Parasitophorous Duct Research Articles

Plug for the parasitophorous duct: a solution of two conundra

BackgroundWe present two conundra in the biology of intraerythrocytic malaria parasite: how an apparent open parasitophorous duct provide direct access of only a select set of serum proteins to the parasitophorous vacuole, and how proteases mediate membrane lysis to allow merozoite egress.SolutionWe posit the existence of a parasitophorous vacuolar duct plug that is originally formed from a tight junction (or parts thereof) between merozoite apical surface and red blood cell plasma membrane, which by moving over the parasite surface towards the posterior end draws the parasite into the host cell interior, and by remaining at the passage orifice provides a location of transporter(s) for import of serum proteins into parasitophorous vacuole and an opening for merozoite egress upon its dissolution/dismantling through protease(s) action.ConclusionThis notion obviates the need of a distinct intact parasitophorous vacuolar membrane, which in the proposed model is an extension of the red blood cell membrane but still forms an intracellular compartment for parasite growth and development. The model is testable using existing high-resolution electron and X-ray tomography tools.

Intracellular calcium and pH conditions of cultured cells infected with Eimeria bovis or E. separata.

Loading of Eimeria bovis-infected Vero cells with membrane-permeant acetoxymethyl esters (AM-esters) of ion-sensitive dyes provided us with a noninvasive method for investigation of the permeability of the parasitophorous vacuole membrane (PVM) and simultaneous measurement of Ca2+ and H+ concentrations in different compartments of the infected cells. The distribution patterns of the cleaved membrane-impermeant dyes argue against the existence of nonselective pores in the PVM. There is also no indication of a parasitophorous duct connecting the vacuolar space with extracellular media. The pH inside the parasitophorous vacuole (PV) was lower than that in the cytoplasm of the host cell or the parasite, whereas the [Ca2+] in these compartments did not differ significantly. In HT29 cells infected with E. separata for 24 h the Ca2+ response to extracellular adenosine triphosphate (ATP) was significantly reduced, indicating influences on the host cell's intracellular signaling.

Transport and trafficking in malaria-infected erythrocytes

D espite almost a century of effort aimed at eradication, malaria remains one of the world's major health problems. The disease is caused by the eukaryotic apicomplexan parasite Plasrnodium spp. The most virulent of the four species known to infect humans is Plasmodium falciparum, and we will focus on this species alone. The pathology of the disease results from the multiplication of asexual parasites in red blood cells (RBCs) and the sequestration of these infected cells in the organs of the vertebrate host. The metabolic processes leading to parasite maturation at this stage of the life cycle are poorly understood. There have been many recent studies on the mechanisms of uptake of metabolites in vitro by asexual parasites, the most recent of which is by Haldar's group I and attempts to characterize an uptake route that could constitute a target for either chemotherapy or drug delivery. Asexual stages of the parasite mature in RBCs within a parasitophorous vacuole (PV) formed during merozoite invasion. It has long been recognized that the parasite imports host cell haemoglobin in a macropinocytotic process involving an invagination of the parasite plasmalemma with host cell cytoplasm at a specialized structure termed the micropore 2 (Fig. 1). However, not all nutrients and growth factors necessary for parasite maturation are provided by the digestion of haemoglobin: in addition, several exogenous substrates, including glucose, assorted dicarboxylic acids, purines and specific amino acids 3,4 that are essential for parasite maturation, are imported from the extracellular milieu 3,s. Thus, although the host RBC provides a protected environment, the intracellular location of the parasite requires imported and exported solutes to cross a series of three unit membranes: the RBC plasmalemma, the PV membrane and the parasite plasmalemma. The mechanisms by which malaria parasites obtain nutrients have been the subject of much controversy and debate during the past few years. There have been reports of three potential mechanisms for uptake: (1) by parasite-induced permeability changes in the RBC plasmalemma (for reviews, see Refs 3,6); (2) by a direct connection known as a parasitophorous duct between the parasite, and the external milieu7,8; and (3) by the involvement of a tubovesicular membrane (TVM) network in directed transport across the RBC cytoplasm *,3,9. Parasiteinduced permeability changes in RBC plasma membranes, which occur 12-20 h postinvasion, have been described by several groups (reviewed in Refs 3,6). The existence and possible role of the parasitophorous duct is still controversial and lacks independent corroboration. Indeed, various ukrastructural studies using electron-dense tracers and labelling techniques ~°,~1 have so far failed to find any evidence for a direct connection between the exterior of the RBC and any of the intracellular compartments. Even the most recent paper by the Taraschi group 12, which claims to provide ultrastructural evidence for the presence of the duct, does not actually give illustrations showing membrane continuity between the TVIVl and the RBC plasmalemma (as the

Presence of the parasitophorous duct in Plasmodium falciparum and P. vivax parasitized Saimiri monkey red blood cells

Although the exchange of metabolites between the intraerythrocytic malaria parasite and the external medium has been studied extensively, the transport of molecules across the erythrocyte cytoplasmic membrane and cytoplasm and the parasitophorous vacuolar membrane needs to be investigated more fully to be completely understood. Recently, the concept of the parasitophorous duct, establishing a continuity between the environment and the vacuolar space surrounding the intraerythrocytic parasite, has been suggested to provide an explanation of how macromolecules can cross two membranes in a cell devoid of an endocytic system. This concept is highly controversial and has been suspected to be an in vitro artefact. In this article, Bruno Pouvelle and Jürg Gysin present evidence of the existence of the parasitophorous duct in Saimiri monkey Plasmodium falciparum- and P. vivax-infected erythrocytes, with a series of ex vivo experiments showing stage and species dependent variations of the characteristics of this structure.

Characterization of macromolecular transport pathways in malaria-infected erythrocytes

We have previously provided evidence for a pathway in Plasmodium falciparum-infected erythrocytes, coined the parasitophorous duct pathway, which provides serum (macro)molecules direct access to intraerythrocytic parasites [1]. The present study addresses the purity of the fluorescent macromolecules used to define the duct pathway and provides ultrastructural evidence for its presence. The fluorescent tracers used to characterize transport remain intact during their incubation with infected erythrocytes. Transport of macromolecules in the external medium or host cell cytosol to the intracellular parasites is shown to occur by two distinct pathways. Fluorescent dextrans in the erythrocyte cytosol are ingested by the parasite via a specialized organelle, the cytostome, and are transported to the parasite food vacuole. Transport through this pathway occurs throughout the asexual life cycle. By contrast, fluorescent dextrans in the external medium bypass the erythrocyte cytosol, and are internalized by the parasite by a process resembling fluid-phase endocytosis. Serial sections of mature parasites fixed and stained by various methods for transmission electron microscopy reveal areas of apparent membrane continuity between the erythrocyte membrane and the parasitophorous vacuolar membrane that surrounds the parasite, that could leave the parasites exposed to the external medium. Using carboxylate and amidine-modified fluorescent latex spheres and laser scanning confocal microscopy, macromolecules up to 50–70 nm in diameter are found to have direct access to intraerythrocytic parasites. This size exclusion is consistent with the dimensions of the parasitophorous duct pathway revealed by electron microscopy. This investigation reports for the first time the existence of two, distinct macromolecular transport pathways in malaria-infected erythrocytes.

Efflux of 6-deoxy- d-glucose from Plasmodium falciparum-infected erythrocytes via two saturable carriers

Glucose transport in human erythrocytes infected with the malaria parasite, Plasmodium falciparum, has been studied using 6-deoxy- d-glucose (6DOG) as a non-metabolised glucose analogue. Inhibition studies using cytochalasin B, a powerful inhibitor of the erythrocyte glucose transporter, GLUT1, indicate that in the infected red blood cell (IRBC), glucose is transported via a saturable carrier. However, inhibition is not as complete as in the uninfected erythrocyte. The synergistic inhibition effect of 6DOG entry by niflumic acid, an inhibitor of the non-specific malaria-induced pore, in the presence of cytochalasin B suggests that some glucose may also enter the infected erythrocytes through the pore, if entry via the carrier is blocked. The time course of 6DOG efflux from infected erythrocytes in the presence of cytochalasin B did not follow simple first-order kinetics. To elucidate the kinetic mechanism of 6DOG efflux from the infected erythrocytes, the concentration dependence of efflux was determined. Eight two-compartment kinetic models were simulated, involving first-order pore diffusion and carrier-mediated saturable diffusion in two systems, one ductless and one assuming the existence of a parasitophorous duct. The only two models showing reasonable fits to the efflux data each involve two saturable carriers. It is likely that one of the saturable carriers is associated with the parasite itself. Evidence that the parasite carrier has different inhibitor sensitivities from that of GLUT1 is presented.

Characterization of the Enhanced Transport of L- and D-Lactate into Human Red Blood Cells Infected with Plasmodium falciparum Suggests the Presence of a Novel Saturable Lactate Proton Cotransporter

Human erythrocytes parasitized with the malarial protozoan Plasmodium falciparum showed rates of L-lactate, D-lactate, and pyruvate uptake many fold greater than control cells. Thus it was necessary to work at 0 degrees C to resolve true initial rates of transport. Studies on the dependence of the rate of transport on substrate concentration implied the presence in parasitized cells of both a saturable mechanism blocked by alpha-cyano-4-hydroxycinnamate (CHC) and a nonsaturable mechanism insensitive to CHC. The former was dominant at physiological substrate concentrations with Km values for pyruvate and D-lactate of 2.3 and 5.2 mM, respectively, with no stereoselectivity for L- over D-lactate. CHC was significantly less effective as an inhibitor of lactate transport in parasitized erythrocytes than in uninfected cells, whereas p-chloromercuribenzenesulfonate, a potent inhibitor in control cells, gave little or no inhibition of lactate transport into parasitized erythrocytes. Inhibition of transport into infected cells was also observed with phloretin, furosemide, niflumic acid, stilbenedisulfonate derivatives, and 5-nitro-2-(3-phenylpropylamino)benzoic acid at concentrations similar to those that inhibit the lactate carrier of control erythrocytes. These compounds were more effective inhibitors of the rapid transport of chloride into infected cells than of lactate transport, whereas CHC was more effective against lactate transport. This implies that different pathways are involved in the parasite-induced transport pathways for lactate and chloride. The transport of L-lactate into infected erythrocytes was also inhibited by D-lactate, pyruvate, 2-oxobutyrate, and 2-hydroxybutyrate. The intracellular accumulation of L-lactate at equilibrium was dependent on the transmembrane pH gradient, suggesting a protogenic transport mechanism. Our data are consistent with lactate and pyruvate having direct access to the malarial parasite, perhaps via the proposed parasitophorous duct or some close contact between the host cell and parasite plasma membranes, with transport across the latter by both a proton-linked carrier (CHC-sensitive, saturable, and the major route) and free diffusion of the undissociated acid (CHC-insensitive, unsaturable, and a minor route).

Parasitophorous duct? still more questions than answers

Parasitophorous duct? still more questions than answers

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We have previously provided evidence for a pathway in Plasmodium falciparum-infected erythrocytes, coined the parasitophorous duct pathway, which provides serum (macro)molecules direct access to intraerythrocytic parasites [1]. The present study addresses the purity of the fluorescent macromolecules used to define the duct pathway and provides ultrastructural evidence for its presence. The fluorescent tracers used to characterize transport remain intact during their incubation with infected erythrocytes. Transport of macromolecules in the external medium or host cell cytosol to the intracellular parasites is shown to occur by two distinct pathways. Fluorescent dextrans in the erythrocyte cytosol are ingested by the parasite via a specialized organelle, the cytostome, and are transported to the parasite food vacuole. Transport through this pathway occurs throughout the asexual life cycle. By contrast, fluorescent dextrans in the external medium bypass the erythrocyte cytosol, and are internalized by the parasite by a process resembling fluid-phase endocytosis. Serial sections of mature parasites fixed and stained by various methods for transmission electron microscopy reveal areas of apparent membrane continuity between the erythrocyte membrane and the parasitophorous vacuolar membrane that surrounds the parasite, that could leave the parasites exposed to the external medium. Using carboxylate and amidine-modified fluorescent latex spheres and laser scanning confocal microscopy, macromolecules up to 50–70 nm in diameter are found to have direct access to intraerythrocytic parasites. This size exclusion is consistent with the dimensions of the parasitophorous duct pathway revealed by electron microscopy. This investigation reports for the first time the existence of two, distinct macromolecular transport pathways in malaria-infected erythrocytes.

The parasitophorous duct pathway: new opportunities for antimalarial drug and vaccine development.

The parasitophorous duct pathway: new opportunities for antimalarial drug and vaccine development.

Ultrastructural evidence for a parasitophorous duct in human erythrocytes infected with malaria parasites

The human malaria parasite, Plasmodium falciparum, spends all but a few minutes of its 48 hour asexual life cycle in host erythrocytes (RBC). Models of the host-parasite complex, obtained by transmission and freeze-fracture electron microscopy, suggest that the intraerythrocytic parasite is contained in a continous, sealed vacuole (PVM). The parasite has an obligate nutritional requirement for serum fatty acids, lipids and proteins. Since mature erythrocytes do not endocytose, a long standing enigma has been the mechanism by which intracellular parasites obtain access to these (macro)molecules. Using a variety of fluorescent labeled macromolecules and confocal fluorescence microscopy (CFIM), we demonstrated that macromolecules in serum did not cross the erythrocyte or PVM, but rather gained direct access to the aqueous space surrounding the parasite through a parasitophorous duct. Being limited by the resolution of the light microscope, we initiated TEM investigations to identify the duct at the ultrastructural level.

A parasitophorous duct in Plasmodium-infected red blood cells

A parasitophorous duct in Plasmodium-infected red blood cells

A parasitophorous duct in Plasmodium-infected red blood cells — Reply

A parasitophorous duct in Plasmodium-infected red blood cells — Reply

Direct access to serum macromolecules by intraerythrocytic malaria parasites

Trafficking pathways in malaria-infected erythrocytes are complex because the internal parasite is separated from the serum by the erythrocyte and parasitophorous vacuolar membranes. Intraerythrocytic Plasmodium falciparum parasites can endocytose dextrans, protein A and an IgG2a antibody. Here we show that these macromolecules do not cross the erythrocyte or parasitophorous vacuolar membranes, but rather gain direct access to the aqueous space surrounding the parasite through a parasitophorous duct. Evidence for this structure includes visualization of membranes that are continuous between the parasitophorous vacuolar and erythrocyte membranes, and surface labelling of the parasite with fluorescent macromolecules under conditions that block endocytosis. The parasite can internalize by fluid-phase endocytosis macromolecules from the aqueous compartment surrounding it. Thus, surface antigens on trophozoites and schizonts should be considered as targets for antibody-directed parasiticidal agents.