Observations on the Fine Structure of the "Cyst Stage" of Besnoitia jellisoni

SYNOPSIS. Light and electron microscope studies of the "cyst" of Besnoitia jellisoni indicate that it consists of an extracellular wall, a large, sometimes multinucleate, host cell, and an intracellular vacuole containing the parasites. The "cyst" wall has fine fibrils and small dense granules embedded in an election-lucid matrix. The wall may be formed from a secretion of the enclosed host cell. The plasma membrane of the host cell is very irregular, being modified into microvillar or pseudopodial extensions. Small vesicles and invaginations of the plasma membrane indicate mioropinocytosis. The one to several large lobular nuclei lie in a thick area of cytoplasm which is filled with rough endoplasmic reticulum and many mitochondria with lamellar cristae. The parasite-containing vacuole is limited by a vacuolar membrane which has many blebs suggesting a transfer of materials into the vacuole.
The "cyst" organisms are crescentic or piriform and are enclosed by a pellicle consisting of outer and inner membranes. Twenty-two subpellicular fibrils extend longitudinally adjacent to the inner membrane from the anterior polar ring to a posterior ring. A micropyle is situated laterally in the pelliole near the level of the nucleus. A conold and several associated paired organelles are present at the anterior end. Microuemes, more abundant in older organisms, are also present in the anterior portion of the parasite. A Golgi apparatus lies adjacent and anterior to the nucleus. One or more mitochondria with saccular cristae, ovoid glycogen bodies, free ribosomes and occasional vacuoles are also present. Organisms within the "cyst" multiply by endodyogeny.     

Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites?

Together with micronemes and rhoptries, dense granules are specialised secretory organelles of Apicomplexa parasites. Among Apicomplexa, Plasmodium represents a model of parasites propagated by way of an insect vector, whereas Toxoplasma is a model of food borne protozoa forming cysts. Through comparison of both models, this review summarises data accumulated over recent years on alternative strategies chosen by these parasites to develop within a parasitophorous vacuole and explores the role of dense granules in this process. One of the characteristics of the Plasmodium erythrocyte stages is to export numerous parasite proteins into both the host cell cytoplasm and/or plasma membrane via the vacuole used as a step trafficking compartment. Whether this feature can be correlated to few storage granules and a restricted number of dense granule proteins, is not yet clear. By contrast, the Toxoplasma developing vacuole is decorated by abundantly expressed dense granule proteins and is characterised by a network of membranous nanotubes. Although the exact function of most of these proteins remains currently unknown, recent data suggest that some of these dense granule proteins could be involved in building the intravacuolar membranous network. Conserved expression of the Toxoplasma dense granule proteins throughout most of the parasite stages suggests that they could also be key elements of the cyst formation.     

Further morphological studies on the behavior of Trypanosoma rangeli in the hemocytes of Rhodnius prolixus

The hemocytes of Rhodnius prolixus were analyzed during the course of infection with the protozoan Trypanosoma rangeli. The following cell types were identified: prohemocyte, plasmatocyte, adipocyte, granular cell and oenocytoid. The number of these cells changes during the infection course thus indicating a cell response to infection of R. prolixus by T. rangeli. Transmission electron microscopy showed that plasmatocytes were able to ingest epimastigote forms of the parasite, which were then found within a parasitophorous vacuole. Amorphous material was seen within the vacuole suggesting that fusion of host cell lysosomes with the vacuole took place. Intravacuolar parasites in process of digestion were observed. In addition, reaction product indicative of the presence of acid phosphatase was observed in parasite-containing vacuoles. No dividing parasites were seen within the vacuole in contrast to what was observed outside the host cells.     

Extraintestinal stages fo Isospora ohioensis from dogs in mice

The development of Isospora ohioensis was studied in mice by feeding tissues of mice inoculated with oocysts to coccidia-free dogs and by the examination of mesenteric lymph nodes using light and electron microscopes. Extraintestinal organs of mice became infectious to dogs within 1 day after ingesting oocysts and remained infectious for at least 211 days after inoculation (DAI). Isospora ohioensis sporozoites were found in lymphoreticular cells of mesenteric lymph nodes of mice from 1-374 DAI. Intracellular sporozoites were located in parasitophorous vacuoles. Sporozoites grew from 5--6 to 11--16 micron in length on the 39th DAI but never lost the 2 crytalloid bodies typical for coccidian sporozoites. PAS-positive granules accumulated gradually in intracellular sporozoites with duration of infection in mice. The appearance of parasitophorous vacuoles varied with duration of infection. Beginning with 7 DAI, the vacuole contained a marginal zone of electron-dense material (up to 0.8 micron wide), giving the appearance of a cyst wall or sheath under the light microscope; a true cyst wall was was not found.     

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.     

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.     

The ultrastructure of the parasitophorous vacuole formed by Leishmania major

Protozoan parasites of Leishmania spp. invade macrophages as promastigotes and differentiate into replicative amastigotes within parasitophorous vacuoles. Infection of inbred strains of mice with Leishmania major is a well-studied model of the mammalian immune response to Leishmania species, but the ultrastructure and biochemical properties of the parasitophorous vacuole occupied by this parasite have been best characterized for other species of Leishmania. We examined the parasitophorous vacuole occupied by L. major in lymph nodes of infected mice and in bone marrow-derived macrophages infected in vitro. At all time points after infection, single L. major amastigotes were wrapped tightly by host membrane, suggesting that amastigotes segregate into separate vacuoles during replication. This small, individual vacuole contrasts sharply with the large, communal vacuoles occupied by Leishmania amazonensis. An extensive survey of the literature revealed that the single vacuoles occupied by L. major are characteristic of those formed by Old World species of Leishmania, while New World species of Leishmania form large vacuoles occupied by many amastigotes.     

Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host

Toxoplasma gondii has a complex life cycle involving definite (cat) and intermediate (all warm blooded animals) hosts. This gives rise to four infectious forms each of which has a distinctive biological role. Two (tachyzoite and merozoite) are involved in propagation within a host and two (bradyzoite and sporozoite) are involved in transmission to new hosts. The various forms can be identified by their structure, host parasite relationship and distinctive developmental processes. In the present in vivo study, the various stages have been evaluated by electron microscopy and immunocytochemistry using a panel of molecular markers relating to surface and cytoplasmic molecules, metabolic iso-enzymes and secreted proteins that can differentiate between tachyzoite, bradyzoite and coccidian development. Tachyzoites were characterised as being positive for surface antigen 1, enolase isoenzyme 2, lactic dehydrogenase isoenzyme 1 and negative for bradyzoite antigen 1. In contrast, bradyzoites were negative for SAG1 but positive for BAG1, ENO1 and LDH2. When stage conversion was followed in brain lesion at 10 and 15 days post-infection, tachyzoites were predominant but a number of single intermediate organisms displaying tachyzoite and certain bradyzoite markers were observed. At later time points, small groups of organisms displaying only bradyzoite markers were also present. A number (9) of dense granule proteins (GRA1-8, NTPase) have also been identified in both tachyzoites and bradyzoites but there were differences in their location during parasite development. All the dense granule proteins extensively label the parasitophorous vacuole during tachyzoite development. In contrast the tissue cyst wall displays variable staining for the dense granule proteins, which also expresses an additional unique cyst wall protein. The molecular differences could be identified at the single cell stage consistent with conversion occurring at the time of entry into a new cell. These molecular differences were reflected in the structural differences in the parasitophorous vacuoles observed by electron microscopy. Stage conversion to enteric (coccidian) development was limited to the enterocytes of the cat small intestine. Although no specific markers were available, this form of development can be identified by the absence of specific tachyzoite (SAG1) and bradyzoite (BAG1) markers although the isoenzymes ENO2 and LHD1 were expressed. There was also a significant difference in the expression of the dense granule proteins. The coccidian stages and merozoites only expressed two (GRA7 and NTPase) of the nine dense granule proteins and this was reflected in significant differences in the structure of the parasitophorous vacuole. The coccidian stages also undergo conversion from asexual to sexual development. The mechanism controlling this process is unknown but does not involve any change in the host cell type or parasitophorous vacuole and may be pre-programmed, since the number of asexual cycles was self-limiting. In conclusion, it was possible using a combination of molecular markers to identify tachyzoite, bradyzoite and coccidian development in tissue sections.     

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.     

Ultrastructure of the cyst of Sarcocystis muris.

Cysts of Sarcocystis muris develop within muscle cells and each is bounded by a parasitophorous vacuole membrane. Closely spaced spherical blebs formed from this membrane extend into the muscle cell cytoplasm. A dense substance fills the cavity of the bleb and occupies the vacuolar space immediately adjacent to the membrane. The remainder of the vacuole is filled with a moderately dense matrix within which the parasites develop. At 40 days after infection only metrocytes are present, characterized by their ovoid shape, lightly stained cytoplasm, amylopectin-like granules, and lack of micronemes. Metrocytes divide by a process resembling endodyogeny and eventually produce bradyzoites. By 78 days after infection, at which time the cyst is infective for cats, the few remaining metrocytes are located at the cyst periphery but most organisms are elongated and contain organalles characteristic for bradyzoites including micronemes, dense granules, and amylopectin. Structures indicative of division were not seen in bradyzoites. Rhoptries are few in number. Numerous vesicles of smooth endoplasmic reticulum accumulate in the cytoplasm of muscle cells adjacent to the periphery of the enlarging cyst but significant destruction of muscle fibers containing cysts with viable organisms was not seen in specimens fixed between 40 and 325 days after infection. Unusual lamellar structures were seen in some parasitized muscle cells and intracystic tubules occurred in some cysts.     

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.     

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.     

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.     

Spontaneous cystogenesis in vitro of a Brazilian strain of Toxoplasma gondii

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation in the life cycle of the parasite have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK₂ epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK₂ cells after 4 days of infection, when 72.3 ± 15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the fully conversion of parasites presenting the typical characteristics of bradyzoites as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also detected. Besides, the scanning microscopy revealed that the intracyst matrix tubules were shorter than those from the parasitophorous vacuole intravacuolar network and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle.     

Ultrastructure of tachyzoites, bradyzoites and tissue cysts of Neospora caninum

Dividing tachyzoites of Neospora caninum were 4 x 3 microns and had ultrastructural characteristics typical for the cyst-forming coccidia. Unusual ultrastructural characteristics of fully-formed tachyzoites included no micropores, 8-12 anterior and 4-6 posterior rhoptries, and a few posterior micronemes. Most tachyzoites were located free in the host cell cytoplasm; only a few occurred within a parasitophorous vacuole. Parasite multiplication appeared to be rapid because most organisms were in various stages of endodyogeny. Neural tissue cysts of N. caninum were 24.3 x 19.2 microns and contained 50-200 bradyzoites (7.3 x 1.5 microns), which lacked micropores. The cyst wall was 0.74-1.12 microns thick and consisted of the primary cyst wall (the parasitophorous vacuole membrane) and a thick granular layer with electron-dense vesicles.     

Intestinal expression of H+ V-ATPase in the mosquito Aedes albopictus is tightly associated with gregarine infection

Vacuolar ATPase (V-ATPase) is a family of ATP-dependent proton pumps expressed on the plasma membrane and endomembranes of eukaryotic cells. Acidification of intracellular compartments, such as lysosomes, endosomes, and parasitophorous vacuoles, mediated by V-ATPase is essential for the entry by many enveloped viruses and invasion into or escape from host cells by intracellular parasites. In mosquito larvae, V-ATPase plays a role in regulating alkalization of the anterior midgut. We extracted RNA from larval tissues of Aedes albopictus, cloned the full-length sequence of mRNA of V-ATPase subunit A, which contains a poly-A tail and 2,971 nucleotides, and expressed the protein. The fusion protein was then used to produce rabbit polyclonal antibodies, which were used as a tool to detect V-ATPase in the midgut and Malpighian tubules of mosquito larvae. A parasitophorous vacuole was formed in the midgut in response to invasion by Ascogregarina taiwanensis, confining the trophozoite(s). Acidification was demonstrated within the vacuole using acridine orange staining. It is concluded that gregarine sporozoites are released by ingested oocysts in the V-ATPase-energized high-pH environment. The released sporozoites then invade and develop in epithelial cells of the posterior midgut. Acidification of the parasitophorous vacuoles may be mediated by V-ATPase and may facilitate exocytosis of the vacuole confining the trophozoites from the infected epithelial cells for further extracellular development.     

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.     

Ultrastructure of Tachyzoites, Bradyzoites and Tissue Cysts of Neospora caninum

. Dividing tachyzoites of Neospora caninum were 4x3 μm and had ultrastructural characteristics typical for the cyst-forming coccidia. Unusual ultrastructural characteristics of fully-formed tachyzoites included no micropores, 8–12 anterior and 4–6 posterior rhoptries, and a few posterior micronemes. Most tachyzoites were located free in the host cell cytoplasm; only a few occurred within a parasitophorous vacuole. Parasite multiplication appeared to be rapid because most organisms were in various stages of endodyogeny. Neural tissue cysts of N. caninum were 24.3 × 19.2 μm and contained 50–200 bradyzoites (7.3 × 1.5 μm), which lacked micropores. The cyst wall was 0.74–1.12 μm thick and consisted of the primary cyst wall (the parasitophorous vacuole membrane) and a thick granular layer with electron-dense vesicles.     

Histological and Ultrastructural Studies of the Interaction of Toxoplasma gondii Tachyzoites with Mouse Omentum in Experimental Infection1

Mouse omentum was studied after intraperitoneal challenge with tachyzoites of Toxoplasma gondii. Parasites inhabit omental histiocytes, fibroblasts, mesothelial cells, and free peritoneal macrophages. Recently infected cells showed enhanced metabolic and functional activity. Villous projections of the parasitophorous vacuole wall appeared, usually opposite the anterior pole of the parasite. In mesothelial cells, projections formed terminal swellings not observed in other infected cells. Activation of host cells was followed by reduction of the density of the cytoplasmic matrix, autophagosome formation, and intracellular edema, indicating the damage. The wall of the parasitophorous vacuole loses the supporting host cell endoplasmic reticulum that was attached to the vacuole just after entrance of the parasite into the cell. Then lysis of the parasitophorous vacuole and complete cell destruction occurs. The growth of parasites in undamaged cells does not coincide with the inflammatory response. Inflammation of the peritoneum develops only after the start of mass destruction of infected cells. Thus tachyzoites of Toxoplasma exert significant pathogenic effects by their ability to activate the host cell, causing lysis of the parasitophorous vacuole and subsequent destruction of the entire cell.     

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.