Effects of chloroquine on the feeding mechanism of the intraerythrocytic human malarial parasite Plasmodium falciparum

Ultrastructural investigations of P. falciparum cultivated in vitro in human erythrocytes revealed new features of the feeding mechanism of the parasite. Mature trophozoites and schizonts take up a portion of the host cytosol by endocytosis which is restricted to cytostomes and which involves the invagination of both parasitophorous and parasite membranes. The resulting endocytic vesicles, surrounded by two concentric membranes, migrate towards the central food vacuole membrane. The external membrane of the endocytic vesicles apposes that of the food vacuole, leading to the internalization of vesicles bounded by a single membrane into the vacuole space where they are rapidly degraded. We conclude from this sequence of events that endocytic vesicles fuse with the food vacuole. Treatment of infected cells with therapeutic concentrations of chloroquine inhibited the last step of the feeding process, i.e. vacuolar degradation. This was manifested by the accumulation within the vacuolar space of intact vesicles bounded by single membranes. The implications of these findings for the antimalarial activity of chloroquine are discussed.     

Microtubules, Microfilaments, and Membranes in Phagocytosis: Structure and Function of the Oral Apparatus of the Ciliate Climacostomum virens

Climacostomum virens uses oral membranelles to drive suspended food particles into its buccal cavity. The cavity leads to a buccal tube which extends into the cell by as much as half a cell length. The inner end of this tube is delimited by a haplokinety (two rows of basal bodies). Internal to this zone is the cytostome and cytopharynx where food vacuoles form. The buccal tube is encircled by a ring of fibrous material, the cytostomal cord, in the region of the cytostome immediately below the haplokinety. Ribbons of postciliary microtubules extend from the kinetosomes of the haplokinety, attach to the cytopharyngeal membrane, and pass under the cytostomal cord. They become broader and expand into the cytoplasm. Cytopharyngeal vesicles pass between the microtubular ribbons and fuse with the cytopharyngeal membrane to generate membrane for forming food vacuoles. The cytopharyngeal vesicles contain a material which is secreted into the forming food vacuoles. Ciliates continue to feed after incubation in a medium containing cycloheximide, indicating that they draw on a pre-existing pool of membrane when forming the food vacuole.     

Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum

A family of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole. To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II-GFP chimera. By taking advantage of green fluorescent protein fluorescence in live parasites, the ultrastructural resolution of immunoelectron microscopy, and inhibitors of trafficking and PM maturation, we have investigated the biosynthetic path leading to mature PM II in the food vacuole. Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.     

Acidification of the malaria parasite's digestive vacuole by a H+-ATPase and a H+-pyrophosphatase

As it grows within the human erythrocyte, the malaria parasite, Plasmodium falciparum, ingests the erythrocyte cytosol, depositing it via an endocytotic feeding mechanism in the "digestive vacuole," a specialized acidic organelle. The digestive vacuole is the site of hemoglobin degradation, the storage site for hemozoin (an inert biocrystal of toxic heme), the site of action of many antimalarial drugs, and the site of proteins known to be involved in antimalarial drug resistance. The acidic pH of this organelle is thought to play a critical role in its various functions; however, the mechanisms by which the pH within the vacuole is maintained are not well understood. In this study, we have used a combination of techniques to demonstrate the presence on the P. falciparum digestive vacuole membrane of two discrete H(+) pumping mechanisms, both capable of acidifying the vacuole interior. One is a V-type H(+)-ATPase, sensitive to concanamycin A and bafilomycin A(1). The other is a H(+)-pyrophosphatase, which was inhibited by NaF and showed a partial dependence on K(+). The operation of the H(+)-pyrophosphatase was dependent on the presence of a Mg(2+)-pyrophosphate complex, and kinetic experiments gave results consistent with free pyrophosphate acting as an inhibitor of the protein. The presence of the combination of a H(+)-ATPase and a H(+)-pyrophosphatase on the P. falciparum digestive vacuole is similar to the situation in the acidic tonoplasts (vacuoles) of plant cells.     

Effects of Chloroquine on the Feeding Mechanism of the Intraerythrocytic Human Malarial Parasite Plasmodium falciparum1

Ultrastructural investigations of P. falciparum cultivated in vitro in human erythrocytes revealed new features of the feeding mechanism of the parasite. Mature trophozoites and schizonts take up a portion of the host cytosol by endocytosis which is restricted to cytostomes and which involves the invagination of both parasitophorous and parasite membranes. The resulting endocytic vesicles, surrounded by two concentric membranes, migrate towards the central food vacuole membrane. The external membrane of the endocytic vesicles apposes that of the food vacuole, leading to the internalization of vesicles bounded by a single membrane into the vacuolar space where they are rapidly degraded. We conclude from this sequence of events that endocytic vesicles fuse with the food vacuole. Treatment of infected cells with therapeutic concentrations of chloroquine inhibited the last step of the feeding process, i.e. vacuolar degradation. This was manifested by the accumulation within the vacuolar space of intact vesicles bounded by single membranes. The implications of these findings for the antimalarial activity of chloroquine are discussed.     

Phagosome fusion vesicles of paramecium. I. Thin-section morphology

Ultrastructural studies of the digestive system of Paramecium caudatum focusing on the first 5 min of digestive-vacuole age reveal a set of vesicles, named phagosome fusion vesicles (PFVs), which fuse with the digestive vacuole just after the vacuoles are released from the cytopharynx and concomitant with vacuole acidification. Serial thin-sections of vacuoles labeled with horseradish peroxidase (HRP) and/or latex beads in pulse-chase studies were observed. PFVs, irregularly shaped, electron-translucent vesicles ranging from a small diameter to over 1 micro, are first seen in the region of the cytopharynx where they bind to the nascent vacuole membrane. Within 30 sec of vacuole release the PFVs fuse with the vacuole where they remain for a brief time connected to the vacuole by a narrow annulus. HRP-reaction product is found in vacuoles but not in PFVs before PFVs fuse with the vacuoles. After fusion with PFVs HRP is quickly inactivated. Tubular extensions of vacuole membrane then form between the fused PFVs. By 3 to 5 min both PFVs and tubules disappear from the vacuole surface and lysosomes appear in their place. We believe the tubules are pinched off as PFV membrane is being added to the vacuole. Microfilaments coat the membrane during all these dynamic events. Since the pH of the vacuole becomes acid during the first few minutes, we are now looking for a direct correlation between PFV fusion and acidification.     

Fine Structural Observations of the Erythrocytic Stages of Plasmodium chabaudi Landau, 1965*

SYNOPSIS. Electron microscopic examination of Plasmodium chabaudi in mouse erythrocytes revealed many characteristics resembling those observed in other mammalian malarial parasites. A double unit membrane surrounds the trophozoite cytoplasm which contains many ribonucleoprotein particles, a limited amount of endoplasmic reticulum and membraned organelles including sausage-shaped vacuoles and multilaminated membraned bodies. More or less circular double membraned vacuoles, possibly cross sections of the sausage-shaped vacuoles, are common. Typical protozoan mitochondria are lacking. The limiting membrane of the merozoites is triple-layered. Paired organelles and small dense bodies are found in the merozoites along with dense granular masses in the nuclei. Trophozoites have cytostomal structures as well as invaginations of the plasma membrane at sites where no cytostomes are evident. Digestion appears to occur in single membrane-bound vesicles which contain one to several pigment grains. P. chabaudi frequently contains multiple food vacuoles and has polymorphism manifested in part by the presence of cytoplasmic extensions and of nuclei with a variety of shapes. Several apparently free forms are noted, often accompanied by a thin rim of host cytoplasm. “Appliqué” forms are common among the trophozoites as are forms in which 2 or more trophozoites are joined together. Finally, alterations in the host cytoplasm resembling the socalled Maurer's clefts are frequent. Ferritin-containing vacuoles also appear in the host cell.     

Etude ultrastructurale comparée du processus de dégradation de l'hémoglobine par P. berghei (Vincke et Lips, 1948) en fonction de l'état de maturité de la cellule hǒte1

By serial sectioning and 3D reconstruction we have been able to demonstrate that the type of system for hemoglobin digestion in two strains of Plasmodium berghei, N and RC, is dependent on the maturity of the host cell. In parasites growing in erythrocytes, both systems for the endocytosis of hemoglobin—micropinocytosis and the cytostomal system (i.e. a cytostome budding a cytostomal tube that releases food vacuoles)—are fully functional and produce a great quantity of residual pigment. Parasites growing in reticulocytes have a disrupted cytostomal system; no tube is formed and only food vacuoles are visible in their cytoplasm. Residual pigment is smaller in size and in quantity. The reduced quantity of pigment in reticulocytes is explained by our observation of the exocytosis of pigment. We propose a hypothesis that relates the process of degradation of hemoglobin to the maturity of the host cell and a possible mechanism of protection against chloroquine, a drug known for its affinity for malarial pigment.     

Comparative ultrastructural study of the process of hemoglobin degradation by P. berghei (Vincke and Lips, 1948) as a function of the state of maturity of the host cell

By serial sectioning and 3D reconstruction we have been able to demonstrate that the type of system for hemoglobin digestion in two strains of Plasmodium berghei, N and RC, is dependent on the maturity of the host cell. In parasites growing in erythrocytes, both systems for the endocytosis of hemoglobin-micropinocytosis and the cytostomal system (i.e. a cytostome budding a cytostomal tube that releases food vacuoles)-are fully functional and produce a great quantity of residual pigment. Parasites growing in reticulocytes have a disrupted cytostomal system; no tube is formed and only food vacuoles are visible in their cytoplasm. Residual pigment is smaller in size and in quantity. The reduced quantity of pigment in reticulocytes is explained by our observation of the exocytosis of pigment. We propose a hypothesis that relates the process of degradation of hemoglobin to the maturity of the host cell and a possible mechanism of protection against chloroquine, a drug known for its affinity for malarial pigment.     

Targeting the plasmepsin 4 orthologs of Plasmodium sp. with "double drug" inhibitors

Plasmepsin 4 (PM4) is a digestive vacuole enzyme found in all Plasmodium species examined to date. While P. falciparum has three additional aspartic proteinases in its digestive vacuole in addition to plasmepsin 4, other Plasmodium species have only PM4 in their digestive vacuole. Therefore, PM4 may be a good target for the development of an antimalarial drug. This study presents data obtained with PM4s from several Plasmodium species. Low nanomolar K(i) values have been observed for all PM4s studied.     

Distribution of Acid Phosphatase in Paramecium caudatum: Its Relations with the Process of Digestion

SYNOPSIS. The distribution of acid phosphatase was investigated at the ultrastructural level in Paramecium caudatum. Acid phosphatase occurs in endoplasmic reticulum, Golgi apparatus, food vacuoles, autophagic vesicles, vacuolar and dense bodies. Some slight deposits are also seen in the mitochondria.
These observations point out that this hydrolase activity is related to digestive processes. The enzyme, originating from the endoplasmic reticulum and Golgi apparatus reaches the food vacuole or autophagic vesicle likely via the reticulum. The digestion of the bacteria or of the enclosed organelle gives rise to electronopaque material which is later found in dense bodies. These dense bodies are likely secondary lysosomes and it is possible that they may fuse with the young food vacuole or with autophagic vesicles.     

Phagosome fusion vesicles of Paramecium. II. Freeze-fracture evidence for membrane replacement

Phagosome fusion vesicles (PFVs), a new population of relatively large granules in Paramecium caudatum which fuse with the first stage of digestive vacuoles (DV-I) shortly after these vacuoles are released from the cytopharynx (their site of formation), have been studied by using the freeze-fracture technique. Identification of PFVs is possible in the resulting replicas at all sites where they are commonly found in thin sections, at the cytopharynx, bound but not fused with nascent digestive vacuoles and fused with released vacuoles in the cell's posterior end. These PFVs have membranes which do not resemble the membranes of the forming digestive vacuole membrane or the discoidal vesicle membranes from which vacuole membrane is derived. Their smooth E-fracture face with only 50 to 100 intramembrane particles (IMPs) per micrometers 2 and particulate P-face (approximately 2500 IMPs/micrometers) do resemble the second vacuole stage (DV-II) which is characterized by a smaller diameter and acid pH. Evidence is presented for PFV fusion with the DV-I and for membrane replacement, at least in part, as the DV-I becomes a DV-II. Membrane replacement entails first adding PFVs to the DV-I and then removing the original discoidal vesicle-derived membrane as tubules as the vacuole condenses. Implications of the possible role of PFVs in forming intravacuolar symbiotic relationships are also discussed.     

Paramecium Phagosome Membrane: From Oral Region to Cytoproct and Back Again1

Ten years of research on digestive vacuoles (phagosomes) of Paramecium caudatum have revealed sequential changes both within the vacuole lumen as well as within the surrounding membrane. Four vacuole stages can be recognized by a combination of thin section and freeze-fracture ultrastructural features. Three sets of vesicles (discoidal vesicles, acidosomes, and lysosomes) fuse with the vacuole, each at a predetermined stage, to bring about these membrane and physiological changes. At various times membrane is removed as vesicles from the vacuole surface, which has the effect of regulating vacuole size. Membrane recycling, membrane replacement, and specific membrane to membrane recognition all appear to be operating during the digestive cycle. Details of these events are summarized in this address and a number of unanswered questions suggest areas for future research.     

A cytochemical ultrastructural study of the lysosomal system of different species of malaria parasites

We have used ultrastructural techniques in different malarial species to demonstrate a lysosomal system. First, we have tried to localize acid phosphatase, a typical lysosomal label. Its activity was localized in the endoplasmic reticulum and in endocytic vesicles, and in dense-cored vesicles near the digestive vacuoles, especially in Plasmodium falciparum (FCR3 strain). Then, we have studied the different cellular compartments of the malarial parasite by the zinc iodide-osmium tetroxide technique that heavily contrasted the cellular compartments of the parasite. This experiment led to the observation of a profound rearrangement of the endoplasmic reticulum, especially in P. berghei. A very atypical but functional Golgi apparatus was demonstrated in all the growing stages of the parasite and lysosome-like vesicles were observed, showing a structure very similar to those of the coated vesicles of a true Golgi complex. The presence of these organelles are in favor of the existence of a lysosomal system and of the endogenicity of some enzymes involved hemoglobin degradation.     

Digestion in the peritrich ciliateOphrydium versatile

Summary Digestion in the peritrich ciliateOphrydium versatile O.F.M. involves a complex sequence of intracytotic and exocytotic membrane fusion and recycling events. Food particulates are concentrated in the lower cytopharynx which forms a fusiform-shaped food vacuole. Upon release from the cytopharynx, this food vacuole begins to condense, concentrating the food particulates. Excess membrane is removed intracytotically. These released membranes pieces form discoidal vesicles which are recycled to the base of the cytopharynx, thus providing additional membrane for subsequent food vacuole formation. In the condensed food vacuole, digestion proceeds; hydrolytic enzymes are delivered to the food vacuole via rough endoplasmic reticulum and/or by the cup-shaped coated vesicles (CSCV). As these vesicles fuse with the food vacuole, the food vacuole enlarges, digestion proceeds and an electron-dense membrane coat appears along the luminal surface of the food vacuole. Prior to defecation, the food vacuole undergoes a final condensation; irregularly-shaped, electron dense, single-membrane bound vesicles are cut-off intracytotically from the old food vacuole. These vesicles undergo condensation and invagination to form the cup-shaped coated vesicles (CSCV) which fuse with younger food vacuoles.     

Digestive system membranes: freeze-fracture evidence for differentiation and flow in Paramecium

Freeze-fractured membranes of digestive vacuoles of randomly feeding Paramecium caudatum exhibit dramatic differences in intramembrane particle (IMP) number and distribution on both E- and P-fracture faces. By pulse-feeding latex spheres to cells we have demonstrated that these differences are related to the age of the digestive vacuoles, and that the membranes of such vacuoles undergo a specific sequence of changes during the digestive cycle. Young digestive vacuoles (DV-I; less than or equal to 6 min), nascent vacuoles still connected to the cytopharynx, and discoidal vesicles, from which vacuole membrane is derived, all have a highly particulate E face and a less particulate P face. As early as 3 min after feeding, a second category of digestive vacuoles (DV-II) can be recognized, which are both considerably smaller in diameter and lack particles on their E face. These findings suggest that the endocytic removal of DV-I membrane material associated with the formation of DV-II vacuoles involves a concomitant and selective removal of E-face particles, as essentially no changes are seen in the density of P-face particles on the two types of vacuoles. Beginning at 10 min the first DV-III vacuoles are encountered. These are both larger than the DV-II vacuoles and possess very prominent E-face particles, which resemble those on the E face of the numerous lysosomes bordering the digestive vacuoles. DV-III vacuoles also exhibit a substantial increase in P-face particles. These membrane changes closely parallel, and are probably correlated with, the physiological events occurring within the vacuole lumen: concentration of food, killing of prey, and digestion. Calculations of the amount of membrane removed from DV-I to form DV-II and of the increase in membrane surface area during the transition from DV-II to DV-III indicate that as much as 90% of the initial phagosome (DV-I) membrane can be removed before digestion begins. The enlargment of DV-II must be caused by fusion with adjacent lysosomes which also contribute the new populations of IMPs to the DV- III membrane. The appearance of numerous endocytic structures on older DV-III vacuoles suggests that membrane is retrieved from DV-III before defecation.     

A Cytochemical Ultrastructural Study of the Lysosomal System of Different Species of Malaria Parasites

ABSTRACT. We have used ultrastructural techniques in different malarial species to demonstrate a lysosomal system. First, we have tried to localize acid phosphatase, a typical lysosomal label. Its activity was localized in the endoplasmic reticulum and in endocytic vesicles, and in dense-cored vesicles near the digestive vacuoles, especially in Plasmodium falciparum (FCR₃ strain). Then, we have studied the different cellular compartments of the malarial parasite by the zinc iodide-osmium tetroxide technique that heavily contrasted the cellular compartments of the parasite. This experiment led to the observation of a profound rearrangement of the endoplasmic reticulum, especially in P. berghei. A very atypical but functional Golgi apparatus was demonstrated in all the growing stages of the parasite and lysosome-like vesicles were observed, showing a structure very similar to those of the coated vesicles of a true Golgi complex. The presence of these organelles are in favor of the existence of a lysosomal system and of the endogenicity of some enzymes involved hemoglobin degradation.     

Timing of perialgal vacuole membrane differentiation from digestive vacuole membrane in infection of symbiotic algae Chlorella vulgaris of the ciliate Paramecium bursaria

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomori's staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomori's thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomori's staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella.     

Evaluation of pH during cytostomal endocytosis and vacuolar catabolism of haemoglobin in Plasmodium falciparum

The DV (digestive vacuole) of the malaria parasite, Plasmodium falciparum, is the site of Hb (haemoglobin) digestion and haem detoxification and, as a consequence, the site of action of CQ (chloroquine) and related antimalarials. However, the precise pH of the DV and the endocytic vesicles that feed it has proved difficult to ascertain. We have developed new methods using EGFP [enhanced GFP (green fluorescent protein)] to measure the pH of intracellular compartments. We have generated a series of transfectants in CQ-sensitive and -resistant parasite strains expressing GFP chimaeras of the DV haemoglobinase, plasmepsin II. Using a quantitative flow cytometric assay, the DV pH was determined to be 5.4-5.5. No differences were detected between CQ-sensitive and -resistant strains. We have also developed a method that relies on the pH dependence of GFP photobleaching kinetics to estimate the pH of the DV compartment. This method gives a pH estimate consistent with the intensity-based measurement. Accumulation of the pH-sensitive probe, LysoSensor Blue, in the DV confirms the acidity of this compartment and shows that the cytostomal vesicles are not measurably acidic, indicating that they are unlikely to be the site of Hb digestion or the site of CQ accumulation. We show that a GFP probe located outside the DV reports a pH value close to neutral. The transfectants and methods that we have developed represent useful tools for investigating the pH of GFP-containing compartments and should be of general use in other systems.     

宁夏枸杞柱头和萌发花粉中钙分布特征

用焦锑酸钾沉淀法对宁夏枸杞柱头和花粉中的钙离子分布进行了研究.结果显示,宁夏枸杞柱头表皮有一覆盖层,其中有许多含钙沉淀颗粒的小泡,当花粉落到柱头后从覆盖层中吸水,在萌发孔的表面上聚集了较多的钙沉淀颗粒.同时,花粉内部出现许多含钙的小液泡,使花粉体积增大,内部产生膨压,花粉萌发;生长在覆盖层中的花粉管顶端穿过覆盖层小泡时,附近聚集了较多的钙沉淀颗粒,在花粉管壁上也附着较多的细小钙沉淀颗粒.萌发的花粉粒中由大液泡占据,在其亚顶端的细胞质中,聚集较多钙沉淀颗粒的线粒体膨大形成了一些含钙沉淀颗粒的小液泡,由这些小液泡融合形成的大液泡,将花粉管细胞质挤到其顶端,使其极性生长.这是首次发现在植物柱头覆盖层中有钙离子的现象,从体内证明了钙离子在花粉萌发过程中的现象.讨论了枸杞柱头组织中钙的分布和花粉管的萌发与生长的关系.