Trafficking of STEVOR to the Maurer's clefts in Plasmodium falciparum-infected erythrocytes

The human malarial parasite Plasmodium falciparum exports proteins to destinations within its host erythrocyte, including cytosol, surface and membranous profiles of parasite origin termed Maurer's clefts. Although several of these exported proteins are determinants of pathology and virulence, the mechanisms and trafficking signals underpinning protein export are largely uncharacterized-particularly for exported transmembrane proteins. Here, we have investigated the signals mediating trafficking of STEVOR, a family of transmembrane proteins located at the Maurer's clefts and believed to play a role in antigenic variation. Our data show that, apart from a signal sequence, a minimum of two addition signals are required. This includes a host cell targeting signal for export to the host erythrocyte and a transmembrane domain for final sorting to Maurer's clefts. Biochemical studies indicate that STEVOR traverses the secretory pathway as an integral membrane protein. Our data suggest general principles for transport of transmembrane proteins to the Maurer's clefts and provide new insights into protein sorting and trafficking processes in 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 conditional export system provides new insights into protein export in Plasmodium falciparum-infected erythrocytes

The human malarial parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within its host erythrocyte, including the cytoplasm, the plasma membrane and membrane profiles of parasite origin termed Maurer's clefts. While there is some information regarding the signals that allot proteins for export, the trafficking route itself has remained largely obscure, partly due to technical limitations in following protein trafficking with time. To overcome these shortcomings, we have established a conditional protein export system in P. falciparum, based on the previously described conditional aggregation domain (CAD domain) that self-aggregates in the endoplasmic reticulum in a manner that is reversible by the addition of a small molecule. By fusing the CAD domain to the first 80 amino acids of STEVOR and full-length PfSBP1, we were able to control export of a soluble and a transmembrane protein to the erythrocyte cytosol and the Maurer's clefts respectively. The conditional export system allowed us to study the temporal sequence of events of protein export and identify intermediate steps. We further explored the potential of the conditional export system in identifying factors that interact with exported proteins en route. Our data provide evidence for a physical interaction of exported proteins with the molecular chaperone PfBiP during early export steps.     

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.     

Role of the Plasmodium Export Element in Trafficking Parasite Proteins to the Infected Erythrocyte

The intracellular survival of Plasmodium falciparum within human erythrocytes is dependent on export of parasite proteins that remodel the host cell. Most exported proteins require a conserved motif (RxLxE/Q/D), termed the Plasmodium export element (PEXEL) or vacuolar targeting sequence (VTS), for targeting beyond the parasitophorous vacuole membrane and into the host cell; however, the precise role of this motif in export is poorly defined. We used transgenic P. falciparum expressing chimeric proteins to investigate the function of the PEXEL motif for export. The PEXEL constitutes a bifunctional export motif comprising a protease recognition sequence that is cleaved, in the endoplasmic reticulum, from proteins destined for export, in a PEXEL arginine- and leucine-dependent manner. Following processing, the remaining conserved PEXEL residue is required to direct the mature protein to the host cell. Furthermore, we demonstrate that N acetylation of proteins following N-terminal processing is a PEXEL-independent process that is insufficient for correct export to the host cell. This work defines the role of each residue in the PEXEL for export into the P. falciparum -infected erythrocyte.     

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.     

Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-infected erythrocytes

Malaria parasites export proteins beyond their own plasma membrane to locations in the red blood cells in which they reside. Maurer's clefts are parasite-derived structures within the host cell cytoplasm that are thought to function as a sorting compartment between the parasite and the erythrocyte membrane. However, the genesis of this compartment and the signals directing proteins to the Maurer's clefts are not known. We have generated Plasmodium falciparum-infected erythrocytes expressing green fluorescent protein (GFP) chimeras of a Maurer's cleft resident protein, the membrane-associated histidine-rich protein 1 (MAHRP1). Chimeras of full-length MAHRP1 or fragments containing part of the N-terminal domain and the transmembrane domain are successfully delivered to Maurer's clefts. Other fragments remain trapped within the parasite. Fluorescence photobleaching and time-lapse imaging techniques indicate that MAHRP1-GFP is initially trafficked to isolated subdomains in the parasitophorous vacuole membrane that appear to represent nascent Maurer's clefts. The data suggest that the Maurer's clefts bud from the parasitophorous vacuole membrane and diffuse within the erythrocyte cytoplasm before taking up residence at the cell periphery.     

Characterization of the pathway for transport of the cytoadherence-mediating protein, PfEMP1, to the host cell surface in malaria parasite-infected erythrocytes

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of antigenically diverse proteins is expressed on the surface of human erythrocytes infected with the malaria parasite P. falciparum, and mediates cytoadherence to the host vascular endothelium. In this report, we show that export of PfEMP1 is slow and inefficient as it takes several hours to traffic newly synthesized proteins to the erythrocyte membrane. Upon removal by trypsin treatment, the surface-exposed population of PfEMP1 is not replenished during subsequent culture indicating that there is no cycling of PfEMP1 between the erythrocyte surface and an intracellular compartment. The role of Maurer's clefts as an intermediate sorting compartment in trafficking of PfEMP1 was investigated using immunoelectron microscopy and proteolytic digestion of streptolysin O-permeabilized parasitized erythrocytes. We show that PfEMP1 is inserted into the Maurer's cleft membrane with the C-terminal domain exposed to the erythrocyte cytoplasm, whereas the N-terminal domain is buried inside the cleft. Transfer of PfEMP1 to the erythrocyte surface appears to involve electron-lucent extensions of the Maurer's clefts. Thus, we have delineated some important aspects of the unusual trafficking mechanism for delivery of this critical parasite virulence factor to the erythrocyte surface.     

Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features

During intraerythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane-bound compartments, known as Maurer's clefts, outside the confines of its own plasma membrane. The Maurer's compartments are thought to be a crucial component of the machinery for protein sorting and trafficking; however, their ultrastructure is only partly defined. We have used electron tomography to image Maurer's clefts of 3D7 strain parasites. The compartments are revealed as flattened structures with a translucent lumen and a more electron-dense coat. They display a complex and convoluted morphology, and some regions are modified with surface nodules, each with a circular cross-section of approximately 25 nm. Individual 25 nm vesicle-like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurer's clefts are connected to the red blood cell membrane by regions with extended stalk-like profiles. Immunogold labelling with specific antibodies confirms differential labelling of the Maurer's clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurer's clefts and parasite membranes and the host plasma membrane.     

Trafficking determinants for PfEMP3 export and assembly under the Plasmodium falciparum-infected red blood cell membrane

During the maturation of intracellular asexual stages of Plasmodium falciparum parasite-encoded proteins are exported into the erythrocyte cytosol. A number of these parasite proteins attach to the host cell cytoskeleton and facilitate transformation of a disk-shaped erythrocyte into a rounded and more rigid infected erythrocyte able to cytoadhere to the vasculature. Knob formation on the surface of infected erythrocytes is critical for this cytoadherence to the host endothelium. P. falciparum proteins have been identified that localize to the parasite-infected erythrocyte membrane: the variant cytoadherence ligand erythrocyte membrane protein 1 (PfEMP1), the knob-associated histidine-rich protein (KAHRP) and the erythrocyte membrane protein 3 (PfEMP3). In this study, we have generated parasites expressing PfEMP3-green fluorescent protein chimeras and identified domains involved in entry to the secretory pathway, export across the parasitophorous vacuolar membrane and attachment to Maurer's clefts and the erythrocyte membrane. Solubility assays, fluorescence photobleaching experiments and immunogold electron microscopy suggest that the exported chimeric proteins are trafficked in a complex rather than in vesicles. This study characterizes elements involved in the tight but transient binding of PfEMP3 to Maurer's clefts and shows that the same elements are necessary for correct assembly under the erythrocyte membrane.     

Maurer's cleft organization in the cytoplasm of plasmodium falciparum-infected erythrocytes: new insights from three-dimensional reconstruction of serial ultrathin sections

Maurer's clefts are single-membrane-limited structures in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum. The currently accepted model suggests that Maurer's clefts act as an intermediate compartment in protein transport processes from the parasite across the cytoplasm of the host cell to the erythrocyte surface, by receiving and delivering protein cargo packed in vesicles. This model is mainly based on two observations. Firstly, single-section electron micrographs have shown, within the cytoplasm of infected erythrocytes, stacks of long slender membranes in close vicinity to round membrane profiles considered to be vesicles. Secondly, proteins that are transported from the parasite to the erythrocyte surface as well as proteins facilitating the budding of vesicles have been found in association with Maurer's clefts. Verification of this model would be greatly assisted by a better understanding of the morphology, dimensions and origin of the Maurer's clefts. Here, we have generated and analyzed three-dimensional reconstructions of serial ultrathin sections covering segments of P. falciparum-infected erythrocytes of more than 1 microm thickness. Our results indicate that Maurer's clefts are heterogeneous in structure and size. We have found Maurer's clefts consisting of a single disk-shaped cisternae localized beneath the plasma membrane. In other examples, Maurer' clefts formed an extended membranous network that bridged most of the distance between the parasite and the plasma membrane of the host erythrocyte. Maurer's cleft membrane networks were composed of both branched membrane tubules and stacked disk-shaped membrane cisternae that eventually formed whorls. Maurer's clefts were visible in other cells as a loose membrane reticulum composed of scattered tubular and disk-shaped membrane profiles. We have not seen clearly discernable isolated vesicles in the analyzed erythrocyte segments suggesting that the current view of how proteins are transported within the Plasmodium-infected erythrocyte may need reconsideration.     

Genetic ablation of a Maurer's cleft protein prevents assembly of the Plasmodium falciparum virulence complex

The malaria parasite Plasmodium falciparum assembles knob structures underneath the erythrocyte membrane that help present the major virulence protein, P. falciparum erythrocyte membrane protein-1 (PfEMP1). Membranous structures called Maurer's clefts are established in the erythrocyte cytoplasm and function as sorting compartments for proteins en route to the RBC membrane, including the knob-associated histidine-rich protein (KAHRP), and PfEMP1. We have generated mutants in which the Maurer's cleft protein, the ring exported protein-1 (REX1) is truncated or deleted. Removal of the C-terminal domain of REX1 compromises Maurer's cleft architecture and PfEMP1-mediated cytoadherance but permits some trafficking of PfEMP1 to the erythrocyte surface. Deletion of the coiled-coil region of REX1 ablates PfEMP1 surface display, trapping PfEMP1 at the Maurer's clefts. Complementation of mutants with REX1 partly restores PfEMP1-mediated binding to the endothelial cell ligand, CD36. Deletion of the coiled-coil region or complete deletion of REX1 is tightly associated with the loss of a subtelomeric region of chromosome 2, encoding KAHRP and other proteins. A KAHRP-green fluorescent protein (GFP) fusion expressed in the REX1-deletion parasites shows defective trafficking. Thus, loss of functional REX1 directly or indirectly ablates the assembly of the P. falciparum virulence complex at the surface of host erythrocytes.     

Maurer's clefts: a novel multi-functional organelle in the cytoplasm of Plasmodium falciparum-infected erythrocytes

Discovered in 1902 by Georg Maurer as a peculiar dotted staining pattern observable by light microscopy in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum, the function of Maurer's clefts have remained obscure for more than a century. The growing interest in protein sorting and trafficking processes in malarial parasites has recently aroused the Maurer's clefts from their deep slumber. Mounting evidence suggests that Maurer's clefts are a secretory organelle, which the parasite establishes within its host erythrocyte, but outside its own confines, to route parasite proteins across the host cell cytoplasm to the erythrocyte surface where they play a role in nutrient uptake and immune evasion processes. Moreover, Maurer's clefts seem to play a role in cell signaling, merozoite egress, phospholipid biosynthesis and, possibly, other biochemical pathways. Here, we review our current knowledge of the ultrastructure of Maurer's clefts, their proteinaceous composition and their function in protein trafficking.     

The Maurer's cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes

During the intra-erythrocytic development of Plasmodium falciparum, the parasite modifies the host cell surface by exporting proteins that interact with or insert into the erythrocyte membrane. These proteins include the principal mediator of cytoadherence, P. falciparum erythrocyte membrane protein 1 (PfEMP1). To implement these changes, the parasite establishes a protein-trafficking system beyond its confines. Membrane-bound structures called Maurer's clefts are intermediate trafficking compartments for proteins destined for the host cell membrane. We disrupted the gene for the membrane-associated histidine-rich protein 1 (MAHRP1). MAHRP1 is not essential for parasite viability or Maurer's cleft formation; however, in its absence, these organelles become disorganized in permeabilized cells. Maurer's cleft-resident proteins and transit cargo are exported normally in the absence of MAHRP1; however, the virulence determinant, PfEMP1, accumulates within the parasite, is depleted from the Maurer's clefts and is not presented at the red blood cell surface. Complementation of the mutant parasites with mahrp1 led to the reappearance of PfEMP1 on the infected red blood cell surface, and binding studies show that PfEMP1-mediated binding to CD36 is restored. These data suggest an important role of MAHRP1 in the translocation of PfEMP1 from the parasite to the host cell membrane.     

Functional Evaluation of Plasmodium Export Signals in Plasmodium berghei Suggests Multiple Modes of Protein Export

The erythrocytic stage development of malaria parasites occurs within the parasitophorous vacuole inside the infected-erythrocytes, and requires transport of several parasite-encoded proteins across the parasitophorous vacuole to several locations, including the cytosol and membrane of the infected cell. These proteins are called exported proteins; and a large number of such proteins have been predicted for Plasmodium falciparum based on the presence of an N-terminal motif known as the Plasmodium export element (PEXEL) or vacuolar transport signal (VTS), which has been shown to mediate export. The majority of exported proteins contain one or more transmembrane domains at the C-terminus and one of three types of N-terminus domain architectures. (1) The majority, including the knob-associated histidine rich protein (KAHRP), contain a signal/hydrophobic sequence preceding the PEXEL/VTS motif. (2) Other exported proteins, including the P. berghei variant antigen family bir and the P. falciparum skeleton binding protein-1, do not appear to contain a PEXEL/VTS motif. (3) The P. falciparum erythrocyte membrane protein-1 (PfEMP1) family lacks a signal/hydrophobic sequence before the motif. These different domain architectures suggest the presence of multiple export pathways in malaria parasites. To determine if export pathways are conserved in plasmodia and to develop an experimental system for studying these processes, we investigated export of GFP fused with N- and C-terminus putative export domains in the rodent malaria parasite P. berghei. Export was dependent on specific N- and C-terminal domains. Constructs with a KAHRP-like or bir N-terminus, but not the PfEMP1 N-terminus, exported GFP into the erythrocyte. The C-terminus of a P. falciparum variant antigen rifin prevented GFP export by the KAHRP-like N-terminus. In contrast, GFP chimeras containing KAHRP-like N-termini and the PfEMP1 C-terminus were exported to the surface of erythrocytes. Taken together, these results suggest that proteins with KAHRP-like architecture follow a common export pathway, but that PfEMP1s utilize an alternative pathway. Functional validation of common putative export domains of malaria parasites in P. berghei provides an alternative and simpler system to investigate export mechanisms.     

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.     

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.     

Delivery of the malaria virulence protein PfEMP1 to the erythrocyte surface requires cholesterol-rich domains

The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or "rafts" have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-beta-cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurer's clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking.     

The Plasmodium falciparum Maurer's clefts in 3D

In 1902, the German physician Georg Maurer discovered a dotted staining pattern within the cytoplasm of Plasmodium falciparum infected erythrocytes that, according to the tradition at the time, was named in his honour. The significance of Georg Maurer's discovery remained unrecognized for almost a century. Only recently are Maurer's clefts appreciated as a novel type of secretory organelle. Established by the malaria parasite within its host cell, Maurer's clefts play an essential role in directing proteins from the parasite to the erythrocyte surface. In this issue of Molecular Microbiology, Hanssen et al. report on the three dimensional structure of Maurer's clefts, as determined by electron tomography. The data presented suggest that Maurer's clefts are connected to both the parasitophorous vacuolar and the erythrocyte plasma membrane, however, no continuum exists that would allow lipids or proteins to freely flow between these three compartments. This seminal work, which stands in the tradition of Georg Maurer's original discovery, represents a milestone in our understanding of the structure and function of this fascinating organelle.