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.     

Metabolic interconnection between the human malarial parasite Plasmodium falciparum and its host erythrocyte. Regulation of ATP levels by means of an adenylate translocator and adenylate kinase

The metabolic inter-relationships between malarial parasites and their host erythrocytes are poorly understood. They have been investigated hitherto mostly by observing parasite behavior in erythrocyte variants, in metabolically altered erythrocytes, or in cell-free in vitro systems. We have studied the interconnection between the bioenergetic metabolism of host and parasite through compartment analysis of ATP in Plasmodium falciparum-infected human red blood cells, using Sendai virus-induced host cell lysis. ATP concentrations in host and parasite compartments were found to be equal. Inhibitors of mitochondrial activity reduce ATP levels to a similar extent in host and parasite compartments, although only the parasite contains functional mitochondria. It is shown that equalization of ATP levels is brought about by means of an adenylate translocator, probably localized at the parasite plasma membrane, in conjunction with adenylate kinase activity detected both in host and parasite compartments. The translocator is inhibited by compounds which are known to inhibit specifically the translocator of the inner membrane of mammalian mitochondria, with identical inhibitory constants. Addition of these inhibitors to intact infected cells causes a rapid depletion of ATP in the host compartment and a parallel increase in the parasite, suggesting that the parasite supplies ATP to its host cell rather than the reverse.     

The malaria parasite progressively dismantles the host erythrocyte cytoskeleton for efficient egress

Plasmodium falciparum is an obligate intracellular pathogen responsible for worldwide morbidity and mortality. This parasite establishes a parasitophorous vacuole within infected red blood cells wherein it differentiates into multiple daughter cells that must rupture their host cells to continue another infectious cycle. Using atomic force microscopy, we establish that progressive macrostructural changes occur to the host cell cytoskeleton during the last 15 h of the erythrocytic life cycle. We used a comparative proteomics approach to determine changes in the membrane proteome of infected red blood cells during the final steps of parasite development that lead to egress. Mass spectrometry-based analysis comparing the red blood cell membrane proteome in uninfected red blood cells to that of infected red blood cells and postrupture vesicles highlighted two temporally distinct events; (Hay, S. I., et al. (2009). A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med. 6, e1000048) the striking loss of cytoskeletal adaptor proteins that are part of the junctional complex, including α/β-adducin and tropomyosin, correlating temporally with the emergence of large holes in the cytoskeleton seen by AFM as early ~35 h postinvasion, and (Maier, A. G., et al. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134, 48-61) large-scale proteolysis of the cytoskeleton during rupture ~48 h postinvasion, mediated by host calpain-1. We thus propose a sequential mechanism whereby parasites first remove a selected set of cytoskeletal adaptor proteins to weaken the host membrane and then use host calpain-1 to dismantle the remaining cytoskeleton, leading to red blood cell membrane collapse and parasite release.     

An electron microscopic study of Babesia microti invading erythrocytes

Summary Intracellular sporozoan parasites invade the host cell through the invagination of the plasma membrane of the host and a vacuole is formed which accommodates the entering parasite. The vacuole may disappear and the invaginated membrane of the host then becomes closely apposed to that of the parasite's own membrane. As a result the parasite is covered by two membranes. Members of the class Piroplasmea differ from other Sporozoa in that their trophozoites are covered by a single membrane. By screening numerous sections of intraerythrocytic Babesia microti belonging to the class Piroplasmea, it was found that merozoites of Babesia enter the erythrocytes of hamsters in the same way as those of other Sporozoa. When a merozoite touches the red blood cell with its anterior end it becomes attached to the membrane of the host, which starts to invaginate and a parasitophorous vacuole is formed. The vacuolar space disappears rapidly and the membrane of the vacuole and that of the parasite become closely adjacent. At this stage the parasite is surrounded by two plasma membranes. The outer membrane derived from the invaginated host membrane disintegrates quickly and the parasite is left with a single membrane throughout its life span.Supported by Grant AI 08989 from the U.S. Public Health Service. The excellent technical assistance of Ms. Renata Klatt is gratefully acknowledged     

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.     

Invasion of Babesia microti into Epithelial Cells of the Tick Gut1

During feeding a peritrophic membrane (PM) is formed in the gut of the tick Ixodes dammini, dividing the lumen of the gut into an ecto- and endoperitrophic space. Babesia and all food particles ingested with the blood meal by the tick are retained in the endoperitrophic space, the lumen proper. Only Babesia equipped with a highly specialized organelle, the arrowhead, are able to pass the PM and enter the ectoperitrophic compartment. During the crossing of the PM the arrowhead loses its density, suggesting that enzymes released from it dissolve the polymers in the PM, making passage of the parasite through this barrier possible. In the ectoperitrophic space the arrowhead of Babesia touches the epithelial cell. At the point of contact the membrane of the host cell starts to invaginate, and simultaneously the arrowhead's fine structure loses its highly organized pattern. The growing host membrane encircles the parasite and the arrowhead diminishes progressively in size. When the piroplasm is inside the host cell, the arrowhead can no longer be found. During invasion the host membrane often touches the parasite's plasma membrane at the site of a coiled structure, and the host membrane becomes ruptured and the nearby host cytoplasm appears to be lysed. Babesia inside the host cell is covered solely by its own plasma membrane; the invaginated host membrane is missing. It is postulated that the latter disintegrates during invasion by the parasite through the action of enzymes from the coiled structure. The parasite is surrounded by a halo of homogeneous material deriving most probably from the lysed host cytoplasm.     

Hostile takeover by Plasmodium: reorganization of parasite and host cell membranes during liver stage egress

The protozoan parasite Plasmodium is transmitted by female Anopheles mosquitoes and undergoes obligatory development within a parasitophorous vacuole in hepatocytes before it is released into the bloodstream. The transition to the blood stage was previously shown to involve the packaging of exoerythrocytic merozoites into membrane-surrounded vesicles, called merosomes, which are delivered directly into liver sinusoids. However, it was unclear whether the membrane of these merosomes was derived from the parasite membrane, the parasitophorous vacuole membrane or the host cell membrane. This knowledge is required to determine how phagocytes will be directed against merosomes. Here, we fluorescently label the candidate membranes and use live cell imaging to show that the merosome membrane derives from the host cell membrane. We also demonstrate that proteins in the host cell membrane are lost during merozoite liberation from the parasitophorous vacuole. Immediately after the breakdown of the parasitophorous vacuole membrane, the host cell mitochondria begin to degenerate and protein biosynthesis arrests. The intact host cell plasma membrane surrounding merosomes allows Plasmodium to mask itself from the host immune system and bypass the numerous Kupffer cells on its way into the bloodstream. This represents an effective strategy for evading host defenses before establishing a blood stage infection.     

Cryptosporidium parvum attachment to and internalization by human biliary epithelia in vitro: a morphologic study

To explore the mechanisms by which Cryptosporidium parvum infects epithelial cells, we performed a detailed morphological study by serial electron microscopy to assess attachment to and internalization of biliary epithelial cells by C. parvum in an in vitro model of human biliary cryptosporidiosis. When C. parvum sporozoites initially attach to the host cell membrane, the rhoptry of the sporozoite extends to the attachment site; both micronemes and dense granules are recruited to the apical complex region of the attached parasite. During internalization, numerous vacuoles covered by the parasite's plasma membrane are formed and cluster together to establish a preparasitophorous vacuole. This preparasitophorous vacuole comes in contact with host cell membrane to form a host cell-parasite membrane interface, beneath which an electron-dense band begins to appear within the host cell cytoplasm. Simultaneously, host cells display membrane protrusion along the edge of the host cell-parasite membrane interface, resulting in the formation of a mature parasitophorous vacuole that completely covers the parasite. During internalization, vacuole-like structures appear in the apical complex region of the attached sporozoite, which bud out into host cells. A tunnel directly connecting the parasite to the host cell cytoplasm forms during internalization and remains when the parasite is totally internalized. Immunoelectron microscopy showed that sporozoite-associated proteins were localized along the dense band and at the parasitophorous vacuole membrane. These morphological observations provide evidence that secretion of parasite apical organelles and protrusion of host cell membrane play an important role in the attachment and internalization of host epithelial cells by C. parvum.     

Import of host delta-aminolevulinate dehydratase into the malarial parasite: identification of a new drug target

The parasite Plasmodium berghei imports the enzyme delta-aminolevulinate dehydratase (ALAD), and perhaps the subsequent enzymes of the pathway from the host red blood cell to sustain heme synthesis. Here we have studied the mechanism of this import. A 65-kDa protein on the P. berghei membrane specifically bound to mouse red blood cell ALAD, and a 93-amino-acid fragment (ALAD-DeltaNC) of the host erythrocyte ALAD was able to compete with the full-length enzyme for binding to the P. berghei membrane. ALAD-DeltaNC was taken up by the infected red blood cell when added to a culture of P. falciparum and this led to a substantial decrease in ALAD protein and enzyme activity and, subsequently, heme synthesis in the parasite, resulting in its death.     

Blocking effect of a biotinylated protease inhibitor on the egress of Plasmodium falciparum merozoites from infected red blood cells

The malaria parasite Plasmodium falciparum invades human red blood cells. Before infecting new erythrocytes, the merozoites have to exit their host cell to get into the blood plasma. Knowledge about the mechanism of egress is scarce, but it is thought that proteases are basically involved in this step. We have introduced a biotinylated dibenzyl aziridine-2,3-dicarboxylate (bADA) as an irreversible cysteine protease inhibitor to study the mechanism of merozoite release and to identify the proteases involved. The compound acts on parasite proteins in the digestive vacuole and in the host cell cytosol, as judged by fluorescence microscopy. The inhibitor blocks rupture of the host cell membrane, leading to clustered merozoite structures, as evidenced by immunoelectron microscopy. Interestingly, bADA did not prevent rupture of the parasitophorous vacuole membrane (PVM) that surrounds the parasite during the period of intraerythrocytic maturation. The compound appears to be a valuable template for the development of inhibitors specific for individual plasmodial proteases, which would be useful tools to dissect the molecular mechanisms underlying the process of merozoite release and consequently to develop potent antimalarial drugs.     

Vesicle-mediated transport of membrane and proteins in malaria-infected erythrocytes

Malaria parasites during intraerythrocytic development change the ultrastructure, biophysics, and the antigens of the host red blood cell membrane. Parasite-encoded proteins are associated with, inserted into, or secreted across the infected erythrocyte membrane. Since parasites of the genus Plasmodium are eukaryotic cells, it must be assumed that they possess essentially eukaryotic modes of vesicle-mediated transport and translocation of proteins and membranes. Numerous studies have demonstrated vesicular structures in the cytoplasm of malaria-infected red blood cells and an assortment of parasite proteins associated with the different vesicles, membranes, and membrane-defined compartments. Some parasite polypeptides remain trapped between the parasite and the parasitophorous vacuole membranes PVM, whereas others are associated with morphologically distinct membrane-limited vesicles and vacuoles. Some of these same parasite protein antigens also associate with the erythrocyte membrane or with parasite-induced ultrastructural modifications in the membrane of the parasitized red blood cells. This implies that intracellular transport occurs in malaria-infected erythrocytes, a capacity that uninfected red blood cells normally lose upon enucleation. The specific locations of parasite antigens within the infected cell also implys the existence of targeting signals in the translocated parasite polypeptides and perhaps transport-mediating proteins. The genes corresponding to some of these translocated proteins have been sequenced. Typical (and in some cases atypical) signal peptide sequences occur, as well as a number of sequences that may result in posttranslational modifications. How or if these features figure in to the translocation across, and targeting to a particular membrane compartment of the intraerythrocytic parasite remains unknown.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Cellular architecture of Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum is a protozoan parasite that is responsible for the most pathogenic form of human malaria. The particular virulence of this parasite derives from its ability to develop within the erythrocytes of its host and to subvert their function. The intraerythrocytic parasite devours haemoglobin, and remodels its host cell to cause adhesion to blood vessel walls. Ultrastructural studies of P. falciparum have played a major role in defining its cell architecture and in resolving cell biology controversies. Here we review some of the early studies and describe some recent developments in electron microscopy techniques that have revealed information about the organization of the parasite in the blood stage of development. We present images of P. falciparum at different stages of the life cycle and highlight some of the plasmodium-specific organelles, the haemoglobin digestive apparatus and the membrane structures that are elaborated in the host cell cytoplasm to traffic virulence proteins to the erythrocyte surface. We describe methods for whole cell ultrastructural imaging that can provide three-dimensional views of intraerythrocytic development.     

Actin‐mediated plasma membrane plasticity of the intracellular parasite Theileria annulata

Pathogen–host interactions are modulated at multiple levels by both the pathogen and the host cell. Modulation of host cell functions is particularly intriguing in the case of the intracellular Theileria parasite, which resides as a multinucleated schizont free in the cytosol of the host cell. Direct contact between the schizont plasma membrane and the cytoplasm enables the parasite to affect the function of host cell proteins through direct interaction or through the secretion of regulators. Structure and dynamics of the schizont plasma membrane are poorly understood and whether schizont membrane dynamics contribute to parasite propagation is not known. Here we show that the intracellular Theileria schizont can dynamically change its shape by actively extending filamentous membrane protrusions. We found that isolated schizonts bound monomeric tubulin and in vitro polymerized microtubules, and monomeric tubulin polymerized into dense assemblies at the parasite surface. However, we established that isolated Theileria schizonts free of host cell microtubules maintained a lobular morphology and extended filamentous protrusions, demonstrating that host microtubules are dispensable both forthe maintenance of lobular schizont morphology and for the generation of membrane protrusions. These protrusions resemble nanotubes and extend in an actin polymerization‐dependent manner; using cryo‐electron tomography, we detected thin actin filaments beneath these protrusions, indicating that their extension is driven by schizont actin polymerization. Thus the membrane of the schizont and its underlying actin cytoskeleton possess intrinsic activity for shape control and likely function as a peri‐organelle to interact with and manipulate host cell components.     

Altered membrane permeability: a new approach to malaria chemotherapy

During its development: in the host erythrocyte, the malarial parasite causes profound alterations in the permeability of the host cell membrane. Nucleoside transport pathways, which are induced by the parasite in the host erythrocyte membrane, have properties significantly different from those of the host cell. Here, Annette Gero and Joanne Upston review the current knowledge o f the parasite-induced transporters and show that they can be used to selectively direct cytotoxic compounds into the parasite-infected cell, thereby indicating their chemotherapeutic potential.     

Trypanosoma cruzi: mechanisms for entry into host cells

Infective trypomastigote stages of the obligate intracellular protozoan parasite Trypanosoma cruzi are capable of entering virtually any mammalian cell in vitro. Entry is a complex process, involving initial parasite attachment to surface moieties of the target cell, internalization of the parasite via formation of a vacuole, and finally disruption of the vacuolar membrane to permit access of the parasite to the host cell cytoplasm. Attachment requires parasite metabolic energy. At sites of parasite entry recruitment of host cell lysosomes may occur, and lysosomal membrane components contribute prominently to formation of the parasitophorous vacuole. Parasite escape from the vacuole depends upon vacuolar acidification and is mediated by the coordinated action of a parasite-derived neuramindase/trans-sialidase that is capable of desialylating host-derived vacuolar membrane constituents, and a parasite-derived trans-membrane pore-forming protein. Dissection of the entry process at both the organellar and molecular level is providing fundamental and complementary insights into microbial pathogenesis and cell biology.     

Interactions between Toxoplasma gondii and its mammalian host cells

Toxoplasma is a protozoan parasite that is uniquely adapted for invading and surviving within a wide range of host cells. The parasite actively invades the cell, forming a novel vacuole that originates from the host cell plasma membrane. The vacuole membrane is rapidly modified to remove host cell proteins and this compartment subsequently resists fusion with all other host cell endocytic compartments. Shortly after invasion, the parasite secretes a variety of proteins by a process of regulation exocytosis and elaborates an extensive array of membranous tubules that form a network connecting with the vacuolar membrane. Understanding the formation and modification of this unique vacuole may reveal novel mechanisms for subverting host cell endocytic pathways that lead to intracellular survival.     

Lipid compartmentalization in erythrocytes parasitized by Plasmodium spp

Although reasonably well protected from the host immune system by the erythrocyte membrane, the intraerythrocytic malaria parasite has to make that membrane compatible with its own requirements for development and multiplication. The development of Plasmodium spp brings about major changes in the lipid composition of the host cell membrane, as well as in its physical properties. The parasite itself has a lipid composition that differs from that of the host cell and an intense lipid trafficking seems to occur between intracellular parasite and host cell membrane. Here, Ana Paula Sim?es, Ben Roelofsen and Jos Op den Kamp discuss how, despite serious methodological limitations and the existence of some conflicting results, an overall picture of lipid compartmentalization within the parasitized erythrocyte is perceived.     

Apicomplexa in mammalian cells: trafficking to the parasitophorous vacuole

Most Apicomplexa reside and multiply in the cytoplasm of their host cell, within a parasitophorous vacuole (PV) originating from both parasite and host cell components. Trafficking of parasite-encoded proteins destined to membrane compartments beyond the confine of the parasite plasma membrane is a process that offers a rich territory to explore novel mechanisms of protein–membrane interactions. Here, we focus on the PVs formed by the asexual stages of two pathogens of medical importance, Plasmodium and Toxoplasma . We compare the PVs of both parasites, with a particular emphasis on their evolutionary divergent compartmentalization within the host cell. We also discuss the existence of peculiar export mechanisms and/or sorting determinants that are potentially involved in the post-secretory targeting of parasite proteins to the PV subcompartments.