ABSTRACT
BACKGROUND AND OBJECTIVES: Blood transfusions can transmit parasitic infections, such as those caused by Plasmodium (malaria), Trypanosoma cruzi (Chagas' disease), and Babesia (babesiosis). A higher degree of blood transfusion safety would be reached if methods were available for inactivating such parasites. MATERIALS AND METHODS: We evaluated the effectiveness of photosensitization using lipophilic pheophorbide and red light illumination to eradicate red blood cells infected with Plasmodium falciparum, and with Babesia divergens, in whole blood. Fluorescence microscopy and conventional fluorometry showed the specific accumulation of pheophorbide derivatives in the RBC infected with either parasite, compared with uninfected RBC. The effectiveness of different derivatives in eradicating infected RBC was first estimated in parasite cultures. RESULTS: The best photosensitizer was the N-(4-butanol) pheophorbide derivative (Ph4-OH) at 0.2 microM concentration and 5-min illumination. In whole blood, the eradication of RBC infected with B. divergens and P. falciparum was obtained with 2 microM Ph4-OH and 10 and 20 min illumination, respectively. Under these conditions of photosensitization, low levels of RBC hemolysis were noted even after 2 weeks of storage at 4 degrees C and a subsequent 48-hour incubation at 37 degrees C. No reduction of negative charges on treated RBC was noted and no increase in methemoglobin content. CONCLUSIONS: In plasma, Ph4-OH is mainly transported by high-density lipoproteins (HDL). This high affinity for HDL may explain the selective accumulation of lipophilic pheophorbide derivatives in the intracellular parasites. Photosensitization with pheophorbide derivatives may be a promising approach to inactivation of transfusion-transmissible parasites and viruses in blood bank units.
Subject(s)
Babesia/drug effects , Babesiosis/prevention & control , Chlorophyll/analogs & derivatives , Erythrocytes/parasitology , Malaria, Falciparum/prevention & control , Photosensitizing Agents/pharmacology , Plasmodium falciparum/drug effects , Animals , Babesia/physiology , Babesia/radiation effects , Babesiosis/blood , Babesiosis/transmission , Blood Preservation , Chlorophyll/blood , Chlorophyll/pharmacology , Hemolysis/drug effects , Hemolysis/radiation effects , Humans , Light , Lipoproteins, HDL/metabolism , Malaria, Falciparum/blood , Malaria, Falciparum/transmission , Photochemistry , Plasmodium falciparum/physiology , Plasmodium falciparum/radiation effects , Transfusion ReactionABSTRACT
The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors lovastatin and simvastatin inhibit the in vitro intraerythrocytic development of Plasmodium falciparum and Babesia divergens, with concentrations inhibiting parasite growth by 50% in the ranges of 10 to 20 and 5 to 10 micrograms.ml-1, respectively. For P. falciparum, the 50% inhibitory concentrations were in the same range whatever the chloroquine susceptibility of the strains tested (strain F32/Tanzania [chloroquine susceptible] or FcB.1/Columbia [resistant]). The stage-dependent susceptibility of P. falciparum to simvastatin was studied by subjecting synchronized cultures to 6-h pulses of drug throughout the 48-h erythrocytic life cycle. The most important inhibitory effects were observed between the 12th and 30th hours of the cycle, corresponding to the trophozoite stage. This period precedes the S phase and the nuclear divisions. Parasites in the newly formed ring stage (time zero to the 6th hour of the cycle) and the schizont stage (30th to 48th hour of the cycle) were weakly or not susceptible to simvastatin pulses.
Subject(s)
Babesia/growth & development , Erythrocytes/parasitology , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Lovastatin/analogs & derivatives , Plasmodium falciparum/growth & development , Animals , Babesia/drug effects , Erythrocytes/drug effects , Humans , In Vitro Techniques , Lovastatin/pharmacology , Plasmodium falciparum/drug effects , SimvastatinSubject(s)
Babesia/growth & development , Culture Media, Serum-Free/pharmacology , Lipoproteins, HDL/pharmacology , Parasitology/methods , Plasmodium falciparum/growth & development , Animals , Babesia/drug effects , Babesia/metabolism , Erythrocytes/parasitology , Humans , Lipid Metabolism , Lipoproteins, LDL/pharmacology , Lipoproteins, VLDL/pharmacology , Phosphatidylcholines/metabolism , Plasmodium falciparum/drug effects , Plasmodium falciparum/metabolismABSTRACT
A two-fold increase in the amount of phospholipids was observed in Babesia divergens infected human red blood cells. In vitro incubation with [32P]-phosphorus and [3H]-glycerol demonstrated that B divergens has the ability to synthesize the phospholipid backbone. On the other hand, the low incorporation of [14C]acetate indicated the absence of a de novo fatty acid synthesis and suggested the necessity of an exogenous lipid source for the parasite. Several intra-erythrocytic growth cycles of B divergens could be achieved in vitro, using a serum-free medium supplemented only with fractions of human high density lipoproteins (HDL). At an HDL concentration of 0.5 mg/ml (protein concentration) and with a 1% starting parasitaemia, parasite growth was similar to that observed under standard culture conditions with 10% human serum, at least for the first 24 h, a time equivalent to three parasite erythrocytic life-cycles. Lipid transfer from HDL to the intra-erythrocytic parasites was demonstrated by uptake and exchange of fluorescent NBD-phosphatidylcholine (NBD-PC) loaded HDL at different temperatures. Kinetic experiments with [3H]-oleyl-PC-loaded HDL demonstrated a unidirectional transfer of lipids from radiolabelled HDL to the parasite; partial conversion of PC to phosphatidylethanolamine (PE) was also observed. In the semi-defined medium, the HDL fraction appeared to be the major source of lipids for the growth of B divergens in human erythrocytes.
Subject(s)
Babesia/metabolism , Babesiosis/metabolism , Erythrocytes/metabolism , Lipid Metabolism , Lipoproteins, HDL/metabolism , Acetates/metabolism , Animals , Babesia/drug effects , Babesia/growth & development , Erythrocytes/parasitology , Fatty Acids/metabolism , Humans , Lipoproteins, HDL/pharmacology , Microscopy, Fluorescence , Phospholipids/metabolismABSTRACT
Several intraerythrocytic growth cycles of Plasmodium falciparum could be achieved in vitro using a serum free medium supplemented only with a human high density lipoprotein (HDL) fraction (d = 1.063-1.210). The parasitemia obtained was similar to that in standard culture medium containing human serum. The parasite development was incomplete with the low density lipoprotein (LDL) fraction and did not occur with the VLDL fraction. The lipid traffic from HDL to the infected erythrocytes was demonstrated by pulse labeling experiments using HDL loaded with either fluorescent NBD-phosphatidylcholine (NBD-PC) or radioactive [3H]palmitoyl-PC. At 37 degrees C, the lipid probes rapidly accumulated in the infected cells. After incubation in HDL medium containing labeled PC, a subsequent incubation in medium with either an excess of native HDL or 20% human serum induced the disappearance of the label from the erythrocyte plasma membrane but not from the intraerythrocytic parasite. Internalization of lipids did not occur at 4 degrees C. The mechanism involved a unidirectional flux of lipids but no endocytosis. The absence of labeling of P. falciparum, with HDL previously [125I]iodinated on their apolipoproteins or with antibodies against the apolipoproteins AI and AII by immunofluorescence and immunoblotting, confirmed that no endocytosis of the HDL was involved. A possible pathway of lipid transport could be a membrane flux since fluorescence videomicroscopy showed numerous organelles labeled with NBD-PC moving between the erythrocyte and the parasitophorous membranes. TLC analysis showed that a partial conversion of the PC to phosphatidylethanolamine was observed in P. falciparum-infected red cells after pulse with [3H]palmitoyl-PC-HDL. The intensity of the lipid traffic was stage dependent with a maximum at the trophozoite and young schizont stages (38th h of the erythrocyte life cycle). We conclude that the HDL fraction appears to be a major lipid source for Plasmodium growth.
Subject(s)
Erythrocytes/parasitology , Lipoproteins, HDL/metabolism , Plasmodium falciparum/metabolism , Animals , Biological Transport , Chromatography, Thin Layer , Endocytosis , Erythrocytes/metabolism , Fluorescent Antibody Technique , Humans , Immunoblotting , Lipoproteins, LDL/metabolism , Lipoproteins, VLDL/metabolism , Plasmodium falciparum/growth & developmentABSTRACT
Products of the isoprenoid metabolism were identified upon incubations of extracts from Plasmodium falciparum infected red blood cells with [14C] mevalonate. Uninfected erythrocytes and wild type yeast Saccharomyces cerevisiae extracts were used as controls. In parasitized red blood cells as well as in yeast extracts, mevalonate was converted into the biosynthetic isoprenoid precursors of sterol pathway until farnesyl pyrophosphate. In contrast, no mevalonate conversion was observed in uninfected erythrocyte extracts. The isoprenoid metabolism appeared stage-dependent as shown by the increase of radiolabelled farnesyl pyrophosphate amount at the beginning of the schizogonic phase (30-36 hours).
Subject(s)
Erythrocytes/parasitology , Hemiterpenes , Malaria/metabolism , Mevalonic Acid/analogs & derivatives , Organophosphorus Compounds/metabolism , Plasmodium falciparum/metabolism , Polyisoprenyl Phosphates/metabolism , Acetates/metabolism , Animals , Cells, Cultured , Humans , Mevalonic Acid/metabolism , Saccharomyces cerevisiae/metabolism , SesquiterpenesABSTRACT
The effects of serum and human lipoproteins (HDL, VLDL, LDL) were investigated on the intraerythrocytic cycle of P. falciparum using in vitro synchronized cultures. The reinvasion process of erythrocytes by merozoites and the development until the young trophozoite stage are independent of serum components. In the absence of serum, schizogony did not occur. However, addition of serum before the 24th hour of culture in basal medium restores a normal schizogony. Serum replacement by the different lipoprotein fractions showed that only the HDL fraction was able to assure a complete schizogony as well as a normal erythrocyte reinvasion. No division was observed with the apolipoproteins.
Subject(s)
Lipoproteins, HDL/pharmacology , Plasmodium falciparum/cytology , Animals , Apolipoproteins/pharmacology , Blood/parasitology , Cell Division/drug effects , Humans , In Vitro Techniques , Lipoproteins, LDL/pharmacology , Lipoproteins, VLDL/pharmacologyABSTRACT
In Bacillus subtilis, the phosphatidate cytidylyltransferase was localized exclusively in the membrane fraction prepared by sucrose density gradient fractionation. A single enzyme could synthesize the two liponucleotides: CDPdiacylglycerol and dCDPdiacylglycerol. Kinetic experiments and isotopic exchange reactions suggested a ping-pong mechanism. Among the nucleosides monophosphate, CMP specifically reduced the synthesis of both liponucleotides. This inhibition was non-competitive and might be involved in regulation of phospholipid synthesis.
Subject(s)
Bacillus subtilis/enzymology , Cytidine Monophosphate/pharmacology , Cytosine Nucleotides/pharmacology , Nucleotidyltransferases/antagonists & inhibitors , Chemical Phenomena , Chemistry , Kinetics , Phospholipids/biosynthesis , Substrate SpecificityABSTRACT
We investigated the lipid composition of vegetative cells of Stigmatella aurantiaca. Four phospholipids were isolated and identified: phosphatidylethanolamine as the main component, phosphatidylglycerol, lysophosphatidylethanolamine in an exceptionally large amount (17%), and phosphatidylinositol (18 to 25%), rare in procaryotic cells. This composition did not change significantly during growth. The fatty acids of total lipids were found to be rather similar to those of other strains of myxobacteria; the main fatty acids found were unsaturated and branched. We noted a different fatty acid pattern for each phospholipid. The presence of unusual alkyl ether linkages, established by chemical hydrolysis and infrared spectroscopy, was unexpected in these bacteria. Diacyl ester, dialkyl ether, and monoacyl-monoalkyl structures were shown in phosphatidylethanolamine and phosphatidylglycerol. Lysophosphatidylethanolamine was essentially a monoacyl form, whereas phosphatidylinositol was a unique dialkyl ether phospholipid.
Subject(s)
Lysophospholipids , Myxococcales/analysis , Phospholipids/analysis , Fatty Acids/analysis , Phosphatidylethanolamines/analysis , Phosphatidylglycerols/analysis , Phosphatidylinositols/analysis , Spectrophotometry, InfraredABSTRACT
In Bacillus subtilis, the fatty acid moiety of the phospholipids was affected differently during growth in the presence of 1.1 M-methanol or 0.7 M-ethanol, though at these concentrations methanol and ethanol had the same effects on growth rate and completely inhibited sporulation. Synthesis of phosphatidylglycerol was also strongly inhibited and the amount of total cell phospholipids was reduced by 50% by both alcohols. The composition of fatty acids, especially the relative concentration of 12-methyltetradecanoic acid, was modified only by ethanol; in bacteria grown in the presence of methanol, changes in fatty acid composition were negligible. In non-sporulating mutants, synthesis of phosphatidylglycerol was much less affected than in the wild-type and synthesis of phosphatidylethanolamine was increased. In these strains, fatty acid composition was also modified by ethanol but unaffected by methanol.
Subject(s)
Bacillus subtilis/metabolism , Ethanol/pharmacology , Lipid Metabolism , Methanol/pharmacology , Bacillus subtilis/drug effects , Bacillus subtilis/growth & development , Fatty Acids/metabolism , Phosphatidylglycerols/metabolism , Phospholipids/metabolismABSTRACT
The growth rate of Bacillus subtilis is lowered but the final cell yield is unchanged when certain concentrations of ethanol are present in the culture medium. At the concentration allowing growth at half-maximal rate, practically no spores are formed. Blockage of spore formation generally occurs at stage 0-I. Sensitivity to ethanol of the capacity to form spores is limited, in a nonsynchronized culture, to a period of at most 45 min around t1. Postexponential events such as excretion of certain enzymes and modification of ribonucleic acid polymerase are altered or suppressed in the presence of ethanol, possibly as the results of a physical change upon the cell membrane. In effect, ethanol is turning wild-type cells into phenocopies of spoO mutants.