140/130/110-kDa complex are transferred to the erythrocyte membrane during invasion. The ring-infected surface antigen (RESA), a 155-kDa polypeptide located in dense granules also associates with the erythrocyte membrane during invasion. Erythrocyte-binding studies have demonstrated that both the 140/130/110-kDa rhoptry complex and RESA bind to inside-out-vesicles (IOVs) prepared from human erythrocytes. The 140/130/110-kDa complex also binds to erythrocyte membranes prepared by hypotonic lysis. These proteins, however, do not bind to intact human erythrocytes. In a heterologous erythrocyte model, both the 140/130/110-kDa complex and RESA are shown to bind directly to mouse erythrocytes. Other studies have shown that RESA associates with spectrin in the erythrocyte cytoskeleton. We have recently developed a liposome-binding assay to demonstrate the lipophilic binding properties of the P. falciparum rhoptry complex of 140/130/110 kDa. The rhoptry complex binds to liposomes containing neutrally, positively, and negatively charged phospholipids. However, liposomes containing phosphatidylethanolamine compete effectively for rhoptry protein binding to mouse erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of erythrocyte membrane proteins on the in vitro invasion of the human malarial parasite (Plasmodium falciparum) into its host cell

The intracellular development of the erythrocytic stage of the malarial parasite (merozoite) is initiated by the attachment of the parasite to the erythrocyte surface. This paper describes an assay system to investigate Plasmodium falciparum merozoite entry into the host cell and reports on three observations regarding this interaction. (a) Merozoites do not invade human erythrocytes treated with either trypsin or neuraminidase, and both enzymes partially cleave glycophorin A, the major erythrocyte surface sialoglycoprotein. (b) A membrane protein fraction containing glycophorin A will, at low concentrations, inhibit the invasion of isolated merozoites into erythrocytes; no other fractions of membrane proteins have appreciable effects on the reinvasion. (c) Merozoites do not reinvade erythrocytes preincubated with F ab' fragments of antibody prepared against glycophorin A. Together, these three observations imply a role for glycophorin A in the attachment of the malarial parasite to the erythrocyte surface.     

Biosynthesis, localization, and macromolecular arrangement of the Plasmodium falciparum translocon of exported proteins (PTEX)

To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA(+) ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ～600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.     

Sequestration and metabolism of host cell arginine by the intraerythrocytic malaria parasite Plasmodium falciparum

Human erythrocytes have an active nitric oxide synthase, which converts arginine into citrulline and nitric oxide (NO). NO serves several important functions, including the maintenance of normal erythrocyte deformability, thereby ensuring efficient passage of the red blood cell through narrow microcapillaries. Here, we show that following invasion by the malaria parasite Plasmodium falciparum the arginine pool in the host erythrocyte compartment is sequestered and metabolized by the parasite. Arginine from the extracellular medium enters the infected cell via endogenous host cell transporters and is taken up by the intracellular parasite by a high‐affinity cationic amino acid transporter at the parasite surface. Within the parasite arginine is metabolized into citrulline and ornithine. The uptake and metabolism of arginine by the parasite deprive the erythrocyte of the substrate required for NO production and may contribute to the decreased deformability of infected erythrocytes.     

A potential novel mechanism for the insertion of a membrane protein revealed by a biochemical analysis of the Plasmodium falciparum cytoadherence molecule PfEMP-1

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is exposed on the surface of infected erythrocytes where it both acts as an important pathogenicity factor in malaria and undergoes antigenic variation as a means of immune evasion. Because the mammalian erythrocyte lacks a protein secretory machinery there has been much interest in elucidating the mechanism whereby this protein is transferred from its site of synthesis within the parasite to its final destination. Current opinion favours a mechanism whereby PfEMP-1 becomes cotranslationally inserted into the endoplasmic reticulum of the parasite and is subsequently transported as an integral part of an erythrocyte cytoplasmic membrane system derived from the parasite. Here we show that the solubility characteristics of this protein during several stages of its transport pathway are inconsistent with this view. Instead we propose that the protein is synthesized as a peripheral membrane protein which only when it arrives at its final destination assumes a transmembrane topology. Even in this state, the extractability of the protein with urea suggest that it is anchored in the membrane by protein-protein rather than by protein-lipid interaction.     

Heterochromatin formation in bistable chromatin domains controls the epigenetic repression of clonally variant Plasmodium falciparum genes linked to erythrocyte invasion

Clonally variant gene expression is a common survival strategy used by many pathogens, including the malaria parasite Plasmodium falciparum. Among the genes that show variant expression in this parasite are several members of small gene families linked to erythrocyte invasion, including the clag and eba families. The active or repressed state of these genes is clonally transmitted by epigenetic mechanisms. Here we characterized the promoters of clag3.1, clag3.2 and eba-140, and compared nuclease accessibility and post-translational histone modifications between their active and repressed states. Activity of these promoters in an episomal context is similar between parasite subclones characterized by different patterns of expression of the endogenous genes. Variant expression is controlled by the euchromatic or heterochromatic state of bistable chromatin domains. Repression is mediated by H3K9me3-based heterochromatin, whereas the active state is characterized by H3K9ac. These marks are maintained throughout the asexual blood cycle to transmit the epigenetic memory. Furthermore, eba-140 is organized in two distinct chromatin domains, probably separated by a barrier insulator located within its ORF. The 5' chromatin domain controls expression of the gene, whereas the 3' domain shares the chromatin conformation with the upstream region of the neighbouring phista family gene, which also shows variant expression.