Enhanced uptake and metabolism of riboflavin in erythrocytes infected with Plasmodium falciparum

Riboflavin deficiency inhibits the growth of malaria parasites both in vitro and in vivo in infected animals and humans. Although the precise mechanisms underlying this inhibition are unknown, they may involve enhanced requirements for riboflavin by parasites. To investigate this possibility, the rate of uptake of [14C]riboflavin and the biosynthesis of FMN and FAD from riboflavin were studied in infected (5-8% parasitemia) and uninfected human erythrocytes. All cells were incubated for 0-3 h at 37 degrees C in phosphate buffered saline containing MgCl2, glucose, and [14C]riboflavin (2.5-7.5 microM). At hourly intervals, samples were removed, centrifuged, washed twice with cold buffer, and lysed before counting the radioactivity. The rate of in vitro biosynthesis of FMN and FAD from riboflavin in erythrocytes was measured by ion exchange chromatography and reverse isotope dilution techniques. Results showed that the rate of riboflavin uptake and the biosynthesis of FMN and FAD were enhanced in erythrocytes with parasitemia as compared with results in unparasitized erythrocytes. Riboflavin uptake in erythrocytes was proportional to the extent of parasitemia and especially to percent of schizonts present in erythrocytes. These studies indicate that the requirement for riboflavin may be greater in the parasite than in the host erythrocyte. This increased riboflavin requirement may be due to rapid multiplication, higher metabolic rate, and extreme vulnerability to oxidative stress of malaria parasites compared with that of host erythrocytes. The differential requirement of riboflavin by the host and the malaria parasite may hold important potential for developing new strategies for malaria chemotherapy.     

Structural alteration of the membrane of erythrocytes infected with Plasmodium falciparum

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30-40 nm in height and 90-100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.     

14C-desferrioxamine B: uptake into erythrocytes infected with Plasmodium falciparum

Plasmodium falciparum-infected human erythrocytes (early trophozoite stages) and non-infected erythrocytes were incubated in 1.7 mM 14C-desferrioxamine B (specific activity 1 microCi/2.6 mg desferrioxamine B). After 270 min the cells were washed and the radioactivity was measured in the cell pellet and, after lysis, in cytoplasm and membranes. The results indicate that Desferrioxamine B can the red blood cell and pass through the parasite membrane and that the parasites are killed by the intracellular action of the chelator.     

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.     

Chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of Plasmodium falciparum infected erythrocytes.

BACKGROUND: Chondroitin-4-sulfate (CSA) was recently described as a Plasmodium falciparum cytoadherence receptor present on Saimiri brain microvascular and human lung endothelial cells. MATERIALS AND METHODS: To specifically study chondroitin-4-sulfate-mediated cytoadherence, a parasite population was selected through panning of the Palo-Alto (FUP) 1 P. falciparum isolate on monolayers of Saimiri brain microvascular endothelial cells (SBEC). Immunofluorescence showed this SBEC cell line to be unique for its expression of CSA-proteoglycans, namely CD44 and thrombomodulin, in the absence of CD36 and ICAM-1. RESULTS: The selected parasite population was used to monitor cytoadherence inhibition/dissociating activities in Saimiri sera collected at different times after intramuscular injection of 50 mg CSA/kg of body weight. Serum inhibitory activity was detectable 30 min after injection and persisted for 8 hr. Furthermore, when chondroitin-4-sulfate was injected into monkeys infected with Palo-Alto (FUP) 1 P. falciparum, erythrocytes containing P. falciparum mature forms were released into the circulation. The cytoadherence phenotype of circulating infected red blood cells (IRBC) was determined before and 8 hr after inoculation of CSA. Before inoculation, in vitro cytoadherence of IRBCs was not inhibited by CSA. In contrast, in vitro cytoadherence of circulating infected erythrocytes obtained 8 hr after CSA inoculation was inhibited by more than 90% by CSA. CONCLUSIONS: In the squirrel monkey model for infection with P. falciparum, chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of parasitized erythrocytes.     

Characteristics of 86Rb+ transport in human erythrocytes infected with Plasmodium falciparum

Human red cells infected in vitro with Plasmodium falciparum showed a significant increase in the rate of both ouabain-sensitive and ouabain-insensitive 86Rb+ influx. The increase in ouabain-insensitive 86Rb+ influx was due, in part, to increased transport via a bumetanide-sensitive system and, in part to transport via a pathway that was absent (or at least inactive) in uninfected cells. The parasite-induced pathway was inhibited by piperine and had a dose response very similar to that of the Gardos channel of uninfected cells but was less sensitive than the Gardos channel to inhibition by quinine.     

Refractoriness of erythrocytes infected with Plasmodium falciparum gametocytes to lysis by sorbitol

Unlike erythrocytes infected with mature asexual parasites of Plasmodium falciparum, those infected with gametoeytes are not lysed by 5% sorbitol solutions. This observation was used to devise a method for producing synchronized cultures of gametocytes, free of asexual stage parasites. The refractoriness to sorbitol suggests that the major anion transport pathway, which appears in the membrane of erythrocytes infected with asexual stage parasites, is not present in cells infected with gametocytes.     

Electron microscopic observations on tracheal epithelia of mice infected with Bordetella bronchiseptica

To clarify the pathogenesis of Bordetella in vivo infection, the tracheal epithelia of mice were examined in detail by electron microscopy at various intervals after intranasal inoculation with graded doses of phase I Bordetella bronchiseptica. In mice infected with a lethal dose (6 to 7 x 10(7) CFU), a remarkable rupture of the cell membranes of cilia and microvilli of the middle trachea was found on day I postinfection. The rupture of the membrane was observed over the entire tracheal epithelia, on day 2 after infection. The affected cilia were constricted at the transitional region and were broken off. In the ciliated cells the adherence of organisms to ciliary apexes and colonization in the interciliary spaces were also remarkable. In both the ciliated and nonciliated epithelial cells, the cytoplasmic vacuolation and pyknosis or karyorrehexis were also notable. In mice infected with one-tenth of the lethal dose, similar findings were seen, but appeared more slowly and the bacteria were not seen attaching to ciliary apexes. In mice receiving one-hundredth of the lethal dose, only mild cilial abnormality such as aggregation of cilia, and slight cytoplasmic vacuolation were found 6 days postinfection. Based on these findings, a possible mechanism of the ciliary damages produced by B. bronchiseptica was postulated.     

Uptake of L-tryptophan by erythrocytes infected with malaria parasites (Plasmodium falciparum)

The initial rates of uptake of L-tryptophan into normal human red blood cells and into cells infected by the malarial parasite Plasmodium falciparum in vitro, were investigated. We find that transport in non-infected cells, which is mediated by the specific saturable T system and the apparently non-saturable L system (Rosenberg, Young and Ellory (1980) Biochim. Biophys. Acta 598, 375-384) is considerably enhanced by blood preservation and culture conditions. This increase is mostly due to an increase in the maximal velocity of the saturable component and of the rate constant of the linear component. Uptake is further enhanced in non-infected cells by factors released from infected cells into the culture medium and, even more so, in infected cells at the advanced stage of intraerythrocytic parasite development. At these stages the susceptibility of the transport system to the non-specific inhibitor phloretin and to the competitive inhibitor phenylalanine, is virtually lost. The effect of the parasite on L-tryptophan uptake by the host cell membrane is exerted only on the maximal velocity of the T system, which is carrying most of the substrate under physiological conditions. The possible implications of these findings to the life of the intraerythrocytic parasite are briefly discussed.     

Plasmodium falciparum: fine structural changes in the cytoskeletons of infected erythrocytes

Following parasitization by Plasmodium falciparum, numerous changes take place in the host erythrocyte membrane. In this study, we used the technique of whole cell mount electron microscopy to determine if the ultrastructure of the erythrocyte cytoskeleton changed following parasitization with knobby and knobless strains of P. falciparum. Using this technique, a network of spectrin filaments (3-10 X 45-120 nm) branching from electron dense junctions (15-25 nm in diameter), the presumed site of bands 4.1 and actin, were visualized. The overall architecture of normal and parasitized erythrocyte cytoskeletons was the same: however, additional patches (35 to 60 nm in size) and aggregates (30 X 150 nm) of electron dense material were present in parasitized skeletons. The ultrastructure of knobby and knobless cytoskeletons was similar, except knobless skeletons usually did not possess the larger aggregates of material. Antigens associated with the erythrocyte cytoskeleton of cells infected with knobby and knobless strains, but not uninfected cells, were demonstrated by indirect immunofluorescence. Results suggest that antigens, associated with the erythrocyte cytoskeleton, may contribute to perturbations in the host erythrocyte membrane.     

Ultrastructural observations on spermatozoa retained within the seminiferous epithelium after treatment with dibutyryl cyclic AMP

Spermiation was inhibited in the Syrian hamster by administering large doses of dibutyryl cyclic AMP. After treatment with dibutyryl cyclic AMP most stage VIII, IX, and X seminiferous tubules contained some mature spermatozoa within the seminiferous epithelium. The acrosomal membranes and plasma membranes of the unreleased spermatozoa remained intact, indicating that the spermatozoa had not been phagocytized by the Sertoli cells. Sertoli-spermatid junctional specializations were usually applied to the heads of the mature spermatozoa. The unreleased spermatozoa often appeared swollen with accumulated fluid located in the subacrosomal space. The accumulation of subacrosomal fluid in the unreleased spermatozoa seems to result from the absence of tubulobulbar complexes. That is, when tubulobular complexes fail to form the normal flow of cytoplasm into the tubulobular complexes is blocked resulting in an accumulation of fluid around the nucleus. Inhibition of spermiation may result from the absence of tubulobulbar complex formation. It is postulated that the tubulobulbar complex functions to transfer a chemical trigger from the maturing spermatid into the Sertoli cell. This chemical trigger may initiate the disappearance of the Sertoli-spermatid junctional specialization and induce spermiation.     

Pfl I-I and Pf332: two giant proteins synthesized in erythrocytes infected with Plasmodium falciparum

Although the malaria parasite develops within erythrocytes, it has to modify the surrounding red blood cell membrane for its intracellular survival and maturation. These changes include the translocation of proteins across the parasite and the parasitophorous vacuole membranes to the host membrane. In this review, Denise Mattei, Katherine Hinterberg and Artur Scherf focus on two distinct giant parasite molecules of unprecedented size (approximately one MDa), called Pf332 and PflI-I, that are synthesized and exported into the cytoplasm of the host cell in the asexual and sexual blood stages of Plasmodium falciparum, respectively. The corresponding genes are located in genetically unstable subtelomeric chromosome regions.     

Stage-specific inhibitory effect of cyclic AMP on asexual maturation and gametocyte formation of Plasmodium falciparum

The effect of cyclic AMP on asexual maturation and gametocyte formation of Plasmodium falciparum grown in vitro was examined over a wide range of concentrations. Cyclic AMP inhibited both processes in a stage-specific manner. Asexual maturation was inhibited from shortly after parasite entry into the red cell through the ring stage. However, trophozoites and schizonts matured normally in the presence of cyclic AMP and produced infectious merozoites. Gametocyte formation was inhibited by 95% when 1.0 mM cyclic AMP was added to synchronously growing parasites in the ring stage of development but was only inhibited by 15% when added in the trophozoite or schizont stages. Cyclic AMP was not found to increase gametocyte formation over a wide range of concentrations.     

Electron microscopic observations on ciliated epithelium of tracheal organ cultures infected with Bordetella bronchiseptica

Using mouse tracheal organ cultures, the pathogenic effect of Bordetella bronchiseptica to epithelial cells was studied by electron microscopy. The ultrastructure of epithelial cells in uninfected tracheal rings was preserved well for longer than 3 days. In mouse tracheal rings infected with graded doses (3 x 10(5) to 10(7) CFU/ml) of phase I B. bronchiseptica, the colonization in the interciliary spaces of ciliated epithelial cells was observed after a 20-hr infection period. The infected tracheal rings showed swelling of nonciliated cells as well as ciliated cells, rupture of cell membrane of cilia, swelling and disappearance of cilia, and atrophic cytomorphosis of epithelial cells. The severity of these changes occurred depending on the infection doses. These changes were essentially similar to those observed previously in the tracheal epithelia of the B. bronchiseptica-infected mice. The usefulness of this in vitro model was suggested for studying the pathogenesis of Bordetella infection.     

Malaria enhances cyclic AMP production by immature erythrocytes in vitro.

Infection with a chloroquine-susceptible line of $\text{Plasmodium}$$\text{berghei}$ NYU-2 enhanced the production of cyclic AMP in response to isoproterenol only in immature mouse erythrocytes, which possess a functional hormone-receptor-adenylate cyclase complex. With 10^?7 M isoproterenol the increases in cyclic AMP concentrations (picomoles per 10⁸ erythrocytes) were 19 for a preparation of mature erythrocytes, 81 for a preparation enriched with immature erythrocytes, 25 for a preparation of infected mature erythrocytes, and 900 for a preparation enriched with infected immature erythrocytes. Beta-adrenergic receptor blockade with propranolol prevented this response to isoproterenol but had no effect on the stimulation produced by prostaglandin E₁. These findings indicate that the malaria parasite enhances the responsiveness of a fundamental regulatory system that is intrinsic to immature erythrocytes.     

Enhanced gametocyte formation in young erythrocytes by Plasmodium falciparum in vitro.

Two gametocyte-forming clones, HB-3 and 3D7, were used. Concentrates of late stage parasites were mixed with bloods containing different proportions of young erythrocytes, and the parasitemia and proportion of gametocytes determined after 2, 3 or 4 days of culture. Significantly more gametocytes were formed in light cells than in heavy cells separated from the same normal blood samples. Up to seven times more gametocytes were formed in reticulocyte-rich bloods from patients with sickle cell anemia than in normal control blood.