The D enantiomers of three naturally occurring antibiotics-cecropin A, magainin 2 amide, and melittin-were synthesized. In addition, the D enantiomers of two synthetic chimeric cecropin-melittin hybrid peptides were prepared. Each D isomer was shown by circular dichroism to be a mirror image of the corresponding L isomer in several solvent mixtures. In 20% hexafluoro-2-propanol the peptides contained 43-75% a-helix. The all-D peptides were resistant to enzymatic degradation. The peptides produced single-channel conductances in planar lipid bilayers, and the D and L enantiomers caused equivalent amounts of electrical conductivity. All of the peptides were potent antibacterial agents against representative Gram-negative and Gram-positive species. The D and L enantiomers of each peptide pair were equally active, within experimental error. Sheep erythrocytes were lysed by both D-and L-melittin but not by either isomer of cecropin A, magainin 2 amide, or the hybrids cecropin A-(1-13)-melittin-(1-13)-NH2 or cecropin A-(1-8)-melittin-(1-18)-NH2. The infectivity of the bloodstream form of the malaria parasite Plasmodium falciparum was also inhibited by the D and L hybrids. It is suggested that the mode of action of these peptides on the membranes of bacteria, erythrocytes, plasmodia, and artificial lipid bilayers may be similar and involves the formation of ion-channel pores spanning the membranes, but without specific interaction with chiral receptors or enzymes.The cecropins (1, 2) and several other antibiotic peptides of the animal kingdom, including defensins (3), magainins (4), and the bee venom toxin melittin (5), are thought to function through the formation of ion channels in lipid membranes. This idea has been based on recent studies of electrical conductivity in artificial lipid bilayers (3,(6)(7)(8), where activity is a function of the structure of the peptide and the composition of the membrane lipids. The bilayer lipids and cell membranes are chiral and contain many asymmetric centers. It has been generally assumed that the chirality of the membrane would require a specific chirality ofthe peptide for it to be active, in much the same way that peptide hormones are required to fit with the conformation of their natural receptors or for a substrate and enzyme to form a tight stereospecific complex. However, we have suggested that these peptide antibiotics can exert their effect without requiring a specific target receptor on the cell membrane (7, 9).The purpose of the present study was to test this assumption by the synthesis of the all-D enantiomers of several natural, all-L peptide antibiotics and some of their active analogs. These D stereoisomers would be expected to assume equivalent, but mirror image, conformations when placed in the same environment as the all-L peptides. If a close molecular contact with the chiral components of the cell membrane is required, the D enantiomers would be expected to be inactive. However, if the interaction of the peptide with the membrane is only between achiral...
Solid phase synthesis was used to produce 5 hybrid peptides containing sequences from the antibacterial peptide, cecropin A, and from the bee venom toxin, melittin. Four of these chimeric peptides showed good antibacterial activity against representative Gram-negative and Gram-positive bacterial species. The best hybrid, cecropin A(l-13)-melittin(l-13) was IOO-fold more active than cecropin A against Staphylococcus uureus. It was also a lo-fold better antimalarial agent than cecropin B or magainin 2. Sheep red cells were lysed by melittin at low concentrations, but not by the hybrid molecules, even at 50 times higher concentrations.Cecropin A; Melittin; (Staphylococcus aureus, Plasmodium falciparium)
We have earlier reported two 26‐residue antibacterial peptides made up from different segments ol'cecropin A (CA) and melittin (M). We now report a substantial reduction in size at the C‐terminal section of the highly active hybrid CA(1–8)M(1–18), leading to a series of 20‐, 18‐ and 15‐residue analogs with antibiotic properties similar to the larger molecule. In particular, the 15‐residue hybrids CA(1–7)M(2–9), CA(1–7)M(4–11) and CA(1–7)M(5–12) are the shortest cecropin‐based peptide antibiotics described so far, with antibacterial activity and spectra similar or better than cecropin A and a 60% reduction in size. Their reduced size and highly α‐helical structure require an alternative mechanism for their interaction with bacterial membranes.
Erythrocytes infected with late asexual stages of Plasmodiumfalciparum and P. fragile may form spontaneous rosettes with uninfected erythrocytes (1-3). Here we present a detailed study of rosette formation in P. falciparum malaria .Materials and Methods Assessment ofRosetting . P. falciparum parasites were cultured in vitro under standard conditions . Infected erythorcytes (Ei) were mixed with acridine orange, and E; that had bound two or more noninfected erythrocytes (E.) were scored as rosetting. Blood samples drawn from patients with a blood smear positive for P. falciparum were washed and cultured in vitro. The number of rosettes was counted after 35 h and expressed as a percentage of the total number of trophozoite-or schizont-containing erythrocytes.Enrichment ofRosetting and Nonrosetting Parasites. Cultures containing mature parasite-infected erythrocytes were layered on Ficoll-Isopaque (FIP) and centrifuged for 5 s at room temperature (RT). The cells that passed through the FIP layer were collected in a pellet, washed twice in RPMI 1640, and cultured as described . For enrichment of nonrosetting E;, the cultures were layered over 60% Percoll and centrifuged (500 g) at RT for 20 min. The layer at the interface was collected and washed twice in RPMI 1640 and cultured as above. Rosette-forming parasites were cloned by limiting dilution and screened for rosetting on day 10-14.EM. Erythrocytes from P. falciparum cultures were fixed with 2 17c glutaraldehyde in 0.05 M phosphate buffer containing 4% sucrose (pH 7.4) for 1 h, followed by embedding and cutting procedures as described by Aikawa et al. (4) .Effect ofEnzymes on Rosetting Parasites. Cultures containing rosetted Ei were washed three times in RPMI 1640, treated with 10 or 100 ug/ml of trypsin for 15 min at 37°C, and subsequently washed . In some experiments, trypsinized E; were cultured in complete medium for observation of reappearance of rosettes . Experiments with neuraminidase were performed as above using 0.2, 1, and 5 U/ml, respectively. Cytoadherent Parasites . The assay for cytoadherence of E; to melanoma cells (ATCC 1585, C32r; American Type Culture Collection, Rockville, MD) was performed as described elsewhere (5) . To enrich for cytoadherent parasites, P. falciparum cultures were incubated with melanoma cells grown in a tissue culture flask at RT for 1-2 h with continuous rotation . The bound erythrocytes were retrieved by flushing the melanoma cells with culture medium .
IgG from a donor clinically immune to Plasmodiumfalciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, <1 ,ug/ml) than the original IgG preparation. falciparum (1, 2). These antigens are released from bursting schizonts or merozoites and are deposited in the RBC membrane in the course of invasion. Antibodies recognizing these antigens are of high prevalence in sera from residents of a holoendemic area of Africa (Liberia) but are also present in many sera of acutely infected patients from different parts of the world. A polypeptide of Mr 155,000 (Pf 155) appears to be primarily responsible for this RBC membrane IF. A similar antigen of the same apparent molecular weight has also been found recently by two other groups (3, 4). To further investigate the function of these antigens and the possible significance of the immune response against them, we have now tested the antibodies reacting with Pf 155 and related minor components for their capacity to inhibit RBC invasion by merozoites in vitro. MATERIAL AND METHODSParasites. Parasites were from a Tanzanian strain of P. falciparum (F 32) isolated in 1978 (5) and cultured in vitro in blood group O RBCs (6).Immune Sera. Five sera (Kinon, IS-6 to -8, X-12) were from Liberians, >15 years old (except X-12, from a 12-yearold boy), living in a P. falciparum holoendemic area in which in adult life clinical illness is rare and parasitemia is kept at a low-grade, mostly subpatent, level despite high sporozoite inoculation rates (7). These donors, designated as clinically immune, had not taken any antimalarial drug for the last 6 weeks (X-12) or longer. The 6 remaining donors had acute P. falciparum infections. Four donors (ASF, MV, JOR, FG) were South Americans from an endemic area of Colombia. Two Swedish patients (YC, HP) were suffering from a first infection acquired in Kenya. IgG was prepared by ammonium sulfate precipitation and ion-exchange chromatography on DEAE-Sephadex (Pharmacia). Immunoglobulin concentrations were determined by ELISA.In Vitro Growth Inhibition Assay. Infected RBCs from P. falciparum cultures (5-10% parasitemia, -70% schizonts) were diluted with normal O RBCs to a parasitemia of 0.5%. They were adjusted to 2% hematocrit with Hepes-buffered (20 mM) RPMI-1640 medium (GIBCO) containing 15% normal human serum, 2 mM glutamine, 25 pg of gentamycin per ml, and 0.2% NaHCO3 [complete tissue culture medium (TC medium)]. In some experiments, synchronized parasite cultures were used. In this case, the original cultures (5-10% parasitemia) were adjusted to 10% hematocrit and layered on top of 2.5 ml of 60% Percoll (Pharmacia) diluted in com...
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