Shirley Kutner

Characterization of permeation pathways appearing in the host membrane of Plasmodium falciparum infected red blood cells.

The host cell membrane of Plasmodium falciparum infected cells becomes permeabilized at the trophozoite stage. A variety of otherwise impermeant substances such as carbohydrates, polyols, amino acids and anions easily gain access to the cytosol of infected cells. Using the isotonic-hemolysis method or uptake of labeled substances, we characterized the new permeation pathways as pores of approximately 0.7 nm equivalent radius. The pores bear a positively charged character which facilitates movement of small anions and excludes cations, so that the ionic composition and osmotic properties of infected cells are not drastically altered. Substances of a molecular size similar to that of disaccharides are fully excluded. Substances of limiting size might be accommodated in the pore, provided they bear a side group of hydrophobic character. The new permeation pathways may provide a vital route for acquisition or release of essential nutrients or catabolites.

Alterations in membrane permeability of malaria-infected human erythrocytes are related to the growth stage of the parasite.

During the intraerythrocytic growth of Plasmodium falciparum in culture, marked changes are observed in the permeability properties of the host cell membrane. Anionic substances otherwise impermeant to normal cells, become highly permeant to infected cells. These changes in permeability become apparent as rings mature into trophozoites and remain throughout schizogony. The permeability changes to anionic substances are not manifested as degradation of band 3, the purported erythrocyte anion transporter. They probably reflect alterations of a more general nature.

Phospholipid and cholesterol uptake by Mycoplasma cells and membranes.

The ability of growing mycoplasma cells and their isolated membranes to take up exogenous phospholipids was correlated with their ability to take up cholesterol. Horse serum or vesicles made of phosphatidylcholine and cholesterol served as lipid donors. Growing cells of five Mycoplasma species took up significant quantities of phosphatidylcholine and sphingomyelin as well as free and esterified cholesterol. In contrast, growing cells of three Acholeplasma species failed to take up any of the exogenous phospholipids, and only incorporated low amounts of free cholesterol and no esterified cholesterol. Hence, the ability of mycoplasmas to take up large quantities of cholesterol appears to be correlated with an ability to take up exogenous phospholipids. Isolated membranes of Mycoplasma capricolum and Acholeplasma laidlawii took up lower amounts of cholesterol than did membranes of growing cells and did not take up phospholipids. Inhibition of M. capricolum growth decreased the ability of the cells to take up exogenous phospholipids and cholesterol. The possibility that the contact between the lipid donors and the membrane involves specific receptors best exposed in actively growing cells is discussed.

On the mode of action of phlorizin as an antimalarial agent in in vitro cultures of plasmodium falciparum

Phlorizin (phloretin-2-beta-glucoside) is a drug which effectively inhibits intraerythrocytic malaria growth in in vitro cultures of Plasmodium falciparum IC50 = 16 +/- 7 microM). Work with synchronously grown cultures indicates that susceptibility to phlorizin is apparent at the trophozoite stage and onward, and that 2-8 hours exposure to the drug causes an irreversible arrest of parasite growth. The drug has also been found to inhibit pores which are induced by the parasite in the host cell membrane (IC50 = 17 +/- 2 microM) and which are apparently essential for intraerythrocytic growth. The effect on the pores is apparent soon after exposure of the cells to the drug and can be reversed, although extensive washing and incubation in culture conditions are required to achieve it. The results of this study indicate that the putative site of action of phlorizin on the pores is on the cytoplasmic surface of the host cell membrane. The drug which normally cannot permeate uninfected red cells, gains access to the cytoplasm via the pores, appearing in the host cell membrane. Those become eventually the target of phlorizin itself. The proposed mechanism of action of phlorizin on malarial growth invokes blockade of the pores, although additional effects of the drug on intraerythrocytic parasites cannot be ruled out.

Selectivity properties of pores induced in host erythrocyte membrane by Plasmodium falciparum. Effect of parasite maturation.

The intraerythrocytic malarial parasite permeabilizes its host cell membrane by inducing pore-like pathways which mediate the passage of nonelectrolytes and anions. In the present work we show that, although the permeability increases with parasite maturation, the selectivity of the pores to various solutes is essentially preserved, suggesting that the number of pores increases without any alteration in their intrinsic solute conductance.

Effective immunization of mice against cutaneous leishmaniasis using an intrinsically adjuvanted synthetic lipopeptide vaccine

Two peptides representing predicted T-cell epitopes of gp63, a major surface glycoprotein of the parasite Leishmania major, were used in vaccines tested in a murine model of cutaneous leishmaniasis. Either subcutaneous or intraperitoneal immunization in saline with a peptide representing gp63 amino acids 467-482 (p467) significantly protected CBA mice against the development of severe cutaneous lesions only when the peptide was intrinsically adjuvanted by covalently adding a lauryl-cysteine moiety (LC-p467) to its amino terminus during synthesis. In marked contrast, administration of p467 alone, cysteinyl-p467 or gp63 protein in saline resulted in some disease exacerbation. Splenic cells of LC-p467 immunized mice stimulated in vitro with LC-p467 displayed strong proliferative responses and secretion of IL-2, IFN-tau and GM-CSF (but not IL-4 and IL-10) suggesting that immunization with the lipopeptide induced the TH1 type cytokine responses associated with cell-mediated immunity. The safety, efficacy, ease of production and standardization of such lipopeptide vaccines suggest that they have significant potential for the development of vaccines for humans against leishmaniasis or other parasitic or viral diseases that require cell-mediated immunity for protection.

Relationships between cell surface protease and acid phosphatase activities ofLeishmania promastigote

A correlation between the ratio of the cell surface protease activity to phosphatase activity and the complexity of the pattern of cell surface exposed polypeptides ofLeishmania promastigotes was demonstrated for various strains grown under similar conditions.The ratio of the cell surface protease activity to acid phosphatase activity was high forL. major andL.b. panamensis and it correlates with the expression of a single polypeptide of 63 KDa on their cell surface. Intermediate and lower ratios of these enzymatic activites relate with more complex radio-iodinated patterns: two main bands inL.b. guyanensis (70 and 58 KDa) andL.b. braziliensis (72 and 60 KDa) and three main bands 65, 50, 27 KDa in allL.m. mexicana strains tested. Evidence is presented that the acid phosphatase located on theL.m. mexicana cell surface is not an artifact due to a secondary absorption of the secreted acid phosphatase from the culture medium.These results confirm theLeishmania antigen cell surface heterogeneity. The implications on the biology ofLeishmania and the clinical manifestation of leishmaniasis are discussed.

Inhibition of P. Falciparum Growth in vitro by Specific Inhibitors of Red Blood Cell Anion Transport

Erythrocytes actively metabolize glucose to lactic acid. Disposal of the latter is accomplished by three passive membrane pathways: a high-affinity low capacity H+-lactate cotransport system specific for L-lactate (I), a low affinity high-capacity transport system involved physiologically in C1-HCO3 exchange (II) and simple diffusion (III). In P. falciparum infected cells, intracellular lactic acid production rises by more than 100 fold. It was our assumption that specific blockade of pathway II should raise intraerythrocytic lactic acid primarily in infected cells and thus affect in vitro parasite growth. Blockade was accomplished by 4,4′-dinitro-2, 2′-stilbene disulfonate (DNDS) or the irreversible 4,4′-diisothiocyano analog DIDS, both nonpenetrating, specific inhibitors of II and by use of phloridzin and furose-mide, two classical but less specific inhibitors of II. In in vitro culture conditions, DNDS inhibited both, system-II mediated anion transport and parasite growth (but not invasion) with ID50 of ca50μM. Similar results were obtained with phloridzin (ID5020μM) and furosemide (ID5050μM). Chemical modification of noninfected cells with DIDS inhibited anion transport (ID500.25μM), arrested growth (ID501.0μM) but did not prevent invasion.