Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Oct;74(10):5487-96.
doi: 10.1128/IAI.01934-05.

Fatty acids from Plasmodium falciparum down-regulate the toxic activity of malaria glycosylphosphatidylinositols

Françoise Debierre-Grockiego  1 , Louis Schofield, Nahid Azzouz, Jörg Schmidt, Cristiana Santos de Macedo, Michael A J Ferguson, Ralph T Schwarz
Affiliations

Fatty acids from Plasmodium falciparum down-regulate the toxic activity of malaria glycosylphosphatidylinositols

Françoise Debierre-Grockiego et al. Infect Immun. 2006 Oct.

Abstract

Plasmodium falciparum malaria kills roughly 2.5 million people, mainly children, annually. Much of this mortality is thought to arise from the actions of a malarial toxin. This toxin, identified as glycosylphosphatidylinositol (GPI), is a major pathogenicity determinant in malaria. A malarial molecule, Pfj, labeled by [3H]glucosamine like the GPIs, was identified as a non-GPI molecule. Here we show that Pfj is able to down-regulate tumor necrosis factor alpha (TNF-alpha) production induced by the GPI of P. falciparum. Mass spectrometry analysis showed that Pfj was not a single molecule but represented a number of molecules. Separation methods, such as cation-exchange chromatography and thin-layer chromatography, were used to isolate and identify the following four main fatty acids responsible for the inhibitory effect on TNF-alpha production: myristic, pentadecanoic, palmitic, and palmitoleic acids. This regulatory effect on cytokine production suggests that there is balanced bioactivity for the different categories of malarial lipids.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Pfj inhibits GPI-induced TNF-α production. (A) TLC scan of [3H]glucosamine-labeled glycolipids of P. falciparum using a Berthold LB 2842 linear analyzer. (B) Macrophages were incubated for 24 h in the presence of Pfj extracted from 106, 107, or 108 P. falciparum trophozoites. LPS (200 ng/ml) was used as a positive control. Medium alone was used as a negative control (lane M). (C) Macrophages were incubated for 24 h in the presence of the P. falciparum GPI Pfα (α) alone or with Pfj extracted from 106 to 5 × 108 trophozoites or with glycolipids extracted from noninfected erythrocytes and corresponding to the Pfj area after TLC (Ej). Pfj and Ej were added 30 min before Pfα was added. Supernatants were assayed for TNF-α production using a sandwich ELISA or for NO production using the Griess assay (D). The values are means ± standard deviations of triplicate samples in a representative experiment.
FIG. 2.
FIG. 2.
Pfj fraction does not contain glycosphingolipids: positive-ion electrospray mass spectra of ganglioside GM1 (A), used as an authentic glycosphingolipid standard to validate the analytical method, and of the Pfj fraction (B). The data were normalized to the intensity of the most abundant [M + 2H]2+ doubly charged GM1 ganglioside ion (m/z 809.9, 100%) in panel A. Only low-intensity, singly charged (nonglycosphingolipid) ions were observed in the Pfj sample.
FIG. 3.
FIG. 3.
Fraction P3 containing fatty acids inhibits GPI-induced TNF-α production. (A) Macrophages were incubated for 24 h in the presence of the six different fractions (P1 to P6) isolated from 2 × 108 trophozoites. (B and C) Macrophages were incubated for 24 h in the presence of Pfα (α) alone or with fractions P1 to P6 (added 30 min before Pfα was added) (B) or with the six fractions extracted from noninfected erythrocytes (E1 to E6 added 30 min before Pfα was added) (C). Medium alone was used as a negative control (lanes M). The values are means ± standard deviations of triplicate samples in a representative experiment.
FIG. 4.
FIG. 4.
Fatty acid analysis of Plasmodium and noninfected red cell fractions. Negative-ion electrospray-mass spectrometry was used to identify the carboxylate ([M-H]−) ions of free fatty acids present in the P3 fraction from P. falciparum (A), the E3 fraction from noninfected erythrocytes (RBC) (B), and the Pfj fraction from P. falciparum (C). The data in panel B were normalized to the intensity of the most intense fatty acid ion (m/z 281) in panel C to illustrate the paucity of fatty acids in the noninfected-erythrocyte-derived fraction.
FIG. 5.
FIG. 5.
Isolation by TLC of the inhibitory fatty acids. (A) Macrophages were incubated for 24 h in the presence of Pfα (α) alone or with the eight different fractions (P3/1 to P3/8) obtained after separation of fraction P3 (from 2 × 108 trophozoites) by TLC. (B) Macrophages were incubated for 24 h in the presence of Pfα alone or with the eight fractions (P3/2/1 to P3/2/8 added 30 min before Pfα was added) obtained after separation of fraction 3/2 by TLC. Medium alone was used as a negative control (lanes M). The values are means ± standard deviations of triplicate samples in a representative experiment. (C) Negative-ion mass spectrum of P. falciparum inhibitory fraction P3/2/8. The annotated ions correspond to [M-H]− fatty acid carboxylate ions with the numbers of carbon atoms and numbers of C=C double bonds indicated. The y axis shows the relative ion intensities. Nominal mass values are indicated on the spectrum, but the accurate mass values were all within 3 ppm of the theoretical masses for the corresponding fatty acid carboxylate ions.
FIG. 6.
FIG. 6.
Myristic, pentadecanoic, palmitic, and palmitoleic acids inhibit GPI-induced TNF-α production. Macrophages were incubated for 24 h with medium alone (open bars), with Pfα alone (black bars), or with myristic acid (A), pentadecanoic acid (B), palmitic acid (C), palmitoleic acid (D), stearic acid (E), or oleic acid (F) at a concentration of 1 μM (light gray bars) or 10 μM (dark gray bars) or the six fatty acids together (G) added 30 min before Pfα was added. The values are means ± standard deviations of triplicate samples in a representative experiment.
FIG. 7.
FIG. 7.
All precursors of the GPI Pfα activate production of TNF-α. Macrophages were incubated for 24 h with GPI Pfα (α) or with its precursors Pfβ (β), Pfγ (γ), Pfδ (δ), Pfɛ (ɛ), Pfζ (ζ), Pfη (η), and Pfθ (θ). The supernatant obtained after GPI precipitation containing non-GPI species was added to macrophages 30 min before Pfα was added (α+S). The values are means ± standard deviations of triplicate samples in a representative experiment. (B) TLC scans of P. falciparum GPI precursors and of P. falciparum contaminating molecules (supernatant), showing their comigration with Pfγ to Pfθ. (C and D) The contaminant molecules migrating with Pfɛ, Pfζ, Pfη, and Pfθ, but not with Pfγ, inhibited production of TNF-α and IL-12 in macrophages (γS,ɛS, ζS, ηS, and θS were added 30 min before Pfγ, Pfɛ, Pfζ, Pfη, and Pfθ were added). Fraction 3, separated from the supernatant using LC-NH2 and LC-WCX cartridges, inhibited production of TNF-α and IL-12 in macrophages (S3 was added 30 min before Pfα was added). Medium alone was used as a negative control (lanes M). (E) TLC of S3. Free fatty acids are rose-violet. Myristic acid (My) was used as a positive control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ansorge, I., D. Jeckel, F. Wieland, and K. Lingelbach. 1995. Plasmodium falciparum-infected erythrocytes utilize a synthetic truncated ceramide precursor for synthesis and secretion of truncated sphingomyelin. Biochem. J. 308:335-341. - PMC - PubMed
    1. Azzouz, N., H. Shams-Eldin, and R. T. Schwarz. 2005. Removal of phospholipid contaminants through precipitation of glycosylphosphatidylinositols. Anal. Biochem. 343:152-158. - PubMed
    1. Bodennec, J., O. Koul, I. Aguado, G. Brichon, G. Zwingelstein, and J. Portoukalian. 2000. A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges. J. Lipid Res. 41:1524-1531. - PubMed
    1. Debierre-Grockiego, F., N. Azzouz, J. Schmidt, J. F. Dubremetz, H. Geyer, R. Geyer, R. Weingart, R. R. Schmidt, and R. T. Schwarz. 2003. Roles of glycosylphosphatidylinositols of Toxoplasma gondii. Induction of tumor necrosis factor-alpha production in macrophages. J. Biol. Chem. 278:32987-32993. - PubMed
    1. Delorenzi, M., A. Sexton, H. Shams-Eldin, R. T. Schwarz, T. Speed, and L. Schofield. 2002. Genes for glycosylphosphatidylinositol toxin biosynthesis in Plasmodium falciparum. Infect. Immun. 70:4510-4522. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources