Evidence for host cells as the major contributor of lipids in the intravacuolar network of Toxoplasma-infected cells

The intracellular parasite Toxoplasma gondii develops inside a parasitophorous vacuole (PV) that derives from the host cell plasma membrane during invasion. Previous electron micrograph images have shown that the membrane of this vacuole undergoes an extraordinary remodeling with an extensive network of thin tubules and vesicles, the intravacuolar network (IVN), which fills the lumen of the PV. While dense granule proteins, secreted during and after invasion, are the main factors for the organization and tubulation of the network, little is known about the source of lipids used for this remodeling. By selectively labeling host cell or parasite membranes, we uncovered evidence that strongly supports the host cell as the primary, if not exclusive, source of lipids for parasite IVN remodeling. Fluorescence recovery after photobleaching (FRAP) microscopy experiments revealed that lipids are surprisingly dynamic within the parasitophorous vacuole and are continuously exchanged or replenished by the host cell. The results presented here suggest a new model for development of the parasitophorous vacuole whereby the host provides a continuous stream of lipids to support the growth and maturation of the PVM and IVN.     

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.     

Overcoating of Toxoplasma Parasitophorous Vacuoles with Host Cell Vimentin Type Intermediate Filaments

The interaction between the Toxoplasma parasitophorous vacuole and vimentin-type intermediate filaments in Vero cells was investigated via immunofluorescence microscopy. A significant rearrangement of host cell vimentin around the Toxoplasma parasitophorous vacuoles occurs throughout the course of infection. Host cell vimentin associates with the parasitophorous vacuoles within an hour after invasion. This vimentin overcoating of the vacuole is initiated at the host cell nuclear surface. During parasite multiplication, vimentin retains a closely defined association with the cytosolic surface of the parasitophorous vacuole. In addition, the vimentin intermediate filaments originating from the host cell nuclear surface are progressively rearranged around the enlarging parasitophorous compartment. During infections, the order of vimentin cytoskeleton is normal throughout the cell and appears redefined only at the vicinity of the parasitophorous vacuole. Depolymerization of the intermediate filaments was achieved with the phosphatase inhibitors okadaic acid and calyculin A. Disruption of the intermediate filament networks resulted in displacement of the parasitophorous vacuoles from the host cell nuclear surface. The data indicate that host cell vimentin binds to the Toxoplasma parasitophorous vacuoles and that the host intermediate filament network serves to dock the parasite compartment to the host cell nuclear surface.     

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.     

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.     

Ultrastructural morphology suggesting a new hypothesis for development of microsporidians seen in Loma salmonae infecting the gills of rainbow and brook trout

In two variants of Loma salmonae that have specificity for rainbow trout Oncorhynchus mykiss (OA variant) and specificity to brook trout Salvelinus fontinalis (SV variant), the parasitophorous vacuole forms at the onset of sporogony. In the OA variant the merogonial stage is bound by a single plasma membrane in direct contact with host cytoplasm. The parasitophorous vacuole formation is initiated by the host cell surrounding the merogonial stages with endoplasmic reticulum (ER) as occurs in autophagy. Of the two host ER membranes surrounding the parasite, one remains in close association with the plasma membrane of the meront, while the other forms the limiting membrane of the vacuole. The sporogonial stage is bounded by two closely apposed membranes, giving the appearance of a thick electron dense plasmalemma. The observations from this study support the novel hypothesis that this microsporidian uses the intracellular process of autophagy to aid formation of a parasitophorous vacuole. The morphology of the SV‐variant is consistent with that of the OA‐variant suggesting that it uses the same mechanism for development.     

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.     

Penetration of Toxoplasma gondii into host cells induces changes in the distribution of the mitochondria and the endoplasmic reticulum.

Fluorescence microscopy, using dyes which specifically label mitochondria, endoplasmic reticulum and the Golgi complex, and transmission electron microscopy, were used to analyze the changes which occur in the organization of these structures during interaction of Toxoplasma gondii with host cells. In uninfected cells the mitochondria are long filamentous structures which radiate from the nuclear region toward the cell periphery. After parasite penetration they become shorter and tend to concentrate around the parasite-containing vacuole (parasitophorous vacuole) located in the cytoplasm of the host cell. The mitochondria of extracellular parasites, but not of those located within the parasitophorous vacuole, were also stained by rhodamine 123. Labeling with DiOC6, which binds to elements of the endoplasmic reticulum, in association with transmission electron microscopy, revealed a concentration of this structure around the parasitophorous vacuole. The membrane lining this vacuole was also stained, suggesting that components of the endoplasmic reticulum are also incorporated into this membrane. The Golgi complex, as revealed by staining with NBD-ceramide and electron microscopy, maintains its perinuclear position throughout the evolution of the intracellular parasitism.     

Localization of a Toxoplasma gondii Rhoptry Protein by Immunoelectron Microscopy During and After Host Cell Penetration

ABSTRACT We immunolocalized a Toxoplasma gondii rhoptry protein (ROP1) before and after parasite host cell invasion of human fibroblasts and TG180 murine sarcoma cells by electron microscopy and immunogold labeling using either a monoclonal antibody (Tg49) or a monospecific rabbit antiserum (α249). At all stages of parasite growth ROP1 was found within the body but rarely within the peduncle of rhoptries, even in those that appeared empty. Immediately after host cell invasion ROP1 was associated with the parasitophorous vacuole membrane. Within hours after invasion the amount of ROP1 immunodetectable on the parasitophorous vacuole membrane was markedly decreased. The localization of ROP1 suggests a role in the early establishment of infection in host cells, consistent with previous work that has indicated that monoclonal antibodies to ROP1 (including the one used in these studies) interfere with the phenomenon of penetration enhancement.     

Proteases and chaperones are the most abundant proteins in the parasitophorous vacuole of Plasmodium falciparum-infected erythrocytes

After invasion of erythrocytes, the human malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole (PV) which forms an interface between the host cell cytosol and the parasite surface. This vacuole protects the parasite from potentially harmful substances, but allows access of essential nutrients to the parasite. Furthermore, the vacuole acts as a transit compartment for parasite proteins en route to the host cell cytoplasm. Recently we developed a strategy to biotin label soluble proteins of the PV. Here, we have paired this strategy with a high-throughput MALDI-TOF-MS analysis to identify 27 vacuolar proteins. These proteins fall into the following main classes: chaperones, proteases, and metabolic enzymes, consistent with the expected functions of the vacuole. These proteins are likely to be involved in several processes including nutrient acquisition from the host cytosol, protein sorting within the vacuole, and release of parasites at the end of the intraerythrocytic cycle.     

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.     

Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole.

Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry-derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein-rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite-containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre-existing, parasite-containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two-step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by TOXOPLASMA: The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.     

Immunoelectron microscopic demonstration of the exocytosis of dense granule contents into the secondary parasitophorous vacuole of Sarcocystis muris (Protozoa, Apicomplexa)

Merozoites of the parasitic protozoon Sarcocystis muris (Apicomplexa) possess three types of characteristic organelles with electron dense contents named rhoptries, micronemes, and dense granules, which are supposed to be involved in the parasite-host cell interactions during and after invasion. Dense granules were purified from a merozoite homogenate by centrifugation on a sucrose density gradient. It was shown by SDS polyacrylamide gel electrophoresis that they contain a major protein of 21 kDa. Polyclonal antibodies raised against this protein were applied to ultrathin frozen and Lowicryl-K4M-embedded sections of the parasite before and after host cell invasion. Dense granules were distinctly labeled by immunogold before and after invasion. After host cell invasion the parasite is enclosed in a secondary parasitophorous vacuole which contains an electron-dense material. This deposition was heavily labeled by anti 21 kDa antibodies which clearly demonstrated that the dense granule contents is released into the secondary parasitophorous vacuole.     

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.     

Localization of a Toxoplasma gondii rhoptry protein by immunoelectron microscopy during and after host cell penetration.

We immunolocalized a Toxoplasma gondii rhoptry protein (ROP1) before and after parasite host cell invasion of human fibroblasts and TG180 murine sarcoma cells by electron microscopy and immunogold labeling using either a monoclonal antibody (Tg49) or a monospecific rabbit antiserum (alpha 249). At all stages of parasite growth ROP1 was found within the body but rarely within the peduncle of rhoptries, even in those that appeared empty. Immediately after host cell invasion ROP1 was associated with the parasitophorous vacuole membrane. Within hours after invasion the amount of ROP1 immunodetectable on the parasitophorous vacuole membrane was markedly decreased. The localization of ROP1 suggests a role in the early establishment of infection in host cells, consistent with previous work that has indicated that monoclonal antibodies to ROP1 (including the one used in these studies) interfere with the phenomenon of penetration enhancement.     

Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC

Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host‐derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole–host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative ‘meront’ stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (< 10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC‐1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno‐EM revealed that the ATP‐delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria‐vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP‐delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite.     

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP₂ and PIP₃ to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.     

Freeze-fracture studies of Cryptosporidium muris.

The attachment site of Cryptosporidium muris to host cells was investigated using the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with the host plasma membrane at the dense band, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was dramatically altered at the above two junctures. The outer parasitophorous membrane showed low IMP-density as compared to the host plasma membrane, although both membranes were continuous. The inner parasitophorous membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP. When the attachment sites of parasites and host cells were fractured, circular-shaped fractured faces were observed on both sites of the parasite and host cell. These exposed faces corresponded to the dense bands and were very similar in size in each parasite.     

The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites

The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane.     

Toxoplasma: guess who's coming to dinner

In this issue of Cell, Coppens and coworkers (Coppens et al., 2006) describe how Toxoplasma gondii, an obligate intracellular parasite, feeds on the host. Coppens et al. provide evidence that the parasite takes host cell endosomes and lysosomes hostage by sequestering them where the parasite resides, within invaginations of the parasitophorous vacuole.