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.     

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 twists and turns of Maurer's cleft trafficking in P. falciparum-infected erythrocytes

The malaria parasite, Plasmodium falciparum, invades the red blood cells (RBCs) of its human host and initiates a series of morphological rearrangements within the host cell cytoplasm. The mature RBC has no endogenous trafficking machinery; therefore, the parasite generates novel structures to mediate protein transport. These include compartments called the Maurer's clefts (MC), which play an important role in the trafficking of parasite proteins to the surface of the host cell. Recent electron tomography studies have revealed MC as convoluted flotillas of flattened discs that are tethered to the RBC membrane, prompting speculation that the MC could, in one respect, represent an extracellular equivalent of the Golgi apparatus. Visualization of both resident and cargo proteins has helped decipher the signals and routes for trafficking of parasite proteins to the MC and beyond.     

3-D analysis of the Plasmodium falciparum Maurer's clefts using different electron tomographic approaches

The human malaria parasite Plasmodium falciparum exports a large number of proteins into its host erythrocyte to install functions necessary for parasite survival. Important structural components of the export machinery are membrane profiles of parasite origin, termed Maurer's clefts. These profiles span much of the distance between the parasite and the host cell periphery and are believed to deliver P. falciparum-encoded proteins to the erythrocyte plasma membrane. Although discovered more than a century ago, Maurer's clefts remain a mysterious organelle with little information available regarding their origin, their morphology or their precise role in protein trafficking. Here, we evaluated different techniques to prepare samples for electron tomography, including whole cell cryo-preparations, vitreous sections, freeze-substitution and chemical fixation. Our data show that the different approaches tested all have their merits, revealing different aspects of the complex structure of the Maurer's clefts.     

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.     

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.     

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.     

New Export Pathway in Plasmodium falciparum‐Infected Erythrocytes: Role of the Parasite Group II Chaperonin,PfTRiC

The export of numerous proteins to the plasma membrane of its host erythrocyte is essential for the virulence and survival of the malaria parasite Plasmodium falciparum. The Maurer's clefts, membrane structures transposed by the parasite in the cytoplasm of its host erythrocyte, play the role of a marshal platform for such exported parasite proteins. We identify here the export pathway of three resident proteins of the Maurer's clefts membrane: the proteins are exported as soluble forms in the red cell cytoplasm to the Maurer's clefts membrane in association with the parasite group II chaperonin (PfTRIC), a chaperone complex known to bind and address a large spectrum of unfolded proteins to their final location. We have also located the domain of interaction with PfTRiC within the amino‐terminal domain of one of these Maurer's cleft proteins, PfSBP1. Because several Maurer's cleft membrane proteins with different export motifs seem to follow the same route, we propose a general role for PfTRiC in the trafficking of malarial parasite proteins to the host erythrocyte.      

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.     

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.     

Novel Plasmodium falciparum Maurer's clefts protein families implicated in the release of infectious merozoites

The pathogenicity of the most deadly human malaria parasite, Plasmodium falciparum, relies on the export of virulence factors to the surface of infected erythrocytes. A novel membrane compartment, referred to as Maurer's clefts, is transposed to the host erythrocyte, acting as a marshal platform in the red blood cell cytoplasm, for exported parasite proteins addressed to the host cell plasma membrane. We report here the characterization of three new P. falciparum multigene families organized in 9 highly conserved clusters with the Pfmc‐2tm genes in the subtelomeric regions of parasite's chromosomes and expressed at early trophozoite stages. Like the PfMC‐2TM proteins, the PfEPF1, 3 and 4 proteins encoded by these families are exported to the Maurer's clefts, as peripheral or integral proteins of the Maurer's cleft membrane and largely exposed to the red cell cytosolic face of this membrane. A promoter titration approach was used to question the biological roles of these P. falciparum‐specific exported proteins. Using the Pfepf1 family promoter, we observed the specific downregulation of all four families, correlating with the inefficient release of merozoites while the parasite intra‐erythrocytic maturation and Maurer's clefts morphology were not impacted.     

Ultrastructure of the erythrocytic stages of Plasmodium malariae

This report describes the fine structure of the erythrocytic stages of Plasmodium malariae. Erythrocytic parasites from a naturally acquired human infection and an experimentally infected chimpanzee were morphologically indistinguishable and structurally similar to other primate malarias. New findings included observations of highly structured arrays of merozoite surface coat proteins in the cytoplasm of early schizonts and on the surface of budding merozoites and the presence of knobs in the membranes of Maurer's clefts. Morphological evidence is presented suggesting that proteins are transported between the erythrocyte surface and intracellular parasites via two routes: one associated with Maurer's clefts for transport of membrane-associated knob material and a second associated with caveolae in the host cell membrane for the import or export of host- or parasite-derived substances through the erythrocyte cytoplasm.     

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.     

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.     

Whole cell imaging reveals novel modular features of the exomembrane system of the malaria parasite, Plasmodium falciparum

During its intra-erythrocytic development Plasmodium falciparum establishes a membrane network beyond its own limiting membrane in the cytoplasm of its host. These membrane structures play an important role in the trafficking of virulence proteins to the erythrocyte surface, however their ultrastructure is only partly defined and there is on-going debate regarding their origin, organisation and connectivity. We have used two whole cell imaging modalities to explore the topography of parasitised erythrocytes. Three-dimensional structured illumination microscopy provides resolution beyond the optical diffraction limit and permits analysis of fluorescently labelled whole cells. Immunoelectron tomography offers the possibility of high resolution imaging of individual ultrastructural features in a cellular context. Combined with serial sectioning and immunogold labelling, this technique permits precise mapping of whole cell architecture. We show that the P. falciparum exported secretory system comprises a series of modular units, comprising flattened cisternae, known as Maurer’s clefts, tubular connecting elements, two different vesicle populations and electron-dense structures that have fused with the erythrocyte membrane. The membrane network is not continuous, pointing to an important role for vesicle-mediated transport in the delivery of cargo to different destinations in the host cell.     

Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes

After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob-associated histidine-rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite-derived structures, known as Maurer's clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.     

Ultrastructure of the Erythrocytic Stages of Plasmodium malariae1

This report describes the fine structure of the erythrocytic stages of Plasmodium malariae. Erythrocytic parasites from a naturally acquired human infection and an experimentally infected chimpanzee were morphologically indistinguishable and structurally similar to other primate malarias. New findings included observations of highly structured arrays of merozoite surface coat proteins in the cytoplasm of early schizonts and on the surface of budding merozoites and the presence of knobs in the membranes of Maurer's clefts. Morphological evidence is presented suggesting that proteins are transported between the erythrocyte surface and intracellular parasites via two routes: one associated with Maurer's clefts for transport of membrane-associated knob material and a second associated with caveolae in the host cell membrane for the import or export of host- or parasite-derived substances through the erythrocyte cytoplasm.     

Protein and lipid trafficking induced in erythrocytes infected by malaria parasites

The human malaria parasite Plasmodium falciparum develops in a parasitophorous vacuolar membrane (PVM) within the mature red cell and extensively modifies structural and antigenic properties of this host cell. Recent studies shed significant new, mechanistic perspective on the underlying processes. There is finally, definitive evidence that despite the absence of endocytosis, transmembrane proteins in the host red cell membrane are imported in to the PVM. These are not major erythrocyte proteins but components that reside in detergent resistant membrane (DRM) rafts in red cell membrane and are detected in rafts in the PVM. Disruption of either erythrocyte or vacuolar rafts is detrimental to infection suggesting that raft proteins and lipids are essential for the parasitization of the red cell. On secretory export of parasite proteins: an ER secretory signal (SS) sequence is required for protein secretion to the PV. Proteins carrying an additional plastid targeting sequence (PTS) are also detected in the PV but subsequently delivered to the plastid organelle within the parasite, suggesting that the PTS may have a second function as an endocytic sorting signal. A distinct but yet undefined peptidic motif underlies protein transport across the PVM to the red cell (although all of the published data does not yet fit this model). Further multiple exported proteins transit through secretory 'cleft' structures, suggesting that clefts may be sorting compartments assembled by the parasite in the red cell.     

Protein unfolding is an essential requirement for transport across the parasitophorous vacuolar membrane of Plasmodium falciparum

Plasmodium falciparum traffics a large number of proteins to its host cell, the mature human erythrocyte. How exactly these proteins gain access to the red blood cell is poorly understood. Here we have investigated the effect of protein folding on the transport of model substrate proteins to the host cell. We find that proteins must pass into the erythrocyte cytoplasm in an unfolded state. Our data strongly support the presence of a protein-conducing channel in the parasitophorous vacoular membrane, and additionally imply an important role for molecular chaperones in keeping parasite proteins in a 'translocation competent' state prior to membrane passage.