The effects of anti-bacterials on the malaria parasite Plasmodium falciparum

Many anti-bacterial drugs inhibit growth of malaria parasites by targeting their bacterium-derived endosymbiotic organelles, the mitochondrion and plastid. Several of these drugs are either in use or being developed as therapeutics or prophylactics, so it is paramount to understand more about their target of action and modality. To this end, we measured in vitro growth and visualized nuclear division and the development of the mitochondrion and apicoplast in Plasmodium falciparum treated with five drugs targeting bacterial housekeeping pathways. This revealed two distinct classes of drug effect. Ciprofloxacin, rifampicin, and thiostrepton had an immediate effect: slowing parasite growth, retarding organellar development and preventing nuclear division. Classic delayed-death, in which the drug has no apparent effect until division and reinvasion of a new host by the daughter merozoites, was only observed for two drugs: clindamycin and tetracycline. These cells had apparently normal division and segregation of organelles in the first cycle but severe defects in apicoplast growth, subtle changes in the mitochondrion and a failure to complete cytokinesis during the second cycle. In two cases, the drug response in P. falciparum directly conflicted with reported responses for the related parasite Toxoplasma gondii, suggesting significant differences in apicoplast biology between the two parasites.     

Calcium regulation in the intraerythrocytic malaria parasite Plasmodium falciparum

The regulation of intracellular Ca(2+) in the intraerythrocytic form of the human malaria parasite, Plasmodium falciparum, was investigated using parasites 'isolated' from their host cells by saponin-permeabilisation of the erythrocyte membrane. The isolated parasites maintained tight control over their resting cytosolic Ca(2+) concentration which ranged from approximately 100 nM in the absence of extracellular Ca(2+) to approximately 700 nM in the presence of 1 mM extracellular Ca(2+). The parasite has two functionally discrete intracellular Ca(2+) stores. One is an 'endoplasmic reticulum (ER)-like' store, the other an 'acidic store'. The ER-like store was discharged by cyclopiazonic acid (CPA), an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) of animal and plant cells, but not by thapsigargin (TG), a more specific inhibitor of SERCAs of animal cells. The acidic store was discharged by nigericin and by NH(4)(+). The amount of Ca(2+) in the ER-like store increased with increasing extracellular Ca(2+) concentration, whereas the amount of Ca(2+) in the acidic store did not. Ca(2+) released from the ER-like store by CPA was cleared from the parasite cytosol by uptake into the acidic store (over a range of extracellular Ca(2+) concentrations), consistent with the acidic store serving as a Ca(2+) reservoir within the intracellular parasite.     

Biosynthesis of glycosphingolipids de-novo by the human malaria parasite Plasmodium falciparum

Glycolipids are important components of cellular membranes involved in various biological functions. In this report we describe the identification of the de-novo synthesis of glycosphingolipids by intraerythrocytic, asexual stages of the malaria parasite, Plasmodium falciparum. Parasite-specific glycolipids were identified in organic solvent extracts of parasites metabolically labeled with tritiated serine and glucosamine and characterised as sphingolipids based on their insensitivity towards alkaline treatment. While the de-novo synthesis of parasite glycosphingolipids was affected by fumonisin B1, threo-PPMP, cyclo-serine and myriocin, these well established inhibitors of de-novo ceramide biosynthesis were unable to arrest the intraerythrocytic development of the parasites in culture.     

Glucose-6-phosphate dehydrogenase activity of the malaria parasite Plasmodium falciparum

Schizonts of Plasmodium falciparum, grown either in normal or glucose-6-phosphate dehydrogenase (G6PDH) deficient human red cells, contain an electrophoretically slow-moving form of G6PDH. The slow mobility of the G6PDH in non-dissociating polyacrylamide gels is due to its large size (Mr ca. 450,000) rather than to its charge. The activity of this enzyme was less than 10% of normal red cell G6PDH. These characteristics of the parasite-associated G6PDH were unaltered when parasites were grown in red cells from a G6PDH A+B+ heterozygote or following the introduction of a heterologous G6PDH into resealed ghosts. Differential absorption of the parasite-associated and red cell G6PDHs was demonstrated with antisera containing antibodies to red cell G6PDH. These studies show that a novel form of G6PDH is associated with P. falciparum in normal red cells without the requirements for induction by one or several cycles of multiplication in G6PDH deficient red cells.     

Characterisation of a delta-COP homologue in the malaria parasite,Plasmodium falciparum

The mature human erythrocyte is a simple haemoglobin-containing cell with no internal organelles and no protein synthesis machinery. The malaria parasite invades this cell and develops inside a parasitophorous vacuole (PV). The parasite exports proteins into the erythrocyte to bring about extensive remodelling of its adopted cellular home. Plasmodial homologues of two COPII proteins, PfSar1p and PfSec31p, are exported to the erythrocyte cytosol where they appear to play a role in the trafficking of proteins across the erythrocyte cytoplasm [Eur. J. Cell Biol. 78 (1999) 453; J. Cell Sci. 114 (2001) 3377]. We have now characterised a homologue of the COPI protein, delta-COP. A recombinant protein corresponding to 90% of the Pfdelta-COP sequence was used to raise antibodies. The affinity-purified antiserum recognised a protein with an apparent M(r) of 58 x 10(3) on Western blots of malaria parasite-infected erythrocytes but not on blots of uninfected erythrocytes. Pfdelta-COP was shown to be largely insoluble in non-ionic detergent, possibly suggesting cytoskeletal attachment. Confocal immunofluorescence microscopy of parasitised erythrocytes was used to show that, in contrast to the COPII proteins, Pfdelta-COP is located entirely within the parasite. The location of Pfdelta-COP partly overlaps that of the endoplasmic reticulum (ER)-located protein, PfERC, and partly that of the trans-Golgi-associated protein, PfRab6. Treatment of ring-stage plasmodium-infected erythrocytes with brefeldin A (BFA) inhibited development of the ER structure within the parasite cytosol and prevented the trafficking of the P. falciparum erythrocyte membrane protein-1, PfEMP1, to the erythrocyte cytosol. The Pfdelta-COP and PfSec31p populations each appear to be associated with the restricted ER structure in brefeldin-treated rings. When more mature stage parasites were treated with BFA, erythrocyte cytosol-located populations of parasite proteins were not reorganised, however, the overlap between Pfdelta-COP and PfERC in parasite cytosol was more complete suggesting a possible redistribution of the Golgi compartment into the ER. These data support the suggestion that both COPI and COPII proteins are involved in the trafficking of proteins within the parasite cytoplasm. However, only COPII proteins are exported to the erythrocyte cytosol to establish a vesicle-mediated protein trafficking pathway to the erythrocyte membrane.     

Metabolism of glutamine in erythrocytes infected with the human malaria parasite: Plasmodium falciparum

The metabolism of glutamine was studied in erythrocytes infected with Plasmodium falciparum, comparatively to normal cells, in presence or not of DON (6-diazo-5-oxo-L-norleucine) or acivicin, two glutamine antagonists which have been shown to inhibit the growth of P. falciparum in vitro. Extracellular glutamine was partially converted into glutamate using two routes corresponding to gamma-glutamyl transpeptidase (GGT) and glutaminase activities. In cells infected with mature trophozoites, the observed enhancement of the glutamine influx and of the glutamate formation was consistent with the enhancement of GGT and glutaminase activities.     

The kinetics of uptake and accumulation of 3,6-bis-omega-diethylamino-amyloxyxanthone by the human malaria parasite Plasmodium falciparum

Malarial parasites rely on the digestion of hemoglobin during the intra-erythrocytic stage. The enzymatic degradation of hemoglobin yields amino acids for parasite survival, and free heme which is detoxified by conversion to an aggregate of dimeric heme known as hemozoin. Xanthones have been found to subvert this process by formation of soluble drug-heme complexes. We have optimized the simple hydroxyxanthone structure to include side chains with protonatable nitrogen atoms to enhance interaction with the propionate groups of heme and to target the drug to the parasite digestive vacuole. One member of this optimized class of compounds, 3,6-bis-omega-diethylaminoamyloxyxanthone (C5), was used as a prototype for mechanistic studies. By HPLC analysis we demonstrate that the drug accumulates in the digestive vacuole from 5 to approximately 33,000 microM within 1 h of exposure to parasitized red cells. Confocal fluorescence microscopy was used to visualize the accumulation process directly and to document the colocalization of the drug with the acidophilic dye, LysoTracker Red.     

Characterisation of a repetitive DNA sequence from the malaria parasite,Plasmodium falciparum

A repetitive DNA fragment cloned from the malaria parasite, Plasmodium falciparum, has been analysed. It contains a 21 base pair sequence which occurs in multiple tandem repeats. Two clusters of the same repeat are found in opposite orientations on the same DNA fragment. The repetitive DNA provides an additional way to distinguish between different strains of parasite by hybridisation to genomic blots and may serve as a species-specific probe for diagnosis.     

Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum trophozoites were isolated by mechanical rupture of infected human erythrocytes followed by a series of differential centrifugation steps. After lysis with sonication, the 100 000 x g supernatant of parasites and uninfected host cells was used to determine the specific activities of a number of enzymes involved in purine and pyrimidine metabolism. P. falciparum possessed the purine salvage enzymes: adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyltransferase (PRTase), xanthine PRTase, adenine PRTase, adenosine kinase. The last two enzymes, however, were present at much lower activity levels. Hypoxanthine was converted (presumably via IMP) into adenine and guanine nucleotides only in the presence both of supernatant and membrane fractions of P. falciparum. Two enzymes involved in the de novo synthesis of pyrimidines, orotic acid PRTase, and orotidine 5'-phosphate decarboxylase, were present in parasite extracts as were the enzymes for pyrimidine nucleotide phosphorylation: UMP-CMP kinase, dTMP kinase, nucleoside diphosphate kinase. Xanthine oxidase, CTP synthetase, cytidine deaminase and several kinases for the salvage of pyrimidine nucleosides were not detected in the parasites. Both phosphoribosyl pyrophosphate synthetase and uracil PRTase were present but at low activity levels. Human erythrocytes displayed similar but not identical enzyme patterns. Enzyme specific activities, however, were generally much lower than those of the corresponding parasite enzymes.