Fine Structure of Schizonts and Merozoites of Eimeria nieschulzi*

SYNOPSIS. Schizonts of E. nieschulzi lie in a vacuole within the host cell. After nuclear division the cell membrane invaginates forming merozoites. Differentiation of the pellicle and other organelles occurs while merozoites are still attached to the schizont cytoplasm. Merozoites have a pellicle thickened at the anterior end to form a polar ring. Radiating posteriorly from the ring, directly beneath the pellicle, are about 25 microtubules. Within the polar ring is a dense conoid. Extending posteriorly from within the conoid is a paired organelle. The paired organelle varies in size and shape in each generation of merozoites. Numerous toxonemes occupy the anterior half of the merozoites. Two paranuclear bodies are present in 1st generation merozoites. One or 2 granular bodies were seen in the anterior end of 2nd generation merozoites. In 3rd generation merozoites 6 or more granular bodies were seen anterior to the nucleus. Each merozoite has a single nucleus containing diffuse chromatin material. Elongate mitochondria and glycogen granules are present. The vacuole surrounding mature merozoites contains residual cytoplasm of the schizont and some granular material. Microvilli project into the vacuole from the host cell membrane.     

Investigations into the fire structure of the asexual developmental stages of Frenkelia in the liver of the bank vole (author's transl)]

Bank voles (Clethrionomys glareolus) were infected by stomach tube with Frenkelia sporocysts from the faeces of buzzards (Buteo buteo). The voles were sacrificed at regular intervals and their livers examined electronmicroscopically. Seven days p.i. developmental stages of Frenkelia could be detected in liver parenchymal cells. The youngest schizonts detected are enveloped by a pellicle consisting of two membranes. This pellicle, which is in direct contact with the host cell mitochondria, shows marked invaginations which increase with the development of the schizont. A parasitophorous vacuole is not detectable. In developing schizonts numerous sections through nuclei with nucleic spindles and merozoite anlagen (dome-shaped) structures) are visible. It is not clear whether there are several nuclei or a section through one large and lobed nucleus. Within the merozoite anlagen the conoid and the subpellicular microtubules are formed first. By the prolongation of the dome-shaped structures towards the posterior pole, the nucleus and the other newly formed cell organelles are incorporated into the forming merozoite. The posterior pole of the merozoite still remains open at this stage of development. With increasing differentiation the merozoites become lancet-shaped, their apical poles bing always directed towards the periphery of the schizont. The outer membrane of the pellicle of the schizont forms the outer part of the pellicle of the merozoites by invaginating around them. At this stage of development the inner membrane of the pellicle of the schizont is no longer detectable. Thus the typical pellicle of the motile stages of sporozoaonsisting of three membranes is formed. In the centre of the merozoites which lie freely in the liver cell a residual body is present. The host cell reacts against the parasites by forming a thick border of mitochondria and distinct endoplasmic reticulum.     

An Ultrastructural Study of Eimeria iroquoina Molnar & Fernando, 1974 in Experimentally Infected Fathead Minnows (Pimephales promelas,Cyprinidae) 3. Merogony1

Fathead minnows, Pimephales promelas, raised from eggs in the laboratory, were experimentally infected with oocysts of Eimeria iroquoina from either P. promelas or the common shiner, Notropis cornutus. Within intestinal epithelial cells, trophozoites thought to be derived from the sporozoites contained a prominent electron-dense refractile body. Merozoites dedifferentiated into trophic forms by losing components of their apical complex and pellicle. The inner membrane components of the pellicle appeared discontinuous, and the micronemes became enclosed within vacuoles. Prior to merozoite formation, multinucleate meronts were limited by a single membrane. Golgi complexes were associated with the nuclei of this stage. Merozoites were formed by ectomerogony in one generation and by endomerogony in the final generation. In both forms of merogony the final nuclear division was coupled with the onset of differentiation of the merozoites and featured eccentric mitotic spindles associated with centrocones located within the nuclear envelope and with the precursors of the apical complex. A Golgi complex was closely associated with the nucleus and apical tip of the forming merozoite. Unlike other Eimeria species, the complete pellicle of the merozoites of the final asexual generation of E. iroquoina was formed within the cytoplasm of the meront, without association with the limiting membrane, thus, all pellicular components are synthesized de novo. The inner membranes of the pellicle initially appeared as longitudinal strips, each of which was associated with a pair of the 22–24 subpellicular microtubules. Mature meronts of the final asexual generation averaged 9 μm in diameter and produced 13–16 merozoites. With the exception of the internal completion of the pellicle of the final generation merozoites, the basic processes of merogony in fish Eimeria species are similar to those recorded in terrestrial hosts.     

Etude Ultrastructurale des Mérozoites et de la Schizogonie des Coccidies (Eimeriina): Eimeria magna (Perard 1925) de I'Intestin des Lapins et E. tenella (Railliet et Lucet, 1891) des Coecums des Poulets

SYNOPSIS. An electron microscopic study is made of merozoites and schizogony of Eimeria magna and Eimeria tenella from rabbits and chickens infected 5 days before fixation.
The merozoite outer layer is formed by a unit membrane lined by a dense osmiophilic layer. A micropyle is present. The apical complex of the cell is constituted by a conoid surmounted by 2 rings and surrounded by another from which about 26 subpellicular, tubular fibrils start. Two "rhoptries" (= toxonemes) go thru the conoid to the apex of cell. Rare sarconemes (= convoluted tubes) are disseminated in the anterior part of merozoites. A nucleus with nucleolus, Golgi apparatus, mitochondria, endoplasmic reticulum, lipid globules and glucidic grains were observed.
Schizogony starts by the formation of a multinucleated schizont which has a centriolar structure. The new merozoites appear as evaginations of the schizont's membrane. Cellular organelles (conoid, rhoptries, micropyle, sarconemes) differentiate and the nuclei enter the diverticula of the schizont. Then the development of merozoites proceeds by "external budding".
The ultrastructural similarities between the merozoites of Eimeria and the endodyocytes of Toxoplasmea, appear to us to be extremely interesting and indicate a close relationship between the Toxoplasmea and the Coccidia.     

Ultrastructural Study of Schizogony in Eimeria callospermophili*

SYNOPSIS The development of 1st generation schizonts of Eimeria callospermophili was studied with cell cultures and with experimentally infected host animals, Spermophilus armatus. Sporozoite-shaped schizonts each had 5-10 nuclei and all of the organelles of the sporozoite; each nucleus had a nucleolus and an associated Golgi apparatus. In stages immediately preceding merozoite formation, an intranuclear spindle apparatus with conical polar areas were observed near the outer margin of each nucleus. Two centrioles, each having 9 single peripheral tubules and one central tubule, were observed near each pole in some specimens. Merozoite formation began internally, with anlagen of 2 merozoites developing near each nucleus. The inner membrane of the merozoites first appeared as 2 dense thickenings adjacent to the polar cones and centrioles; subpellicular microtubules appeared simultaneously. Two anterior annuli and the conoid formed between the 2 thickenings. Vesicles, possibly of Golgi origin, were located next to the forming inner membrane. As the forming merozoites underwent elongation, a rhoptries anlage, a Golgi apparatus, refractile bodies, and mitochondria were incorporated into each. Sporozoite-shaped schizonts with merozoite anlagen transformed into spheroid or ovoid schizonts; at this time the conoid, rhoptries, micronemes, and the inner membrane of the pellicle gradually disappeared; several small refractile bodies were formed from the larger one. When development was about 1/3 complete, the immature merozoites began to grow outward from the surface of the schizont. In this phase of development, the single surface membrane of the schizont became the outer membrane of the merozoite's pellicle, and additional organelles, including the nucleus, were incorporated. Finally, the merozoites became pinched off, leaving a residual body. Development in cell cultures and host tissues was similar. This type of schizogony, previously undescribed in Eimeria, is compared with corresponding stages of development in other species of Eimeria and Sporozoa.     

贝氏隐孢子虫在北京鸭体内发育的超微结构研究

贝氏隐孢子虫各期虫体均位于宿主粘膜上皮细胞的带虫空泡中。在虫体与上皮细胞接触处,虫体表膜反复折迭形成营养器。子孢子或裂殖子与粘膜上皮细胞接触后,逐步过渡为球形的滋养体;滋养体经2—3次核分裂、产生含4或8个裂殖子的两代裂殖体,裂殖体以外出芽方式产生裂殖子;裂殖子无微孔,顶端表皮形成3—4个环嵴,裂殖子进一步发育成为配子体;大配子体含有两种类型的成囊体。小配子呈楔形,无鞭毛和顶体,有一个致密的长椭圆形细胞核,小配子表膜内侧有9根膜下微管;孢子化卵囊内含四个裸露的子孢子和一个大残体。本文是有关鸭体内隐孢子虫超微结构的首次报导。     

Electron microscopic research on Cryptosporidium. I. The asexual stages in the development of Cryptosporidium parvum

Ultrastructural studies were conducted on asexual developmental stages of C. parvum in the ileal fragment of the intestine of 10-11 day old rats experimentally infected with oocysts isolated from calf feces. A young trophozoite is covered with the typical trimembranous apicomplexan pellicle. As the parasite grows, the inner complex of its apical pellicle, facing the host enterocyte, is seen to reduce up to a unit membrane to make a complex multimembranous "feeding organelle" which is in contact with a thick electron dense band bordering the host-parasite interface. It looks likely that no micropores or any other feeding structures exist in the parasite. Unlike, the opposite body part of the trophozoite, facing the lumen of the intestine, preserves its trimembranous pellicle. Two merozoite generations were followed. In addition to numerous ribosomes, rhoptries, micronemes, and trimembranous pellicle, subpellicular microtubules were observed in the segmenting merozoites. The merogony follows the pattern of ectomeric schizogony. However, no details of nuclear division were detected. The whole cytoplasm of the mother meront is completely used up for the merozoite formation without any residual mass to be left.     

Structure and invasive behaviour of Plasmodium knowlesi merozoites in vitro.

The structure and invasive behaviour of extracellular erythrocytic merozoites prepared by a cell sieving method have been studied with the electron microscope. Free merozoites contain organelles similar to those described in late schizonts of Plasmodium knowlesi. Their surface is lined by a coat of short filaments. On mixing with fresh red cells, merozoites at first adhere, then cause the red cell surface to invaginate rapidly, often with the formation of narrow membranous channels in the red cell interior. As the merozoite enters the invagination it forms an attachment by its cell coat to the rim of the pit, and finally leaves this coat behind as it is enclosed in a red cell vacuole. Dense, rounded intracellular bodies then move to the merozoite periphery, and apparently rupture to cause further localized invagination of the red cell vacuole. The merozoite finally loses its rhoptries, the pellicle is reduced to a single membrane and the parasite becomes a trophozoite. Invasion is complete by 1 min after adhesion, and the trophozoite is formed by 10 min.     

Ultrastructural study of developmental stages of Mattesia dispora (Neogregarinorida: Lipotrophidae), a parasite of the flour moth Ephestia kuehniella (Lepidoptera)

The ultrastructure of merozoites, gamonts and oocysts of the neogregarine Mattesia dispora and their development in larvae of the flour moth Ephestia kuehniella were studied by electron microscopy. The apical complex of free macronuclear merozoites was very distinct in micrographs of sections, the polar rings being especially prominent. Two gamonts associated in head-to-head syzygy and the apical complexes served as the contact point during pairing. At this stage the rhoptries became reduced and the conoid widened. The gamonts had a foam-like appearance in the light microscope. Paired gamonts formed an envelope and developed into a gametocyst, within which the gamonts were separated by a distinct border. Four gametes and two residual cells developed inside the gametocyst. The gametes were covered with a single membrane. The gametes fused in pairs to form two spherical zygotes, each covered by two membranes and with one large nucleus. The external layer appeared more undulated than the inner one. A single membrane covered each residual cell. Walls were formed around both zygotes to produce two oocysts. Each mature oocyst was lemon-shaped with polar plugs and eight peripheral sporozoites, which had a pellicle similar to that of the merozoites, lay beneath the thick oocyst wall.     

Plasmodium falciparum: merozoite invasion in vitro in the presence of chloroquine.

The fine structure of invasion of human erythrocytes by merozoites of the malaria parasite Plasmodium falciparum was observed in vitro. The invasion process is similar to that described for P. knowlesi. Merozoites enter apical end first by invagination of the erythrocyte membrane. At the rim of the invagination, where merozoite and erythrocyte are in closest contact, the erythrocyte membrane is thickened. The brushy cell coat of the P. falciparum merozoite appears to be lost at this attachment zone. The part of the merozoite within the erythrocyte invagination has no visible coat. The coat on the portion outside is unaltered. Merozoites can successfully invade erythrocytes after 3 hr in the presence of a concentration of chloroquine harmful to feeding stages.     

Ultrastructure of shizonts and merozoites of Sarcocystis falcatula in the lungs of budgerigars (Melopsittacus undulatus).

Transmission electron microscopy was used to study the ultrastructure of schizogony of Sarcocystis falcatula in the lungs of budgerigars (Melopsittacus undulatus). Schizogony occurred exclusively by endopolygeny within endothelial cells of pulmonary capillaries, venules, and small veins. Early schizonts were elongate with a large nucleus and nucleolus, surrounded by a pellicle consisting of a plasmalemma and an inner single membrane, and contained most of the organelles and inclusion bodies found in merozoites of Sarcocystis species. As development proceeded, schizonts increased in size and conformed to the shapes of the pulmonary blood vessels. As micronemes, dense granules, the conoid, and subpellicular microtubules disappeared, there was an increase in the size and number of mitochondria, Golgi complexes, and Golgi adjuncts (apicoplasts). As the nucleus elongated, there was a progressive increase in the number of spindles located at various intervals along the nuclear envelope. Eventually, 2 merozoites formed internally immediately above each spindle. During endopolygeny, a portion of the nucleus was incorporated into each merozoite bud along with 1 or 2 Golgi adjuncts, a Golgi complex, mitochondria, endoplasmic reticulum, and ribosomes. During merozoite formation, micronemes appeared in close association with the Golgi complex and gradually increased in number. The pellicle invaginated around the merozoites so they budded at the schizont surface leaving behind a small, central residual body. Dense granules appeared after merozoites were completely formed. Schizonts were 24 x 6.8 microm and contained 24-96 merozoites. Merozoites were 5.1 x 1.8 microm and were found free in the pulmonary air passages and pulmonary capillaries and within nearly all cells of the lung except red blood cells.     

Plasmodium simium: Ultrastructure of erythrocytic phase

An electron microscopic study of Plasmodium simium infections in the squirrel monkey has supplied information on the ultrastructure of erythrocytic trophozoites, schizonts, merozoites, and gametocytes, in addition to an unusual form of host cell pathology. In general, the structural features, as well as certain specialized functions, e.g., hemoglobin ingestion and utilization, nuclear and cytoplasmic division, were found to be similar to those described for other malarial parasites. Some striking features were noted, however. A highly asynchronous mode of merozoite production was observed within single segmenting parasites in spite of the overall developmental synchrony displayed by the population as a whole. Secondly, during parasite segmentation, newly formed merozoites are connected to one another, as well as to the parasitophorous membrane, by periodic surface strands. It is speculated that these interparasite bridges serve as structural support to the segmenting parasite. When merozoites are matured fully, these interconnections break, leaving a uniform array of short surface bristles. In addition, a number of different pathological changes in host cell structure have been noted. Localized surface discontinuities appear in region of infected cells where apical regions of developing or fully mature merozoites are abutted against the plasma membrane. These profiles suggest that these specialized apical regions of the merozoite function in release as well as in host cell penetration. More generalized surface pathology occurs within parasitized erythrocytes in the form of surface blebs, surface clefts, and associated cytoplasmic microvesicles. The severity of this pathology increases as the intraerythrocytic parasite matures. Topographically these altered cells have a “berry-like” surface texture which makes them quite distinctive when viewed by scanning electron microscopy.     

The Fine Structure of Some Developmental Stages of Mattesia grandis McLaughlin (Sporozoa, Neogregarinida), a Parasite of the Boll Weevil Anthonomus grandis Boheman

SYNOPSIS Sporozoites, macronuclear schizonts, merozoites and gamonts of Mattesia grandis were examined by electron microscopy. A conoidal complex, consisting of conoid, polar rings and subpellicular microtubules was present in all of these stages. The conoidal complex was similar in structure to the same organelle of other Sporozoa. The conoidal complex in mono- to quadrinucleate macronuclear schizonts is transformed into an organelle similar to the mucron of some eugregarines.
This mucron consists of a specialized area of the cell membrane from which fine fibers extend into a large vacuole situated directly beneath the cell membrane. The top part of the vacuole is encircled by 2 ring-like structures formed by the dilatation of the original apical rings. The vacuole of the mucron contains many anastomosing protrusions of the cytoplasm, suggesting a nutritional role. The mucron disappears when the schizont reaches the multinucleate state. Later the merozoites bud from the surface of the schizont as in the coccidia. Each merozoite again has a conoidal complex, which persists thru the gamont stage and usually serves as the point of contact between 2 gamonts during their pairing.
The presence of a conoidal complex thru a major portion of the life cycle, its transformation into a mucron and the mode of formation of merozoites indicate that the Neogregarinida combine the fine structure characters of both the Eugregarinida and the Eucoccida, thereby suggesting a phylogenetic relationship between these sporozoans, with the neogregarines as a link between eugregarines and coccidia.     

Plasmodium lophurae: the ultrastructure of the exoerythrocytic stages

The fine structure of the exoerythrocytic stages of Plasmodium lophurae was studied. in specimens grown in tissue cultures of avian cells. Specimens were prepared for sectioning by a method which minimizes disturbance and permits precise selection and orientation specimens.Plasmodium lophurae is similar in many aspects to P. fallax. Merozoites are highly specialized and differentiated. Analysis of their ultrastructure revealed the polar complex to be a specialization of the pellicular envelope and its associated underlying microtubules. The polar rings may simply be a modification of the inner membrane of the pellicle and not discrete structures as previously reported. The electron-dense polar organelles are separated on morphological grounds into three groups: the large paired organelles and the small dense bodies which are both linked to microducts, and the transitional bodies, a third organelle being reported for the first time. Transitional bodies are without microducts, occur in fully mature merozoites and persist only for a short period. All three of these organelles appear to be related to and possibly even derived from internal membrane systems and ribosomes. The apolar end of the merozoite contains the mitochondrion and its associated spherical body. Detailed study of the latter shows it to be cylindrical.Upon entering the host cell, the parasite adds a third membrane at the interface between it and the cell. The merozoite becomes spherical and undergoes transformation into a trophozoite. During this reorganization phase, dedifferentiation occurs and is followed by a rapid growth phase. The end of the growth phase is signaled by the appearance of germinal clefts and nuclear division. The entire process of schizogony culminates in a highly synchronized formation of merozoites.Processes of the limiting membrane forming the host parasite interface were observed extending deply into the cytoplasm of the host cell and often appeared to form bridges between two or more parasites. The significance of this new observation is not yet established.     

Development of Babesiosoma stableri (Dactylosomatidae; Adeleida; Apicomplexa) in its Leech Vector (Batracobdella picta) and the Relationship of the Dactylosomatids to the Piroplasms of Higher Vertebrates

The sporogonic and merogonic development of Babesiosoma stableri Schmittner & McGhee, 1961 within its definitive host and vector, a leech Batracobdella picta (Verrill, 1872), was studied by light and electron microscopy. Gamonts released from frog erythrocytes in the blood meal of the leech associated in syzygy and fused; the gamonts were isogamous and only 1 microgamete was formed. The ultrastructural appearance of the resulting zygote was similar to that of the gamonts, but it was larger. The zygote had an apical complex (including a polar ring, conoid and 2 pre-conoidal rings and micronemes, but no recognizable rhoptries), triple-membraned pellicle, about 40 subpellicular microtubules and prominent stores of amylopectin. Zygotes penetrated the cells of the intestine and underwent sporogony directly within the cytosplasm of the ieech epithelial cell without the formation of a parasitophorous vacuole. Eight sporozoites budded simultaneously around the periphery of an irregularly shaped oocyst. No oocyst wall was formed. Each sporozoite had a complete apical complex (including rhoptries), abundant amylopectin inclusions and a triple-membraned pellicle with about 32 subpellicular microtubules. The sporozoites initiated merogonic replication primarily within the salivary cells of the leech although other tissues, such as muscle, were infected. Each meront produced 4 merozoites by simultaneous budding, forming a cruciform meront typical of the intraerythrocytic development of this parasite. The meront was located directly within the cytoplasm of the host cell. Merozoites, with abundant amylopectin, had a complete apical complex and triple-membraned pellicle with about 40 subpellicular microtubules. The merozoites either initiated a further cycle of replication, or they moved into the ductules of the leech salivary cells which extend to the tip of the proboscis. Observations on gametogenesis. syngamy and sporogony of B. stableri in its leech host indicate that the family Dactylosomatidae should be placed in the suborder Adeleina (Eucoccidiida: Apicomplexa). Babesiosoma stableri was transmitted to uninfected frogs (Rana spp.) by the bite of infected leeches. Prepatent periods ranged from 26 to 38 days at 25° C. Despite a directed search in laboratory reared tadpoles which had each been injected intraperitoneally with 150,000 merozoites, no pre-erythrocytic developmental stages were observed. Similarities in their biology suggest close phylogenetic affinities of the dactylosomatids, and other adeleid blood parasites, with the piroplasms of higher vertebrates.     

THE FINE STRUCTURE OF THE EXOERYTHROCYTIC STAGES OF PLASMODIUM FALLAX

The fine structure of the exoerythrocytic cycle of an avian malarial parasite, Plasmodium fallax, has been analyzed using preparations grown in a tissue culture system derived from embryonic turkey brain cells which were fixed in glutaraldehyde-OsO₄. The mature merozoite, an elongated cell 3- to 4-µ long and 1- to 2-µ wide, is ensheathed in a complex double-layered pellicle. The anterior end consists of a conoid, from which emanate two lobed paired organelles and several closely associated dense bodies. A nucleus is situated in the mid portion of the cell, while a single mitochondrion wrapped around a spherical body is found in the posterior end. On the pellicle of the merozoite near the nucleus a cytostomal cavity, 80 to 100 mµ in diameter, is located. Based on changes in fine structure, the subsequent sequence of development is divided into three phases: first, the dedifferentiation phase, in which the merozoite loses many complex structures, i.e. the conoid, paired organelles, dense bodies, spherical body, and the thick inner layers of the pellicle, and transforms into a trophozoite; second, the growth phase, which consists of many nuclear divisions as well as parallel increases in mitochondria, endoplasmic reticulum, and ribosomes; and third, the redifferentiation and cytoplasmic schizogony phase, in which the specialized organelles reappear as the new merozoites bud off from the mother schizont.     

Ultrastructural Study of Schizogony of Eimeria bovis in Cell Cultures*

SYNOPSIS. First-generation schizogony of Eimeria bovis in bovine cell culture was studied by electron microscopy. The intracellular sporozoite retained its structure for at least 6 days at which time it rounded up and lost its apical complex. Although the refractile body underwent certain morphologic changes, it was retained throughout the parasite's growth. The beginning of mitosis was marked by the formation of a cytoplasmic funnel which traversed the nucleus opening on each side toward a pair of centrioles. Subsequently, there developed an intranuclear spindle. Separation of the daughter nuclei was preceded by the formation of typical centrocones. Differentiation of merozoites was accomplished by exogenesis during the last mitotic division. A dense fiber, interpreted as a link connecting the merozoite anlage with its nucleus, extended from the developing apical complex to the nearest division pole. In the anlage, the inner membrane complex was at first composed of patches associated with pairs of subpellicular microtubules. Rhoptries appeared early in merogenesis, whereas micronemes formed at the time the merozoites detached from the residuum. The level of amylopectin, low in schizonts, rose at the beginning of merozoite formation.     

PfRH2b specific monoclonal antibodies inhibit merozoite invasion

Erythrocyte invasion by merozoite is a multistep process involving multiple ligand–receptor interactions. The Plasmodium falciparum reticulocyte binding protein homologues (PfRHs) consists of five functional members. The differential expression of PfRHs has been linked to the utilization of different invasion pathways by the merozoites as well as a mechanism of immune evasion. PfRHs are expressed at the apical end of merozoite and form interactions with distinct red blood cell (RBC) surface receptors that are important for successful invasion. Here we show that PfRH2b undergoes processing before and during merozoite invasion. The different processed fragments bind to chymotrypsin sensitive RBC surface receptors. We also show that PfRH2b follows the merozoite tight junction during invasion. Monoclonal antibodies (mAbs) inhibit merozoites invasion by blocking tight junction formation. mAbs binding to PfRH2b block merozoites intracellular Ca²⁺ signal necessary for EBA175 surface expression. The data suggests that a conserved function of PfRHs, where their interaction with RBC surface receptors facilitated recruitment of EBA175 and other tight junction proteins necessary for merozoite invasion by modulating merozoite intracellular Ca²⁺ signals.     

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.     

Erythrocyte entry by malarial parasites. A moving junction between erythrocyte and parasite

Invasion of erythrocytes by merozoites of the monkey malaria, Plasmodium knowlesi, was investigated by electron microscopy. The apical end of the merozoite makes initial contact with the erythrocyte, creating a small depression in the erythrocyte membrane. The area of the erythrocyte membrane to which the merozoite is attached becomes thickened and forms a junction with the plasma membrane of the merozoite. As the merozoite enters the invagination in the erythrocyte surface, the junction, which is in the form of a circumferential zone of attachment between the erythrocyte and merozoite, moves along the confronted membranes to maintain its position at the orifice of the invagination. When entry is completed, the orifice closes behind the parasite in the fashion of an iris diaphragm, and the junction becomes a part of the parasitophorous vacuole. The movement of the junction during invasion is an important component of the mechanism by which the merozoite enters the erythrocyte. The extracellular merozoite is covered with a prominent surface coat. During invasion, this coat appears to be absent from the portion of the merozoite within the erythrocyte invagination, but the density of the surface coat outside the invagination (beyond the junction) is unaltered.