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Metabolomic analysis of patient plasma yields evidence of plant-like ?-linolenic acid metabolism in Plasmodium falciparum.

ABSTRACT: Metabolomics offers a powerful means to investigate human malaria parasite biology and host-parasite interactions at the biochemical level, and to discover novel therapeutic targets and biomarkers of infection. Here, we used an approach based on liquid chromatography and mass spectrometry to perform an untargeted metabolomic analysis of metabolite extracts from Plasmodium falciparum-infected and uninfected patient plasma samples, and from an enriched population of in vitro cultured P. falciparum-infected and uninfected erythrocytes. Statistical modeling robustly segregated infected and uninfected samples based on metabolite species with significantly different abundances. Metabolites of the ?-linolenic acid (ALA) pathway, known to exist in plants but not known to exist in P. falciparum until now, were enriched in infected plasma and erythrocyte samples. In vitro labeling with (13)C-ALA showed evidence of plant-like ALA pathway intermediates in P. falciparum. Ortholog searches using ALA pathway enzyme sequences from 8 available plant genomes identified several genes in the P. falciparum genome that were predicted to potentially encode the corresponding enzymes in the hitherto unannotated P. falciparum pathway. These data suggest that our approach can be used to discover novel facets of host/malaria parasite biology in a high-throughput manner.

SUBMITTER: Lakshmanan V

PROVIDER: S-EPMC3490690 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Metabolomic analysis of patient plasma yields evidence of plant-like α-linolenic acid metabolism in Plasmodium falciparum.

Lakshmanan Viswanathan V Rhee Kyu Y KY Wang Wei W Yu Yiting Y Khafizov Kamil K Fiser Andras A Wu Peng P Ndir Omar O Mboup Souleymane S Ndiaye Daouda D Daily Johanna P JP

The Journal of infectious diseases 20120507 2

Metabolomics offers a powerful means to investigate human malaria parasite biology and host-parasite interactions at the biochemical level, and to discover novel therapeutic targets and biomarkers of infection. Here, we used an approach based on liquid chromatography and mass spectrometry to perform an untargeted metabolomic analysis of metabolite extracts from Plasmodium falciparum-infected and uninfected patient plasma samples, and from an enriched population of in vitro cultured P. falciparum ...[more]

PMID: 22566569

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Project description:BackgroundThe intra-erythrocytic development of the malaria parasite Plasmodium falciparum depends on the uptake of a number of essential nutrients from the host cell and blood plasma. It is widely recognized that the parasite imports low molecular weight solutes from the plasma and the consumption of these nutrients by P. falciparum has been extensively analysed. However, although it was already shown that the parasite also imports functional proteins from the vertebrate host, the internalization route through the different infected erythrocyte membranes has not yet been elucidated. In order to further understand the uptake mechanism, the study examined the trafficking of human plasminogen from the extracellular medium into P. falciparum-infected red blood cells.MethodsPlasmodium falciparum clone 3D7 was cultured in standard HEPES-buffered RPMI 1640 medium supplemented with 0.5% AlbuMAX. Exogenous human plasminogen was added to the P. falciparum culture and the uptake of this protein by the parasites was analysed by electron microscopy and Western blotting. Immunoprecipitation and mass spectrometry were performed to investigate possible protein interactions that may assist plasminogen import into infected erythrocytes. The effect of pharmacological inhibitors of different cellular physiological processes in plasminogen uptake was also tested.ResultsIt was observed that plasminogen was selectively internalized by P. falciparum-infected erythrocytes, with localization in plasma membrane erythrocyte and parasite's cytosol. The protein was not detected in parasitic food vacuole and haemoglobin-containing vesicles. Furthermore, in erythrocyte cytoplasm, plasminogen was associated with the parasite-derived membranous structures tubovesicular network (TVN) and Maurer's clefts. Several proteins were identified in immunoprecipitation assay and may be involved in the delivery of plasminogen across the P. falciparum multiple compartments.ConclusionThe findings here reported reveal new features regarding the acquisition of plasma proteins of the host by P. falciparum-infected erythrocytes, a mechanism that involves the exomembrane system, which is distinct from the haemoglobin uptake, clarifying a route that may be potentially targeted for inhibition studies.

Project description:BackgroundFor future eradication strategies of malaria it is important to control the transmission of gametocytes from humans to the anopheline vector which causes the spread of the disease. Sensitive, non-invasive methods to detect gametocytes under field conditions can play a role in monitoring transmission potential.MethodsMicroscopically Plasmodium falciparum-positive patients from Jimma, Ethiopia donated finger-prick blood, venous blood, saliva, oral mucosa and urine samples that were spotted on filter paper or swabs. All samples were taken and stored under equal, standardized conditions. RNA was extracted from the filter paper and detected by real-time QT-NASBA. Pfs16-mRNA and Pfs25-mRNA were measured with a time to positivity to detect gametocyte specific mRNA in different gametocyte stages. They were compared to 18S-rRNA, which is expressed in all parasite stages. Results were quantified via a known dilution series of artificial RNA copies.ResultsNinety-six samples of 16 uncomplicated malaria patients were investigated. 10 (66.7%) of the slides showed gametocyte densities between 0.3-2.9 gametocytes/?l. For all RNA-targets, molecular detection in blood samples was most sensitive; finger-prick sampling required significantly smaller amounts of blood than venous blood collection. Detection of asexual 18S-rRNA in saliva and urine showed sensitivities of 80 and 67%, respectively. Non-invasive methods to count gametocytes proved insensitive. Pfs16-mRNA was detectable in 20% of urine samples, sensitivities for other materials were lower. Pfs25-mRNA was not detectable in any sample.ConclusionsThe sensitivity of non-invasively collected material such as urine, saliva or mucosa seems unsuitable for the detection of gametocyte-specific mRNA. Sensitivity in asymptomatic carriers might be generally even lower. Finger-prick testing revealed the highest absolute count of RNA copies per ?L, especially for Pfs25-mRNA copies. The method proved to be the most effective and should preferably be applied in future transmission control and eradication plans. A rapid test for gametocyte targets would simplify efforts.

Dataset Information

Metabolomic analysis of patient plasma yields evidence of plant-like ?-linolenic acid metabolism in Plasmodium falciparum.

Publications

Metabolomic analysis of patient plasma yields evidence of plant-like α-linolenic acid metabolism in Plasmodium falciparum.

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