Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes

The most virulent form of malaria is caused by waves of replication of blood stages of the protozoan pathogen Plasmodium falciparum. The parasite divides within an intraerythrocytic parasitophorous vacuole until rupture of the vacuole and host-cell membranes releases merozoites that invade fresh erythrocytes to repeat the cycle. Despite the importance of merozoite egress for disease progression, none of the molecular factors involved are known. We report that, just prior to egress, an essential serine protease called PfSUB1 is discharged from previously unrecognized parasite organelles (termed exonemes) into the parasitophorous vacuole space. There, PfSUB1 mediates the proteolytic maturation of at least two essential members of another enzyme family called SERA. Pharmacological blockade of PfSUB1 inhibits egress and ablates the invasive capacity of released merozoites. Our findings reveal the presence in the malarial parasitophorous vacuole of a regulated, PfSUB1-mediated proteolytic processing event required for release of viable parasites from the host erythrocyte.     

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.     

Cellular interactions of Plasmodium liver stage with its host mammalian cell

The Plasmodium liver forms are bridgehead stages between the mosquito sporozoite stages and mammalian blood stages that instigate the malaria disease. In hepatocytes, Plasmodium achieves one of the fastest growth rates among eukaryotic cells. However, nothing is known about host hepatic cell interactions, e.g. nutrient scavenging and/or subversion of cellular functions necessary for Plasmodium development and replication. Plasmodium usually invades hepatocytes by establishing a parasitophorous vacuole wherein it undergoes multiple nuclear division cycles. We show that Plasmodium preferentially develops in the host juxtanuclear region. By comparison with the parasitophorous vacuole of other apicomplexan parasites which associate with diverse host organelles, the Plasmodium parasitophorous vacuole only forms an association with the host endoplasmic reticulum. Intrahepatic Plasmodium actively modifies the permeability of its vacuole to allow the transfer of a large variety of molecules from the host cytosol to the vacuolar space through open channels. In contrast with malaria blood stages, the pores within the parasitophorous vacuole membrane of the liver stage display a smaller size as they restrict the passage of solutes to less than 855Da. These pores are stably maintained during parasite karyokinesis until complete cellularisation. Host-derived cholesterol accumulated at the parasitophorous vacuole membrane may modulate the channel activity. These observations define the parasitophorous vacuole of the Plasmodium liver stage as a dynamic and highly permeable compartment that can ensure the sustained supply of host molecules to support parasite growth in the nutrient-rich environment of liver cells.     

Transmission Electron Microscopy of Meront Development of Eimeria vermiformis Ernst,Chobotar and Hammond, 1971 (Apicomplexa,Eucoccidiorida) in the Mouse,Mus musculus1

First and second generation meronts of Eimeria vermiformis developed in epithelial cells of the crypts of Lieberkühn. They were usually between the host cell nucleus and the basement membrane. Sporozoite organelles dedifferentiated with the first generation meront's development except for the refractile body and the apical complex, which persisted. After several nuclear divisions, the apical complex dedifferentiated further until only micronemes remained attached by a duct system to the plasmalemma. The form of the apical complex was highly variable. Sometimes the duct system was absent and the micronemes were attached directly to the plasmalemma or a dense material on it. Crescent body-like material was often present in the parasitophorous vacuole next to the microneme structure. The microneme structure was not present in second generation meronts but evaginations of the plasmalemma, cytoplasmic outpocketings, and cytoplasmic vesicles were associated with the round granular bodies in the parasitophorous vacuoles. During first generation merogenesis, invaginations from the parasitophorous vacuole formed channels into the meront along which merozoites budded. Micropores were often at the ends of these invaginations. These and other micropores of the meront had a dense U-shaped band for a collar while those of the merozoites had a collar with a double band of dense material that connected to the inner membrane. First generation merozoites budded randomly from the meront, resulting in a residual body that was usually in the middle of the parasitophorous vacuole. Second generation merozoites budded in one direction, resulting in a peripheral residual body and merozoites that were parallel in an oblong parasitophorous vacuole.     

Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte

Escape from the host erythrocyte by the invasive stage of the malaria parasite Plasmodium falciparum is a fundamental step in the pathogenesis of malaria of which little is known. Upon merozoite invasion of the host cell, the parasite becomes enclosed within a parasitophorous vacuole, the compartment in which the parasite undergoes growth followed by asexual division to produce 16-32 daughter merozoites. These daughter cells are released upon parasitophorous vacuole and erythrocyte membrane rupture. To examine the process of merozoite release, we used P. falciparum lines expressing green fluorescent protein-chimeric proteins targeted to the compartments from which merozoites must exit: the parasitophorous vacuole and the host erythrocyte cytosol. This allowed visualization of merozoite release in live parasites. Herein we provide the first evidence in live, untreated cells that merozoite release involves a primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte plasma membrane. We have confirmed, with the use of immunoelectron microscopy, that parasitophorous vacuole membrane rupture occurs before erythrocyte plasma membrane rupture in untransfected wild-type parasites. We have also demonstrated selective inhibition of each step in this two-step process of exit using different protease inhibitors, implicating the involvement of distinct proteases in each of these steps. This will facilitate the identification of the parasite and host molecules involved in merozoite release.     

Invasion and Early Development of Sarcocystis muris (Apicomplexa,Sarcocystidae) in Tissue Cultures1

Ultrastructural observations on the invasion and early development of merozoites (bradyzoites) of Sarcocystis muris in Madin-Darby canine kidney (MDCK) cells are presented. Invading merozoites cause the host cell plasmalemma to invaginate; they form a membrane junction (moving junction) and move into the host cell where they are enclosed in a primary parasitophorous vacuole (PV). Within 30–45 min after becoming intracellular, merozoites begin to vacate the newly established primary PV and move, forming a new membrane junction, into a secondary PV. Simultaneously with the movement of the parasite, the contents of dense granules in the apical part of the merozoites are shed by exocytosis into the lumen of the developing secondary PV. A lamella of the endoplasmic reticulum of the host cell becomes attached to the PV membrane, forming a PV limited by three host cell membranes.     

Invasion and early development of Sarcocystis muris (Apicomplexa, Sarcocystidae) in tissue cultures

Ultrastructural observations on the invasion and early development of merozoites (bradyzoites) of Sarcocystis muris in Madin-Darby canine kidney (MDCK) cells are presented. Invading merozoites cause the host cell plasmalemma to invaginate; they form a membrane junction (moving junction) and move into the host cell where they are enclosed in a primary parasitophorous vacuole (PV). Within 30-45 min after becoming intracellular, merozoites begin to vacate the newly established primary PV and move, forming a new membrane junction, into a secondary PV. Simultaneously with the movement of the parasite, the contents of dense granules in the apical part of the merozoites are shed by exocytosis into the lumen of the developing secondary PV. A lamella of the endoplasmic reticulum of the host cell becomes attached to the PV membrane, forming a PV limited by three host cell membranes.     

Formation of a parasitophorous vacuole in a nonadequate experimental host: Electron microscopical and x-ray microanalytical study

An unusual mechanism of formation of a parasitophorous vacuole as a result of interaction between an invasive stage of a parasite (merozoites of a protozoon, Mattesia dispora) and defense response of an insect host, Galleria mellonella is reported. The entire ontogenesis of parasitophorous vacuole can be divided into five morphologically clearly discernible stages. They differed, e.g., in the contents and distribution of elements at subcellular level, as determined by direct in situ elemental analysis of single organelles (electron microprobe X-ray analysis). The method was used in conjunction with electron microscopy to investigate the relationship between the host and the parasite.     

Life Cycle of Goussia pannonica (Molnar, 1989) (Apicomplexa, Eimeriorina), an Extracytoplasmic Coccidium from the White Bream Blicca bjoerkna

ABSTRACT Life cycle stages of Goussia pannonica from naturally-infected white bream Blicca bjoerkna were studied by light and electron microscopy. Fourteen of the sixteen fish examined were infected, with developmental stages found in all parts of the intestine. Merogonial, gamogonial, and sporogonial stages were localized intracellularly and extracytoplasmically in the microvillous region of enterocytes. They were separated from the gut lumen by closely apposed enterocyte and parasitophorous vacuole membranes. There were two types of extracytoplasmic attachment: 1) monopodial, with a single zone of attachment, and 2) spider-like, with several isolated zones of attachment to the host cell. First-generation merozoites were formed by ectomerogony. Second- or third-generation merozoites were formed by endodyogeny and endopolygeny. Thirty to 50 biflagellated microgametes developed at the periphery of a microgamont. Macrogamonts contained lipid inclusions, amylopectin and dense granules; however, granules comparable to wall-forming bodies type I and II were absent. At the beginning of sporogony, the sporont cytoplasm detached from two layers which subsequently became constituents of the oocyst wall. After the rupture of enterocyte and parasitophorous vacuole membranes, the sporont was released into the water where exogenous sporulation was completed within 48 h. The thin sporocyst wall contained a small longitudinal suture. Sporocyst and oocyts walls were of similar structure.     

In vitro development from sporozoites to first-generation merozoites in Eimeria contorta Haberkorn, 1971. A fine structural study.

The asexual development of Eimeria contorta from sporozoites to first-generation merozoites in tissue culture was investigated with the electron microscope. Sporozoites with a three-layered pellicle, 26 subpellicular microtubules, a conoid, 4-7 rhoptries, and an abundance of micronemes actively entered host cells and showed direct contact to the host cell's cytoplasm. Shortly after penetration, small vacuoles surrounding the parasite merged into a parasitophorous vacuole. Inside this vacuole, sporozoites assumed a definite U-shape before transformation into schizonts took place. This process was characterised by the occurrence of subpellicular microtubules exclusively in the anterior half of the sporozoite, by a degeneration of the 2 inner pellicular membranes, by an outpocketing of the parasite's surface, and by the arrangement of microtubules in clusters. About 25 merozoites were formed at the surface of mature schizonts, to which they remained attached at their posterior pole. A polar ring was present at that area. Anterior and posterior refractile bodies were conspicuous in merozoites and showed close association with mitochondria. The significance of a fibrillar substructure in rhoptries and micronemes is discussed, and special attention is drawn to the pathway of nutrient transport from host cell mitochondria and dictyosomes through intravacuolar folds, parasitophorous vacuole and crescent body into the parasite's food vacuoles.     

Sarcocystis neurona n. sp. (Protozoa: Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis

Sarcocystis neuronan n. sp. is proposed for the apicomplexan taxon associated with myeloencephalitis in horses. Only asexual stages of this parasite presently are known, and they are found within neuronal cells and leukocytes of the brain and spinal cord. The parasite is located in the host cell cytoplasm, does not have a parasitophorous vacuole, and divides by endopolygeny. Schizonts are 5-35 microns x 5-20 microns and contain 4-40 merozoites arranged in a rosette around a prominent residual body. Merozoites are approximately 4 x 1 micron, have a central nucleus, and lack rhoptries. Schizonts and merozoites react with Sarcocystis cruzi antiserum but not with Caryospora bigenetica. Toxoplasma gondii, Hammondia hammondi, or Neospora caninum antisera in an immunohistochemical test.     

Calcium and Hydrogen Ion Concentrations in the Parasitophorous Vacuoles of Epithelial Cells Infected with the Microsporidian Encephalitozoon hellem

ABSTRACT. Microsporidia of the genus Encephalitozoon undergo merogony and sporogony in a parasitophorous vacuole within the host cell. Cultured green monkey kidney cells infected with Encephalitozoon hellem were loaded with the fluorescent dyes fura-2 or BCECF in order to measure intracellular concentrations of calcium and hydrogen ions respectively. Both the parasitophorous vacuole calcium concentration and pH values resembled those of the host cell cytoplasm in infected cells. Calcein entered the parasitophorous vacuole but not other host cell vacuoles or parasite stages within the parasitophorous vacuole. The lack of a pH or calcium concentration gradient across the parasitophorous vacuole membrane and the permeability of this membrane to a large anion such as calcein suggest that the vacuole membrane surrounding E. hellem resembles that surrounding some other intracellular parasites such as Toxoplasma gondii. A potential role is discussed for the parasitophorous vacuole calcium concentration in germination in situ.     

Relationship between the host cell mitochondria and the parasitophorous vacuole in cells infected with Encephalitozoon microsporidia

Encephalitozoon microsporidia proliferate and differentiate within a parasitophorous vacuole. Using the fluorescent probe, calcein, and the mitochondrial probe, MitoTracker-CMXRos, a vital method was developed that confirmed ultrastructural reports that the host cell mitochondria frequently lie in immediate proximity to the parasitophorous vacuole. Morphometry failed to demonstrate any infection-induced increase in host cell mitochondria as there was no correlation between the mitochondrial volume and the extent of infection as judged by the parasitophorous vacuole volume. The total ATP concentration of infected cells did not differ from that of uninfected cells in spite of the increased metabolic demands of the infection. Treatment with 10(-6) M albendazole, more than ten times the antiparasitic IC50 dose, and demecolcine had no subjective effect on the proximity of mitochondria to the parasitophorous vacuole membrane when studied by either transmission electron microscopy or by confocal microscopy even though these drug concentrations affected microtubule structure. Thus, once the association between mitochondria and the parasitophorous vacuole has been established, host cell microtubule integrity is probably not required for its maintenance. It is unlikely that the antimicrosporidial action of albendazole involves physically uncoupling developing parasite stages from host cell organelle metabolic support.     

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.     

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.     

Immunodetection of the microvillous cytoskeleton molecules villin and ezrin in the parasitophorous vacuole wall of Cryptosporidium parvum (Protozoa: Apicomplexa)

Microvilli - actin - villin - ezrin - Cryptosporidium parvum The sporozoites and merozoites of the Apicomplexan protozoan Cryptosporidium parvum (C. parvum) invade the apical side of enterocytes and induce the formation of a parasitophorous vacuole which stays in the brush border area and disturbs the distribution of microvilli. The vacuole is separated from the apical cytoplasm of the cell by an electron-dense layer of undetermined composition. In order to characterize the enterocyte cytoskeleton changes that occur during C. parvum invasion and development, we used both confocal immunofluorescence and immunoelectron microscopy to examine at the C.parvum-enterocyte interface the distribution of three components of the microvillous skeleton, actin, villin and ezrin. In infected cells, rhodamine-phalloidin and anti-villin and anti-ezrin antibodies recognized ring-like structures surrounding the developing parasites. By immunoelectron microscopy, both villin and ezrin were detected in the parasitophorous vacuole wall surrounding the luminal and lateral sides of the intracellular parasite. In contrast, anti-beta and anti-gamma actin antibodies showed no significant labelling of the vacuolar wall. These observations indicate that the parasitophorous vacuole wall contains at least two microvillus-derived components, villin and ezrin, as well as a low amount of F-actin. These data suggest that C.parvum infection induces a rearrangement of cytoskeleton molecules at the apical pole of the host cell that are used to build the parasitophorous vacuole.     

Secreted protein with altered thrombospondin repeat (SPATR) is essential for asexual blood stages but not required for hepatocyte invasion by the malaria parasite Plasmodium berghei

Upon entering its mammalian host, the malaria parasite productively invades two distinct cell types, that is, hepatocytes and erythrocytes during which several adhesins/invasins are thought to be involved. Many surface-located proteins containing thrombospondin Type I repeat (TSR) which help establish host–parasite molecular crosstalk have been shown to be essential for mammalian infection. Previous reports indicated that antibodies produced against Plasmodium falciparum secreted protein with altered thrombospondin repeat (SPATR) block hepatocyte invasion by sporozoites but no genetic evidence of its contribution to invasion has been reported. After failing to generate Spatr knockout in Plasmodium berghei blood stages, a conditional mutagenesis system was employed. Here, we show that SPATR plays an essential role during parasite's blood stages. Mutant salivary gland sporozoites exhibit normal motility, hepatocyte invasion, liver stage development and rupture of the parasitophorous vacuole membrane resulting in merosome formation. But these mutant hepatic merozoites failed to establish a blood stage infection in vivo. We provide direct evidence that SPATR is not required for hepatocyte invasion but plays an essential role during the blood stages of P. berghei.     

Sarcocystis canis n. sp. (Apicomplexa: Sarcocystidae), the etiologic agent of generalized coccidiosis in dogs

Sarcocystis canis n. sp. is proposed for the protozoon associated with encephalitis, hepatitis, and generalized coccidiosis in dogs. Only asexual stages are known in macrophages, neurons, dermal, and other cells of the body. The parasite is located free in the host cell cytoplasm without a parasitophorous vacuole; schizonts divide by endopolygeny. Schizonts are 5-25 x 4-20 microns and contain 6-40 merozoites. Merozoites are approximately 5-7 microns x 1 micron and do not contain rhoptries. The parasite is PAS-negative and reacts with Sarcocystis cruzi antiserum but not with Toxoplasma gondii, Neospora caninum, or Caryospora bigenetica antisera in an immunohistochemical test.     

Fine Structure of Exoerythrocytic Merozoite Formation of Plasmodium berghei in Rat Liver1

The fine structure of exoerythrocytic merogony of Plasmodium berghei was studied after perfusion-fixation of rat livers from 51 h post-inoculation onwards. Meroblast formation was effected by clefts originating from the parasite plasmalemma and by fusion of vacuoles with each other. Invaginations at the periphery resulted in labyrinthine structures providing the parasites with an enormous increase in surface area, which might facilitate exchange of metabolites. When the parasitophorous vacuole membrane collapsed, the newly formed merozoites were lying free in the hepatocytic cytoplasm, which degenerated until the merozoites were sticking together by a stroma, obviously a remnant of the host hepatocyte. Groups of merozoites, still kept together by the spongy stroma, were subsequently released in the bloodstream. At 53 h most of the developmental stages leading to the release of merozoites could be found and thereafter parasite numbers decreased while large granulomas became apparent.