A parasitophorous duct in Plasmodium-infected red blood cells.

One of the more vexing problems confronting cell biologists who study the erythrocytic stages of malaria is: how does a parasite living within a metabolically sluggish erythocyte and enclosed by a host cell membrane with low-velocity transport systems, as well as a parasitophorous vacuolar membrane, obtain the large quantities of materials essential for its successful reproduction? And, what are the pathways whereby plasmodial products, particularly proteins, reach their final destination? The experiments reported by Pouvelle et al. ~ would appear to provide us with some answers. Plusmodium falcipdrum-infected red blood cells incubated at 37°C or 4°C, either with positively charged fluoresceindextrans ( 10 or 70 kDa) or rhodamineprotein A (42kDa), and then examined by confocal fluorescence imaging microscopy, showed labelled intracellular parasites. The fluorescence of prelabelled (with fluoresceinor rhodamine-protein A) malaria-infected red blood cells was almost completely quenched when cells were exposed to anti-fluorescein and anti-rhodamine IgG, respectively, but only when cells had been pre-labelled at 4°C, not 37°C. This suggested involvement of only external membranes, ie. red blood cell and parasitophorous vacuolar and parasite plasma membranes, in the macromolecular uptake process. To identify which membranes in the red blood cell cytoplasm were involved in the pathway, cells were incubated either at 4°C or 37°C with C6NBD-phospholipids or fluorescently labelled latex beads (30 nm) and then the cells were examined for the presence of label. Fluorescence was found associated with tubular extensions bridging the red blood cell and parasitophorous vacuolar membrane. The subsequent fate of the molecules or particles was tracked using confocal fluorescence microscopy after normal or ATP-depleted malariainfected cells were exposed to rhodamine-dextran. Uptake into the parasite was blocked by energy depletion, suggesting parasite acquisition by endocytosis. When parasites were preloaded with rhodamine-dextran at 37°C, label was released from infected red blood cells only when they were not ATP-depleted, indicating plasmodial exocytosis. Pouvelle et al. concluded that these findings provide evidence for the presence of a parasitophorous duct connecting the vacuole surrounding the parasite with the outside of the erythrocyte. 'This system would provide the parasite with an energy-efficient pathway to obtain nutrients and proteinbound fatty acids and lipids from serum.' And, since IgG appeared to traverse the duct, they suggest this could explain how human immune serum may adversely affect the intracellular parasite. Whether the duct represents an invagination of the membrane that persists from the time of merozoite invasion, or is a membranebound channel formed later during parasite development, was not determined. According to the model proposed by Pouvelle et al., trafficking in the channel would be unidirectional: passive diffusional entry into the malaria-infected red blood cell through a parasitophorous duct, movement into the parasitophorous vacuole, molecular uptake by parasite endocytosis and plasmodial product export via exocytosis. The work of Pouvelle et al. is supported, in part, by Grellier et al. 2, who found a stage-dependent, nonendocytotic, unidirectional transfer of high-density lipoprotein (HDL) from the external medium to the intraerythrocytic parasite. They also showed that HDL was associated with 'numerous vesicles (0.21.0F~m diameter) and tubular structures. . , in the erythrocyte cytoplasm. Fluorescent video-microscopy showed movement of the vesicles between the parasitophorous vacuole and the erythrocyte membrane and a flapping movement of tubular structures.' Similar vesicles were observed previously by Elford et ol. 3 when they exposed infected cells to the fluorescent dyes merocyanine and hypericin, although fusion of the vesicles with the red blood cell membrane was never seen. Earlier work on membrane transport in Plasmodium-infected red blood ceils. has described an uptake pathway that involves 0.7 nm pores and a recent reevaluation of the data 4 suggested involvement of non-Stokesian diffusion, with molecules crossing the erythrocyte membrane by diffusion through 'a soft polymer-like network formed by the membrane's hydrocarbon chains'. Based both on consideration of size and properties, this is unlikely to be the portal into the parasitophorous duct. Although the work of Pouvelle et al. did show that several intracellular structures of the P. falciparum-infected red blood cell bound fluorescent labels, in our view, their data do not provide incontrovertible evidence for the existence of a continuous phospholipidlined conduit that links the outside of the red blood cell with the parasitophorous vacuole. Pouvelle et al. did not provide a characterization of the fluorescent labels they used and this may be of critical importance in the interpretation of their confocal microscopic images. For example, a common problem with commercially obtained fluorescent probe conjugates is the instability of the conjugate and the dissociation of the label. Were this to occur, then the presence of free fluorescent label would affect their results since the small hydrophobic fluorescein and rhodamine molecules can easily penetrate the cell membrane and then be redistributed in intracellular membranous structures. Altered regions of the red blood cell membrane, with large concentrations of membranous vesicles and sac-like dilatations, were described more than 20 years ago in P. bergheiand P. falciparum-infected erythrocytes s. Indeed, by transmission electron microscopy, membranous vesicles in the red blood cell cytoplasm of malaria-infected cells have been observed in all species of Plasmodium so far examined 6-8. In P. falciparuminfected red blood cells, after fixation and staining, these vesicles can be seen at the level of the light microscope and are called Maurer's clefts. Since there are no dynamic studies to show whether Maurer's clefts are involved in red blood cell import or export (or are they functionless?), the claim by