Freeze-fracture study of the site of attachment of Cryptosporidium muris in gastric glands.

The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by 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 host plasma membrane, 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 severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.     

Freeze-Fracture Study of the Site of Attachment of Cryptosporidium muris in Gastric Glands

ABSTRACT The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by 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 host plasma membrane, 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 severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.     

Heterogeneous Distribution of Membrane Cholesterol at the Attachment Site of Cryptosporidium muris to Host Cells

ABSTRACT. Distribution of membrane cholesterol at the attachment site of Cryptosporidium muris was investigated by freeze-fracture cytochemistry using a polyene antibiotic filipin. Since the host plasma membrane enveloped C. muris , the inner and outer membranes were continuous with the parasite plasma membrane at the annular ring and with host membrane at the dense band, respectively. Although many filipin-cholesterol complexes were observed on the plasma membrane of host cells and parasites, a line showing no complexes was evident at the above two membrane junctures. These observations indicate that parasitic infection of C. muris altered the organization of membrane cholesterol.     

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.     

Freeze-fracture study of malaria sporozoites: antibody-induced changes of the pellicular membrane.

Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium berghei sporozoites, before and after incubation with immune serum, were studied after freeze-fracture by electron microscopy. There were evenly distributed numerous intramembranous particles (IMP) on the P face of the outer membrane. The E face of the plasma membrane had fewer IMP than its P face. The E face of the intermediate membrane had few IMP and also linear arrays of slightly raised ridges running the length of the parasite. The P face of the intermediate membrane had many IMP aligned along the long axis of the sporozoite. On the P face of the inner membrane, IMP were arranged in very distinct rows conforming to the long axis of the parasite; the E face of this membrane had a few randomly distributed IMP. A prominent change in the sporozoite incubated in immune serum was the appearance of a layer of aggregated particles around the parasite. The P face of the plasma membrane had several clear areas devoid of IMP and IMP aggregates. No changes were seen in the other fractured faces of the pellicle. These observations suggest that immune serum acts only on the P face of the plasma membrane.     

Freeze-Fracture Study of Malaria Sporozoites: Antibody-Induced Changes of the Pellicular Membrane*

Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium berghei sporozoites, before and after incubation with immune serum, were studied after freeze-fracture by electron microscopy. There were evenly distributed numerous intramembranous particles (IMP) on the P face of the outer membrane. The E face of the plasma membrane had fewer IMP than its P face. The E face of the intermediate membrane had few IMP and also linear arrays of slightly raised ridges running the length of the parasite. The P face of the intermediate membrane had many IMP aligned along the long axis of the sporozoite. On the P face of the inner membrane. IMP were arranged in very distinct rows conforming to the long axis of the parasite; the E face of this membrane had a few randomly distributed IMP. A prominent change in the sporozoite incubated in immune serum was the appearance of a layer of aggregated particles around the parasite. The P face of the plasma membrane had several clear areas devoid of IMP and IMP aggregates. No changes were seen in the other fractured faces of the pellicle. These observations suggest that immune serum acts only on the P face of the plasma membrane.     

Cryptosporidia: epicellular parasites embraced by the host cell membrane

The ultrastructure of two gastric cryptosporidia, Cryptosporidium muris from experimentally infected rodents (Mastomys natalensis) and Cryptosporidium sp. 'toad' from naturally infected toads (Duttaphrynus melanostictus), was studied using electron microscopy. Observations presented herein allowed us to map ultrastructural aspects of the cryptosporidian invasion process and the origin of a parasitophorous sac. Invading parasites attach to the host cell, followed by gradual envelopment, with the host's cell membrane folds, eventually forming the parasitophorous sac. Cryptosporidian developmental stages remain epicellular during the entire life cycle. The parasite development is illustrated in detail using high resolution field emission scanning electron microscopy. This provides a new insight into the ultrastructural detail of host-parasite interactions and species-specific differences manifested in frequency of detachment of the parasitophorous sac, radial folds of the parasitophorous sac and stem-formation of the parasitised host cell.     

An ultrastructural comparison of the attachment sites between Gregarina steini and Cryptosporidium muris

Early developmental stages of Gregarina steini Berndt, 1902 from the intestine of Tenebrio molitor larvae were studied by transmission electron microscopy. The formation and structure of the eugregarine attachment site were compared with comparable features found on the feeder organelle of Cryptosporidium muris Tyzzer, 1907, from the stomach of experimentally infected rodents. The similarity of the attachment strategy between both organisms was revealed. The membrane fusion site in G. steini, formed by the trophozoite plasma membrane, host cell plasma membrane and a membrane-like structure limiting the cortical zone of the epimerite, resembles the Y-shaped membrane junction between the host cell plasma membrane, the trophozoite plasma membrane and membrane surrounding the anterior vacuole in C. muris. The anterior vacuole of C. muris appears to be the precursor of the feeder organelle and its structure is very similar to the epimeritic bud and the cortical zone of G. steini trophozoites. In both investigated organisms, the apical complex disappears early during cell invasion. The possibility of the epicellular location of Cryptosporidium on the surface of host cells is discussed.     

Development of Plasmodium chabaudi in Mouse Red Blood Cells: Structural Properties of the Host and Parasite Membranes1

The structure of both the host and parasite membranes during stages in the asexual development of Plasmodium chabaudi in mouse red blood cells is examined by transmission electron microscopy of thin sections and freeze-fracture preparations. The erythrocyte's plasma membrane, the membrane of the parasitophorous vacuole, and the plasma membrane of the parasite exhibit different structural properties in terms of membrane width and the frequency and diameter of the typical intramembrane-particles (IMP) populating the membrane's fracture faces. The difference between the parasitophorous vacuolar membrane and host cell's plasma membrane is remarkable because the vacuolar membrane is formed from an invagination of the erythrocyte's plasma membrane. The vacuolar membrane has significantly reduced frequencies and diameters of IMP's on both faces and reveals a marked temperature response manifesting itself as large IMP-devoid domains emerging on both faces on chilling to 4°C. In contrast, cooling induces only some very small IMP-devoid patches on both faces of the host plasma membrane. Neither of these membranes changes significantly as parasite development progresses. In contrast, the parasite's plasma membrane shows local alterations during its development, forming compaction domains with the nuclear envelope in ca. 30% of the ring-stages and trophozoites. These compaction domains disappear in late uninuclear trophozoites and schizonts. Furthermore, the plasma membrane of the host cell, the vacuolar membrane, and the parasite's plasma membrane do not respond to externally applied Ca²⁺, and their temperature-response remains unaltered during the infection cycle. Thus, modification of these three membranes as a consequence of invasion and development of the parasites, as recently found in the primate malaria caused by P. knowlesi, can be detected neither directly nor indirectly via temperature- and/or Ca²⁺-response in the rodent malaria caused by P. chabaudi.     

Ultrastructure of Cryptosporidium wrairi from the Guinea Pig

SYNOPSIS. The ultrastructure of the known tissue stages of Cryptosporidium wrairi Vetterling, Jervis, Merrill, and Sprinz, 1971 parasitizing the ileum of guinea pigs is described. Young trophozoites are surrounded by 4 unit membranes, the outer 2 of host origin, the inner 2 the pellicle of the parasite. Each trophozoite contains a vesicular nucleus with a large nucleolus. Its cytoplasm contains ribosomes, but eventually fills with cisternae of the rough endoplasmic reticulum. As the trophozoite matures the area of attachment of the parasite to the host cell becomes vacuolated, with vertical membranous folds. It is apparent that the parasite acquires nourishment from the host cell thru this area of attachment. As schizonts develop, (a) multiple nuclei appear, (b) the endoplasmic reticulum enlarges, (c) the attachment zone increases in area, (d) large vacuoles, which develop as endocytotic vesicles in the attachment area, are found in the cytoplasm and (e) the inner unit membrane of the parasite pellicle is resorbed around the sides of the developing schizont. Following nuclear division, merozoites develop from the schizont by budding. Merozoites have an ultrastructure similar to that described for other coccidia except that no mitochondria, micropores, or subpellicular tubules were observed. Merozoites penetrate the epithelial cell causing invagination of the microvillar membrane and lysing it. No unit membrane is formed between the parasite and the host cell. However, the cell produces one or 2 dense bands adjacent to the parasite attachment area. The macrogamete contains a nucleus, endoplasmic reticulum, attachment zone, and large vacuoles. It also contains a variety of granules, some of which are polysaccharide. The immature microgametocyte contains multiple compact nuclei. No mature microgametocytes or zygotes were found.     

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.     

Plasmodium iophurae: cationized ferritin staining, an electron microscope cytochemical method for differentiating malarial parasite and host cell membranes

The surface membranes of erythrocyte-free Plasmodium lophurae and its host cell, the duckling erythrocyte, stain differentially when exposed to cationized ferritin (CF). At low CF concentrations (0.18 mg/ml) only the outer surface of the red cell stains, whereas at the standard concentration (0.7 mg/ml) both the red cell and the parasitophorous vacuolar membranes (PVM) were stained on their outer faces. By using a high CF concentration (3.7 mg/ml), the parasite's plasma membrane (PM) could be distinguished from that of the PVM: The former did not bind CF, whereas the latter was stained on its outer surface. At this level of CF the red cell membrane stained on both faces if these surfaces were exposed to stain. Although the PVM is formed by red cell endocytosis of the parasite, it can be distinguished from the membrane of the erythrocyte as well as that of the PM.     

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.     

Freeze fracture studies on the interaction between the malaria parasite and the host erythrocyte in Plasmodium knowlesi infections.

The freeze fracture technique has been used to study the internal cyto-architecture of the surface membranes of the parasite and erythrocyte in Plasmodium knowlesi infections. Six fracture faces, derived from the plasma membrane and 2 pellicular membranes, have been identified at the surface of the free merozoite. The apposed leaflets of the 2 pellicular membranes show the characteristic features of E fracture faces, a result compatible with the view that the pellicular membranes line a potential cisterna. There is evidence to suggest that there may be changes in the distribution and density of the integral proteins in the merozoite plasma membrane at invasion. Furthermore, vesicles consisting of stacked membranes occur within and around the erythrocyte invagination at invasion; it is suggested that these vesicles are released from the merozoite rhoptries. Formation of the parasitophorous vacuole is accompanied by dramatic changes in the density and distribution of intra-membraneous particles (IMP) in the vacuolar membrane. Initially there is a great reduction in particle numbers, but subsequently the particles reappear and show reversed polarity. The possible causes and implications of these changes are discussed. The intra-erythrocytic parasite synthesizes new transmembrane proteins as development proceeds, and the trophozoite and schizont stages of development are characterized by the appearance of circular, particle-free regions in the parasite plasmalemma. There is a decrease in the density of transmembrane proteins in the erythrocyte plasma membrane during parasite maturation, and the P face IMP show the characteristic features of aggregation.     

Structural similarity of the membrane envelopes of rhizobial bacteroids and the host plasma membrane as revealed by freeze-fracturing.

The freeze-fracture technique was used to study the host plasma membrane and the membrane envelope of bacteroids in rhizobial root nodules of three host-rhizobium combinations. In all three combinations studied, the membrane envelopes of bacteroids are structurally similar to their host plasma membrane. However, the membrane appears to be reversed, because the number and arrangement of particles in the outer fractured face (face A, concave) and in the inner fractured face (face B, convex) of the host plasma membrane are seen, respectively, in the inner fractured face (face B, convex) and in the outer fractured face (face A, concave) of the membrane envelope of the bacteroids at an early stage. This reversion of the membrane surface is consistent with the hypothesis that the membrane envelopes of bacteroids are derived from the host plasma membrane during endocytotic engulfment.     

Electron microscopic observations on alterations in cultured Plasmodium falciparum and infected erythrocytes after treatment with cyclic AMP in vitro

The fine structure of Plasmodium falciparum treated with cyclic AMP in vitro was studied. Cyclic AMP stimulated the appearance of membranous structures in P. falciparum-infected erythrocytes. Two types of membranous structures originating from the host cell were observed: multilaminate membranous structures and multistranded layer-like membranous structures. The multilaminate structures may play a role in gametocytogenesis and the maturation of the gametocyte. The multilaminate structures were either free in the cytoplasm of infected erythrocytes or present in association with the parasitophorous vacuole membrane surrounding immature gametocytes. These structures may originate from the erythrocyte plasma membrane and the parasitophorous vacuole membrane. Other notable findings in P. falciparum treated with cyclic AMP included the presence of loop-like membrane structures protruding from the plasma membrane of the parasite and termination of some plasma membranes of the parasite in dense granular structures.     

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.     

The adherence of human neutrophils and eosinophils to schistosomula: evidence for membrane fusion between cells and parasites

Human neutrophils and eosinophils adhere to the surface of schistosomula of Schistosoma mansoni that have been preincubated with antischistosomular sera with or without complement. Neutrophils are seen to form small (< 0.5 micrometer), heptalaminar and large (5-8 micrometer), pentalaminar fusions with the normal pentalaminar parasite surface membrane. By freeze-fracture techniques, attachment areas 5-8 micrometer in diameter are seen to form between neutrophils and schistosomula. These areas have three zones--an edge and two centrally located areas, one of which is rich and one of which is poor in intramembrane particles (IMPs). The edge zone is continuous around the attachment areas and is usually composed of a skip-fracture that passes out of the schistosomular outer membrane into the inner membrane. In some cases, the edge zone is made up of a string of IMPs. The IMP-rich central areas have an IMP concentration similar to that of unattached neutrophil membranes, are raised off of the surface of the schistosomulum, and have two normal schistosomular membranes underneath indicating that they are indeed unattached. the IMP-poor central areas are composed of a fused or hybrid membrane that is continuous with the neutrophil plasma membrane but that bears the same spatial relationship to the schistosomular inner membrane that the normal outer membrane does. Similar changes are seen in samples prepared with glycerination. Eosinophils generally do not fuse with the schistosomular outer membrane but, instead, discharge their granular contents onto the surface of the schistosomula and appear to adhere to the parasite through this discharged material. It is suggested that schistosomula have a capability to fuse with mammalian cells and that this fusion proceeds from a fusion of the outer leaflets to a fusion of the bilayers, as appears also to be the case in other systems.     

Ultrastructural analysis of the freeze-etched spore envelope of the microsporidian,Nosema apis zander

The outer limiting layer of the spore coat ofNosema apis is relatively smooth. The inner limiting layer shows two fractured faces, the concave face carrying many stud-like projections, 120 nm long and 50 nm high, while the convex face carries numerous depressions which are complementary to the projections. In addition, the convex face bears 7 nm particles. In between the outer and inner limiting layers lies the thick homogeneous portion of spore coat which is comprised of numerous microfibres, each 9 nm in diameter. These microfibres resemble those in the freeze-etched host endocuticle. Next to the inner limiting layer of the spore coat are double spore membranes. The convex faces of these spore membranes have a dense population of particles, each 7 nm in diameter.     

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.