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2.
J Biol Chem ; 283(41): 27636-27643, 2008 Oct 10.
Article in English | MEDLINE | ID: mdl-18694927

ABSTRACT

Biochemical studies in the human malaria parasite, Plasmodium falciparum, indicated that in addition to the pathway for synthesis of phosphatidylcholine from choline (CDP-choline pathway), the parasite synthesizes this major membrane phospholipid via an alternative pathway named the serine-decarboxylase-phosphoethanolamine-methyltransferase (SDPM) pathway using host serine and ethanolamine as precursors. However, the role the transmethylation of phosphatidylethanolamine plays in the biosynthesis of phosphatidylcholine and the importance of the SDPM pathway in the parasite's growth and survival remain unknown. Here, we provide genetic evidence that knock-out of the PfPMT gene encoding the phosphoethanolamine methyltransferase enzyme completely abrogates the biosynthesis of phosphatidylcholine via the SDPM pathway. Lipid analysis in knock-out parasites revealed that unlike in mammalian and yeast cells, methylation of phosphatidylethanolamine to phosphatidylcholine does not occur in P. falciparum, thus making the SDPM and CDP-choline pathways the only routes for phosphatidylcholine biosynthesis in this organism. Interestingly, loss of PfPMT resulted in significant defects in parasite growth, multiplication, and viability, suggesting that this gene plays an important role in the pathogenesis of intraerythrocytic Plasmodium parasites.


Subject(s)
Methyltransferases/metabolism , Phosphatidylcholines/biosynthesis , Plasmodium falciparum/enzymology , Plasmodium falciparum/pathogenicity , Protozoan Proteins/metabolism , Animals , Ethanolamine/metabolism , Gene Deletion , Methylation , Methyltransferases/genetics , Phosphatidylcholines/genetics , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Serine/genetics , Serine/metabolism
3.
Vector Borne Zoonotic Dis ; 8(4): 565-73, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18399780

ABSTRACT

The prevalence of trypanosome infections in tsetse flies in the Chiawa area of Lower Zambezi in Zambia, with endemic trypanosomosis, was determined by a polymerase chain reaction (PCR) method that allowed the detection of trypanosome DNA and determination of the type of animal host fed on by the tsetse fly Glossina pallidipes, using tsetse-derived DNA extracts as templates. Ninety G. pallidipes (82 females and 8 males; 18.3%) of the 492 flies captured by baited biconical traps tested positive for the presence of Trypanosoma brucei species genomic DNA. Of the 90 T. brucei-positive flies, 47 (52.2%) also tested positive for vertebrate mitochondrial DNA. Sequence analysis of the vertebrate mitochondrial DNA amplicons established that they originated from 8 different vertebrate species, namely, human (Homo sapiens), African elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), waterbuck (Kobus ellipsiprymnus), roan antelope (Hippotragus equinus), greater kudu (Tragelaphus strepsiceros), warthog (Phacochoerus africanus), and goat (Capra hircus). Furthermore, to investigate the prevalence of trypanosome infections in domestic goats in the same area where trypanosomes had been detected in tsetse files, a total of 86 goats were randomly selected from 6 different herds. Among the selected goats, 36 (41.9%) were found to be positive for T. brucei species. This combined detection method would be an ideal approach not only for mass screening for infection prevalence in tsetse populations, but also for the prediction of natural reservoirs in areas endemic for trypanosomosis.


Subject(s)
Trypanosoma brucei brucei/isolation & purification , Tsetse Flies/microbiology , Animals , Elephants , Feeding Behavior , Female , Goat Diseases/epidemiology , Goat Diseases/parasitology , Goats , Host-Parasite Interactions , Humans , Male , Prevalence , Ruminants , Swine , Trypanosomiasis, African/parasitology , Trypanosomiasis, African/veterinary
4.
Eukaryot Cell ; 6(9): 1618-24, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17644653

ABSTRACT

During its intraerythrocytic life cycle, the malaria parasite Plasmodium falciparum undergoes dramatic metabolic and morphological changes and multiplies to produce up to 36 new daughter parasites. This rapid multiplication of the parasite requires an active synthesis of new membranes. The major component of these membranes, phosphatidylcholine, is synthesized via two metabolic routes, the CDP-choline pathway, which uses host choline as a precursor, and the plant-like serine decarboxylase-phosphoethanolamine methyltransferase (SDPM) pathway, which uses host serine as a precursor. Here we provide evidence indicating that the activity of the SDPM pathway is regulated by the CDP-choline precursor, choline. We show that the phosphoethanolamine methyltransferase, Pfpmt, a critical enzyme in the SDPM pathway, is down-regulated at the transcriptional level as well as targeted for degradation by the proteasome in the presence of choline. Transcript analysis revealed that PfPMT transcription is repressed by choline in a dose-dependent manner. Immunoblotting, pulse-chase experiments, and immunoprecipitation studies demonstrated that Pfpmt degradation occurs not only in wild-type but also in transgenic parasites constitutively expressing Pfpmt. The proteasome inhibitor bortezomib inhibited choline-mediated Pfpmt degradation. These data provide the first evidence for metabolite-mediated transcriptional and proteasomal regulation in Plasmodium and will set the stage for the use of this system for conditional gene and protein expression in this organism.


Subject(s)
Choline/pharmacology , Methyltransferases/metabolism , Plasmodium falciparum/enzymology , Proteasome Endopeptidase Complex/drug effects , Protozoan Proteins/metabolism , Transcription, Genetic/drug effects , Animals , Boronic Acids/pharmacology , Bortezomib , Cytidine Diphosphate Choline/metabolism , Gene Expression Regulation , Methyltransferases/antagonists & inhibitors , Methyltransferases/genetics , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Protease Inhibitors/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Protein Biosynthesis/drug effects , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , Pyrazines/pharmacology
5.
Eukaryot Cell ; 6(4): 584-91, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17307963

ABSTRACT

Plasmodium falciparum is the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in P. falciparum. Transgenic parasites were generated which express the translation elongation factor PfEF-1beta harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1beta-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1beta, the G-protein PfEF-1alpha, and two new proteins that we named PfEF-1gamma and PfEF-1delta based on their homology to other eukaryotic gamma and delta translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in P. falciparum, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1beta. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen.


Subject(s)
Chromatography, Affinity/methods , Peptide Elongation Factors/isolation & purification , Plasmodium falciparum/chemistry , Protozoan Proteins/isolation & purification , Amino Acid Sequence , Animals , Genes, Protozoan , Mass Spectrometry , Molecular Sequence Data , Multiprotein Complexes/chemistry , Peptide Elongation Factors/chemistry , Plasmids , Protozoan Proteins/chemistry , Recombination, Genetic
6.
Acta Trop ; 99(1): 34-41, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16899209

ABSTRACT

In order to investigate the transmission dynamics of Theileria parva (T. parva) by the brown ear tick, Rhipicephalus appendiculatus (R. appendiculatus), under experimental conditions, detection of T. parva in ticks and cattle was performed by a quantitative real-time PCR assay. A calf inoculated with a T. parva mixture became PCR-positive for T. parva infection on day 8 post-inoculation, and subsequently, nymphal ticks were introduced and maintained to feed on the infected calf for 6 days. Engorged nymphs were collected daily and allowed to molt into adults, and overall, 70.8% (121/171) of the adult ticks acquired the T. parva infection. Furthermore, the T. parva infection rate in ticks under field conditions was monitored by real-time PCR in R. appendiculatus ticks collected from a traditionally managed pastoral land of Zambia, on which Sanga breed cattle are traditionally reared and the area has endemic East Coast fever (ECF). A total of 70 cattle were randomly selected in the same area and 67 (95.7%) were found to be serologically positive for R. appendiculatus tick antigen (RIM36). Twenty-nine (43.3%) of the 67 serologically positive cattle were real-time PCR-positive for T. parva, although no piroplasms could be detected in the blood smears. Unexpectedly, out of 614 R. appendiculatus nymphal and adult ticks collected by flagging vegetation, 4.1% were positive for T. parva DNA. However, since the rate of transmission of T. parva from infected cattle to ticks and vice versa and the serological evidence of exposure to R. appendiculatus ticks in naturally exposed cattle were relatively high, it would be wise in such a case to consider vector control as well as vaccination against ECF as control measures.


Subject(s)
Rhipicephalus/parasitology , Theileria parva/growth & development , Theileriasis/transmission , Tick Infestations/veterinary , Tick-Borne Diseases/veterinary , Animals , Antibodies, Protozoan/blood , Blotting, Western/veterinary , Cattle , DNA, Protozoan/chemistry , DNA, Protozoan/genetics , Female , Male , Polymerase Chain Reaction/veterinary , Theileria parva/genetics , Theileriasis/blood , Tick Infestations/parasitology , Tick-Borne Diseases/blood , Tick-Borne Diseases/transmission
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