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Defensins from Insects and Plants Interact with Fungal Glucosylceramides

2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês

10.1074/jbc.m311165200

1083-351X

Karin Thevissen, Dirk Warnecke, Isabelle François, Martina Leipelt, Ernst Heinz, Claudia Ott, Ulrich Zähringer, Bart P. H. J. Thomma, Kathelijne K.A. Ferket, Bruno P.A. Cammue,

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Growth of the yeast species Candida albicans and Pichia pastoris is inhibited by RsAFP2, a plant defensin isolated from radish seed (Raphanus sativus), at micromolar concentrations. In contrast, gcs-deletion mutants of both yeast species are resistant toward RsAFP2. GCS genes encode UDP-glucose:ceramide glucosyltransferases, which catalyze the final step in the biosynthesis of the membrane lipid glucosylceramide. In an enzyme-linked immunosorbent assay-based binding assay, RsAFP2 was found to interact with glucosylceramides isolated from P. pastoris but not with soybean nor human glucosylceramides. Furthermore, the P. pastoris parental strain is sensitive toward RsAFP2-induced membrane permeabilization, whereas the corresponding gcs-deletion mutant is highly resistant to RsAFP2-mediated membrane permeabilization. A model for the mode of action of RsAFP2 is presented in which all of these findings are linked. Similarly to RsAFP2, heliomicin, a defensin-like peptide from the insect Heliothis virescens, is active on C. albicans and P. pastoris parental strains but displays no activity on the gcs-deletion mutants of both yeast species. Furthermore, heliomicin interacts with glucosylceramides isolated from P. pastoris and soybean but not with human glucosylceramides. These data indicate that structurally homologous anti-fungal peptides present in species from different eukaryotic kingdoms interact with the same target in the fungal plasma membrane, namely glucosylceramides, and as such support the hypothesis that defensins from plants and insects have evolved from a single precursor. Growth of the yeast species Candida albicans and Pichia pastoris is inhibited by RsAFP2, a plant defensin isolated from radish seed (Raphanus sativus), at micromolar concentrations. In contrast, gcs-deletion mutants of both yeast species are resistant toward RsAFP2. GCS genes encode UDP-glucose:ceramide glucosyltransferases, which catalyze the final step in the biosynthesis of the membrane lipid glucosylceramide. In an enzyme-linked immunosorbent assay-based binding assay, RsAFP2 was found to interact with glucosylceramides isolated from P. pastoris but not with soybean nor human glucosylceramides. Furthermore, the P. pastoris parental strain is sensitive toward RsAFP2-induced membrane permeabilization, whereas the corresponding gcs-deletion mutant is highly resistant to RsAFP2-mediated membrane permeabilization. A model for the mode of action of RsAFP2 is presented in which all of these findings are linked. Similarly to RsAFP2, heliomicin, a defensin-like peptide from the insect Heliothis virescens, is active on C. albicans and P. pastoris parental strains but displays no activity on the gcs-deletion mutants of both yeast species. Furthermore, heliomicin interacts with glucosylceramides isolated from P. pastoris and soybean but not with human glucosylceramides. These data indicate that structurally homologous anti-fungal peptides present in species from different eukaryotic kingdoms interact with the same target in the fungal plasma membrane, namely glucosylceramides, and as such support the hypothesis that defensins from plants and insects have evolved from a single precursor. Innate immunity is an ancient defense strategy used by multicellular organisms to control the natural flora and combat pathogens. This strategy involves, among other responses, the production of cationic antimicrobial peptides (AMPs) 1The abbreviations used are: AMPantimicrobial peptideAFPantifungal peptideELISAenzyme-linked immunosorbent assayGLCgas-liquid chromatographyGlcCerglucosylceramide(s)HPLChigh performance liquid chromatographyM(IP)2CmannosyldiinositolphosphorylceramideMSmass spectrometry. that generally have a broad activity spectrum. Until now, only one class of AMPs is found to be conserved among plants, invertebrates, and vertebrates, namely defensins. Defensins are small, highly basic cysteine-rich peptides that share a common three-dimensional structure (for review, see Ref. 1Thomma B.P.H.J. Cammue B.P.A. Thevissen K. Planta. 2002; 216: 193-202Crossref PubMed Scopus (640) Google Scholar). The global fold of plant defensins comprises a cysteine-stabilized αβ motif consisting of an α-helix and a triple-stranded β-sheet, organized in a βαββ architecture and stabilized by four disulfide bridges. Most plant defensins possess antifungal or antibacterial activity in vitro but are noncytotoxic to either mammalian or plant cells. Insect defensins combine an α-helix and a double-stranded β-sheet stabilized by three disulfide bridges, organized in a cysteine-stabilized αβ motif as found in plant defensins (2Bonmatin J.M. Bonnat J.L. Gallet X. Vovelle F. Ptak M. Reichhart J.M. Hoffmann J.A. Keppi E. Legrain M. Achstetter T. J. Biomol. NMR. 1992; 2: 235-256Crossref PubMed Scopus (131) Google Scholar, 3Cornet B. Bonmatin J.M. Hetru C. Hoffmann J.A. Ptak M. Vovelle F. Structure. 1995; 3: 435-448Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). An even higher homology to plant defensins is found for the insect defensin-like peptide heliomicin, which carries a triple-stranded β-sheet in a βαββ fold forming a cysteine-stabilized αβ motif (4Lamberty M. Caille A. Landon C. Tassin-Moindrot S. Hetru C. Bulet P. Vovelle F. Biochemistry. 2001; 40: 11995-12003Crossref PubMed Scopus (69) Google Scholar). In addition to similarities in their global folds, heliomicin and the radish plant defensin RsAFP2 exhibit very similar distributions of hydrophobic residues (4Lamberty M. Caille A. Landon C. Tassin-Moindrot S. Hetru C. Bulet P. Vovelle F. Biochemistry. 2001; 40: 11995-12003Crossref PubMed Scopus (69) Google Scholar) and display similar biological activities: both peptides are antifungal rather than antibacterial (5Lamberty M. Ades S. Uttenweiler-Joseph S. Brookhart G. Bushey D. Hoffmann J.A. Bulet P. J. Biol. Chem. 1999; 274: 9320-9326Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 6Terras F.R. Schoofs H.M. De Bolle M.F. Van Leuven F. Rees S.B. Vanderleyden J. Cammue B.P. Broekaert W.F. J. Biol. Chem. 1992; 267: 15301-15309Abstract Full Text PDF PubMed Google Scholar). antimicrobial peptide antifungal peptide enzyme-linked immunosorbent assay gas-liquid chromatography glucosylceramide(s) high performance liquid chromatography mannosyldiinositolphosphorylceramide mass spectrometry. Most cationic antimicrobial peptides induce membrane permeabilization after initial electrostatic binding to negatively charged phospholipids on the target cell surface. In contrast, plant defensins induce membrane permeabilization through specific interaction with high affinity binding sites on fungal cells (7Thevissen K. Osborn R.W. Acland D.P. Broekaert W.F. J. Biol. Chem. 1997; 272: 32176-32181Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 8Thevissen K. Osborn R.W. Acland D.P. Broekaert W.F. Mol. Plant Microbe Interact. 2000; 13: 54-61Crossref PubMed Scopus (113) Google Scholar, 9Thevissen K. Terras F.R. Broekaert W.F. Appl. Environ. Microbiol. 1999; 65: 5451-5458Crossref PubMed Google Scholar). Via a genetic complementation approach, IPT1 was identified as a gene determining sensitivity toward the dahlia plant defensin Dm-AMP1 in Saccharomyces cerevisiae (10Thevissen K. Cammue B.P. Lemaire K. Winderickx J. Dickson R.C. Lester R.L. Ferket K.K. Van Even F. Parret A.H. Broekaert W.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9531-9536Crossref PubMed Scopus (171) Google Scholar). IPT1 encodes an enzyme involved in the last step of the synthesis of the sphingolipid mannosyldiinositolphosphorylceramide (M(IP)2C) (11Dickson R.C. Nagiec E.E. Wells G.B. Nagiec M.M. Lester R.L. J. Biol. Chem. 1997; 272: 29620-29625Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Strains with a nonfunctional IPT1 allele lacked M(IP)2C in their membranes bound significantly less DmAMP1 compared with the parental S. cerevisiae strain and were highly resistant to DmAMP1-induced membrane permeabilization. Possibly, membrane patches containing sphingolipids act as binding sites for DmAMP1. Interaction between DmAMP1 and these sphingolipids could lead to insertion of the plant defensin into the membrane resulting in membrane destabilization (10Thevissen K. Cammue B.P. Lemaire K. Winderickx J. Dickson R.C. Lester R.L. Ferket K.K. Van Even F. Parret A.H. Broekaert W.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9531-9536Crossref PubMed Scopus (171) Google Scholar, 12Thevissen K. Francois I.E.J.A. Takemoto J.Y. Ferket K.K.A. Meert E.M.K. Cammue B.P.A. FEMS Microbiol. Lett. 2003; 226: 169-173Crossref PubMed Scopus (100) Google Scholar). In the yeast S. cerevisiae, the main sphingolipids are (mannosylated) inositolphosphorylceramides (namely IPC, MIPC, and M(IP)2C). There is growing evidence that fungi maintain two separate pools of ceramides to be used for the synthesis of different sphingolipids (13Leipelt M. Warnecke D. Zähringer U. Ott C. Müller F. Hube B. Heinz E. J. Biol. Chem. 2001; 276: 33621-33629Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 14Toledo M.S. Levery S.B. Straus A.H. Takahashi H.K. J. Lipid Res. 2000; 41: 797-806Abstract Full Text Full Text PDF PubMed Google Scholar, 15Toledo M.S. Levery S.B. Suzuki E. Straus A.H. Takahashi H.K. Glycobiology. 2001; 11: 113-124Crossref PubMed Scopus (46) Google Scholar). Ceramide backbones with very long chain C24 and C26 fatty acids bound to the sphingobase 4-hydroxysphinganine are directed to the synthesis of the inositolphosphoryl-containing sphingolipids (Fig. 1A), whereas ceramide backbones with C16 or C18 fatty acids linked to the sphingobase 9-methyl-4,8-sphingadienine are exclusively used as precursors for biosynthesis of glucosylceramide (GlcCer) (Fig. 1B). GlcCer have never been found in S. cerevisiae under various culture conditions (16Sakaki T. Zähringer U. Warnecke D.C. Fahl A. Knogge W. Heinz E. Yeast. 2001; 18: 679-695Crossref PubMed Scopus (88) Google Scholar). This is in contrast with many fungal and yeast species in which both inositolphosphoryl-containing sphingolipids and significant amounts of GlcCer have been detected (17Warnecke D. Heinz E. Cell. Mol. Life Sci. 2003; 60: 919-941Crossref PubMed Scopus (135) Google Scholar, 18Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1426: 347-357Crossref PubMed Scopus (170) Google Scholar). S. cerevisiae is resistant toward the radish plant defensin RsAFP2 and completely lacks GlcCer, which points toward a possible role of GlcCer in RsAFP2-mediated growth inhibition. To address this hypothesis, RsAFP2 sensitivity of Pichia pastoris and Candida albicans was evaluated. In contrast to S. cerevisiae, membranes of both yeast species contain GlcCer (16Sakaki T. Zähringer U. Warnecke D.C. Fahl A. Knogge W. Heinz E. Yeast. 2001; 18: 679-695Crossref PubMed Scopus (88) Google Scholar, 19Matsubara T. Hayashi A. Banno Y. Morita T. Nozawa Y. Chem. Phys. Lipids. 1987; 43: 1-12Crossref PubMed Scopus (42) Google Scholar). In this paper, we present for the first time evidence that the antifungal plant defensin RsAFP2 and the highly homologous insect defensin-like peptide heliomicin interact with fungal GlcCer in a first step leading to fungal growth arrest. Materials and Microorganisms—The antifungal peptides DmAMP1, RsAFP2, and RsAFP2(Y38G) were isolated as described previously (6Terras F.R. Schoofs H.M. De Bolle M.F. Van Leuven F. Rees S.B. Vanderleyden J. Cammue B.P. Broekaert W.F. J. Biol. Chem. 1992; 267: 15301-15309Abstract Full Text PDF PubMed Google Scholar, 20Osborn R.W. De Samblanx G.W. Thevissen K. Goderis I. Torrekens S. Van Leuven F. Attenborough S. Rees S.B. Broekaert W.F. FEBS Lett. 1995; 368: 257-262Crossref PubMed Scopus (351) Google Scholar, 21De Samblanx G.W. Goderis I.J. Thevissen K. Raemaekers R. Fant F. Borremans F. Acland D.P. Osborn R.W. Patel S. Broekaert W.F. J. Biol. Chem. 1997; 272: 1171-1179Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Nonimmune and anti-RsAFP2 sera from rabbit were used as described previously (6Terras F.R. Schoofs H.M. De Bolle M.F. Van Leuven F. Rees S.B. Vanderleyden J. Cammue B.P. Broekaert W.F. J. Biol. Chem. 1992; 267: 15301-15309Abstract Full Text PDF PubMed Google Scholar). Heliomicin and anti-heliomicin rabbit anti-serum were kindly provided by Dr. P. Bulet (Institut de Biologie Moléculaire et Cellulaire, CNRS, Strasbourg, France) and Dr. J. L. Dimarcq (Entomed, Strasbourg, France), respectively. Anti-GlcCer serum from rabbit (22Brade L. Vielhaber G. Heinz E. Brade H. Glycobiology. 2000; 10: 629-636Crossref PubMed Scopus (25) Google Scholar) was kindly provided by Dr. L. Brade (Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany). Ergosterol, phosphatase-coupled goat anti-rabbit immunoglobulins, and GlcCer isolated from human (Gaucher's spleen) were purchased from Sigma. GlcCer isolated from soybean was purchased from Avanti Polar Lipids (Alabaster, AL). SYTOX Green was obtained from Molecular Probes (Eugene, OR). Yeast strains used in this study are S. cerevisiae strain BY4741 (Invitrogen), P. pastoris strain GS115 (Invitrogen) and the corresponding P. pastoris gcs-deletion strain (13Leipelt M. Warnecke D. Zähringer U. Ott C. Müller F. Hube B. Heinz E. J. Biol. Chem. 2001; 276: 33621-33629Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), C. albicans strain SC5314 CAI4 (23Fonzi W.A. Irwin M.Y. Genetics. 1993; 134: 717-728Crossref PubMed Google Scholar) and the corresponding C. albicans gcs-deletion strain (13Leipelt M. Warnecke D. Zähringer U. Ott C. Müller F. Hube B. Heinz E. J. Biol. Chem. 2001; 276: 33621-33629Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Antifungal Activity Assay—Antifungal activity of protein samples against yeast strains was assayed by microscopic analysis of liquid cultures grown in microtiter plates as described previously (8Thevissen K. Osborn R.W. Acland D.P. Broekaert W.F. Mol. Plant Microbe Interact. 2000; 13: 54-61Crossref PubMed Scopus (113) Google Scholar, 9Thevissen K. Terras F.R. Broekaert W.F. Appl. Environ. Microbiol. 1999; 65: 5451-5458Crossref PubMed Google Scholar, 10Thevissen K. Cammue B.P. Lemaire K. Winderickx J. Dickson R.C. Lester R.L. Ferket K.K. Van Even F. Parret A.H. Broekaert W.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9531-9536Crossref PubMed Scopus (171) Google Scholar). Growth medium used was either PDB (24 g/liter potato dextrose broth, Sigma) or PDB/YPD (24 g/liter PDB; 2 g/liter yeast extract, Difco; 4 g/liter Peptone, Difco; 4 g/liter glucose) supplemented with 50 mm HEPES, pH 7.0. The antagonistic effect of either antisera or RsAFP2(Y38G) on the antifungal activity of RsAFP2 was assayed by incubating 10 μl of an overnight P. pastoris culture in 1 ml of fresh PDB/YPD, pH 7.0, in the presence of either the antagonizing compound or distilled water. After 30 min of preincubation, the antifungal activity of RsAFP2 on these P. pastoris cultures was assayed as described above. SYTOX Green Uptake—Cells of an overnight culture of P. pastoris in PDB/YPD were incubated with RsAFP2 or 0.5% SDS in PDB/YPD, pH 7.0, for 2 h. After incubation, cells were washed with SMF1 (9Thevissen K. Terras F.R. Broekaert W.F. Appl. Environ. Microbiol. 1999; 65: 5451-5458Crossref PubMed Google Scholar) supplemented with 0.25 μm SYTOX Green and 50 mm HEPES, pH 7.0. Hundred-μl aliquots of this cell suspension were incubated with RsAFP2 in white 96-well microtiter plates (PE white; PerkinElmer Life Sciences) for 2 h at room temperature, after which the fluorescence was measured as described previously (9Thevissen K. Terras F.R. Broekaert W.F. Appl. Environ. Microbiol. 1999; 65: 5451-5458Crossref PubMed Google Scholar). Membrane permeabilization induced by 0.5% SDS corresponds to 100% membrane permeabilization. Purification, Quantification, and Analysis of GlcCer—Purification of GlcCer from P. pastoris was performed as described previously (16Sakaki T. Zähringer U. Warnecke D.C. Fahl A. Knogge W. Heinz E. Yeast. 2001; 18: 679-695Crossref PubMed Scopus (88) Google Scholar). GlcCer from soybean and from human (Gaucher's) spleen were purchased from Avanti Polar Lipids and Sigma, respectively. Monogalactosyldiacylglycerol was purified from a crude lecithin fraction from soybean (Glycine max) by thin-layer chromatography (TLC). The purity of the lipids was confirmed by TLC and by straight phase HPLC (data not shown). Quantification of the glycolipids was performed by the anthrone method (24Morris D.L. Science. 1948; 107: 254-257Crossref PubMed Scopus (1155) Google Scholar). Sphingobase composition of GlcCer from P. pastoris and soybean were analyzed by HPLC and HPLC/MS after hydrolysis of the GlcCer and conversion of the sphingobases into 2,4-dinitrophenol derivatives as described previously (25Ternes P. Franke S. Zähringer U. Sperling P. Heinz E. J. Biol. Chem. 2002; 277: 25512-25518Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). The fatty acid composition of the GlcCer was determined by GLC/MS. Total fatty acid analysis of GlcCer was performed with 200-μg aliquots after methanolysis (1.5 ml of 2 m HCl/MeOH, 85 °C, 1 h) in sealed ampoules. The methanolyzed samples were dissolved in water, and the resulting fatty acid methyl esters were extracted three times with 3 ml of chloroform, concentrated to dryness, acetylated with 1 ml of pyridine/acetic anhydride (2:1, v/v; 80 °C, 1 h) concentrated, and analyzed by GLC/MS. GLC/MS analysis was performed with a HP-5MS column (30 m, Hewlett Packard) using a temperature gradient from 150 °C (3 min) to 320 °C at 5 °C min–1. EI and CI mass spectra were recorded as described previously (26Sperling P. Zähringer U. Heinz E. J. Biol. Chem. 1998; 273: 28590-28596Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Microtiter Plate Binding Assay (ELISA)—Interaction of antifungal peptides with GlcCer was evaluated by using an ELISA-based assay as described previously (27Mamelak D. Lingwood C. J. Biol. Chem. 2001; 276: 449-456Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 28Mylvaganam M. Binnington B. Hansen H.C. Magnusson G. Nyholm P.G. Lingwood C.A. Biochem. J. 2002; 368: 769-776Crossref PubMed Google Scholar, 29Mamelak D. Mylvaganam M. Tanahashi E. Ito H. Ishida H. Kiso M. Lingwood C. Carbohydr. Res. 2001; 335: 91-100Crossref PubMed Scopus (30) Google Scholar, 30Toledo M.S. Suzuki E. Levery S.B. Straus A.H. Takahashi H.K. Glycobiology. 2001; 11: 105-112Crossref PubMed Scopus (31) Google Scholar). Stock solutions of all glycolipids were prepared in methanol:chloroform:water (16:16:5, v/v/v) at a concentration of 500 μm. Lipids were applied in 75-μl aliquots to the wells of microtiter plates and allowed to dry overnight at room temperature. All subsequent handling steps were performed at 37 °C. Blocking buffer was 1% (w/v) gelatin (cold fish skin, Sigma) in phosphate-buffered saline, and washing buffer was 10% blocking buffer. Anti-RsAFP2 and anti-heliomicin rabbit antiserum and phosphatase-coupled goat anti-rabbit immunoglobulin were 1,000-fold diluted in washing buffer. Plotted values are means of triplicates adjusted for the plate background. Plate background values are the absorbance readings of methanol: chloroform:water (16:16:5, v/v/v)-coated wells incubated with peptides and antisera. P. pastoris and C. albicans Mutant Strains Lacking GlcCer Are Resistant to RsAFP2—To investigate a possible role of GlcCer in RsAFP2-mediated growth inhibition, mutants of P. pastoris and C. albicans which are completely devoid of GlcCer were used (13Leipelt M. Warnecke D. Zähringer U. Ott C. Müller F. Hube B. Heinz E. J. Biol. Chem. 2001; 276: 33621-33629Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The gcs-deletion mutants and their corresponding parental strains were tested for sensitivity to growth inhibition by RsAFP2. C. albicans and P. pastoris are sensitive to RsAFP2 at concentrations of 1–2 μm and higher, whereas the corresponding gcs-deletion strains are at least 20-fold more resistant to RsAFP2 (Table I). To test whether the GCS gene is also involved in conferring sensitivity toward other plant defensins, the gcs-deletion strains and the corresponding parental strains were tested for sensitivity to growth inhibition by dahlia plant defensin DmAMP1. DmAMP1 is equally active on both gcs-deletion mutants and the corresponding parental strains (Table I). The involvement of GlcCer in the process leading to fungal growth inhibition by RsAFP2 was supported further by the observation that S. cerevisiae and Schizosaccharomyces pombe, two yeast species that lack GlcCer in their membranes (16Sakaki T. Zähringer U. Warnecke D.C. Fahl A. Knogge W. Heinz E. Yeast. 2001; 18: 679-695Crossref PubMed Scopus (88) Google Scholar, 18Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1426: 347-357Crossref PubMed Scopus (170) Google Scholar), are fully resistant to RsAFP2 (Table I). To examine further the involvement of GlcCer in RsAFP2 activity, we envisaged to test RsAFP2 sensitivity of a S. cerevisiae strain expressing an heterologous GCS gene (13Leipelt M. Warnecke D. Zähringer U. Ott C. Müller F. Hube B. Heinz E. J. Biol. Chem. 2001; 276: 33621-33629Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). However, GlcCer analysis of such S. cerevisiae strains showed the presence of novel types of GlcCer components, consisting of a 4-hydroxysphinganine and a very long chain fatty acid (C24 or C26) (13Leipelt M. Warnecke D. Zähringer U. Ott C. Müller F. Hube B. Heinz E. J. Biol. Chem. 2001; 276: 33621-33629Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). GlcCer consisting of the characteristic 4,8-diunsaturated, C9-methyl-branched sphingobase linked to a long chain fatty acid (C16 or C18), as present in membranes of P. pastoris and C. albicans (Fig. 2 and Table II) (16Sakaki T. Zähringer U. Warnecke D.C. Fahl A. Knogge W. Heinz E. Yeast. 2001; 18: 679-695Crossref PubMed Scopus (88) Google Scholar), could not be detected in such GCS-expressing S. cerevisiae strains, which makes this approach unfeasible.Table IAntifungal activity of plant defensins and insect heliomicin toward various yeast speciesYeast speciesMICaMIC values are the minimal concentrations (μm) of the peptides required to inhibit the growth of a yeast strain by 100%. Data are the means of duplicate measurements. Standard errors were typically below 6.5%.RsAFP2DmAMP1HeliomicinμmS. cerevisiae>40bPDB growth medium used.0.32bPDB growth medium used.>20bPDB growth medium used.S. pombe>40bPDB growth medium used.NDcND, not determined.NDcND, not determined.P. pastoris2.0dPDB/YPD growth medium used.1.25dPDB/YPD growth medium used.2.5bPDB growth medium used.P. pastoris gcs-deletion mutant>40dPDB/YPD growth medium used.1.25dPDB/YPD growth medium used.>40bPDB growth medium used.C. albicans2.5bPDB growth medium used.5bPDB growth medium used.2.5bPDB growth medium used.C. albicans gcs-deletion mutant>40bPDB growth medium used.5bPDB growth medium used.>20bPDB growth medium used.a MIC values are the minimal concentrations (μm) of the peptides required to inhibit the growth of a yeast strain by 100%. Data are the means of duplicate measurements. Standard errors were typically below 6.5%.b PDB growth medium used.c ND, not determined.d PDB/YPD growth medium used. Open table in a new tab Table IISphingobase and fatty acid composition of glucosylceramides from P. pastoris and soybeanSphingobaseMol %aRelative amounts in mol % are given. Components of less than 2 mol % are omitted.Fatty acidMol %Ref.P. pastoris 9-Methyl-d18:2Δ4trans,Δ8trans6918:0-(2-OH)85This work and 16Sakaki T. Zähringer U. Warnecke D.C. Fahl A. Knogge W. Heinz E. Yeast. 2001; 18: 679-695Crossref PubMed Scopus (88) Google Scholar d18:2Δ4trans,Δ8trans1916:0-(2-OH)6 d18:1Δ4trans1217:0-(2-OH)620:0-(2-OH)2Soybean d18:2Δ4trans,Δ8trans6216:0-(2-OH)64This work and 48Sullards M.C. Lynch D.V. Merrill Jr., A.H. Adams J. J. Mass Spectrom. 2000; 35: 347-353Crossref PubMed Scopus (100) Google Scholar d18:2Δ4trans,Δ8cis3324:0(2-OH)18 t18:1Δ8cis322:0-(2-OH)1623:0-(2-OH)2Human d18:1Δ4transNDbND, not determined.24:033Sigma; Matreya, Inc.22:02416:01424:11323:09a Relative amounts in mol % are given. Components of less than 2 mol % are omitted.b ND, not determined. Open table in a new tab RsAFP2 Interacts with Fungal GlcCer but Not with Soybean or Human GlcCer—To get more insight in the involvement of fungal GlcCer in RsAFP2-mediated growth inhibition, the interaction between RsAFP2 and fungal GlcCer was assessed. In contrast to plant defensins DmAMP1 and HsAFP1 (7Thevissen K. Osborn R.W. Acland D.P. Broekaert W.F. J. Biol. Chem. 1997; 272: 32176-32181Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 8Thevissen K. Osborn R.W. Acland D.P. Broekaert W.F. Mol. Plant Microbe Interact. 2000; 13: 54-61Crossref PubMed Scopus (113) Google Scholar), binding assays using radiolabeled RsAFP2 could not be performed. Radiolabeling of RsAFP2 with t-butoxycarbonyl-l-[35S]methionine N-hydroxysuccinimidylester interfered with the antifungal action of RsAFP2, resulting in a 5-fold activity decrease. Therefore, we used an ELISA-based binding assay in which glycolipids are coated to the wells of microtiter plates and interacting peptides are detected immunologically, as described previously (27Mamelak D. Lingwood C. J. Biol. Chem. 2001; 276: 449-456Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 28Mylvaganam M. Binnington B. Hansen H.C. Magnusson G. Nyholm P.G. Lingwood C.A. Biochem. J. 2002; 368: 769-776Crossref PubMed Google Scholar, 29Mamelak D. Mylvaganam M. Tanahashi E. Ito H. Ishida H. Kiso M. Lingwood C. Carbohydr. Res. 2001; 335: 91-100Crossref PubMed Scopus (30) Google Scholar). The following glycolipids were purified, characterized, and used in the assay: GlcCer from P. pastoris, human spleen, and soybean, and monogalactosyldiacylglycerols from soybean. These glycolipids were devoid of detectable contaminations as confirmed by TLC and HPLC analysis (data not shown). The structures of the sphingobase and the fatty acid moieties of fungal and plant GlcCer were analyzed by reversed phase HPLC after hydrolysis of the purified GlcCer and conversion of the sphingobases into their 2,4-dinitrophenol derivatives. Whereas soybean GlcCer contain mainly the diunsaturated sphingobases (4E,8E)-sphinga-4,8-dienine (d18:2Δ4trans,Δ8trans) and (4E,8Z)-sphinga-4,8-dienine (d18:2Δ4trans,Δ8cis), the dominating sphingobase of GlcCer from P. pastoris is diunsaturated but carries a methyl group at C-9 (4E,8E)-9-methylsphinga-4,8-dienine (9-methyl-d18:2Δ4trans,Δ8trans) (Fig. 2 and Table II). Two additional glycolipids were used as controls in the assay. GlcCer from human (Gaucher's) spleen containing mainly (4E)-sphing-4-enine (d18:1Δ4trans) (Sigma; Matreya Inc., State College, PA) and monogalactosyldiacylglycerols from soybean, which contain a β-galactosyl headgroup and a diacylglycerol backbone instead of ceramide. The fatty acids of P. pastoris and soybean GlcCer differ only in the carbon chain length. Although soybean GlcCer contain saturated C16,C22, and C24 α-hydroxy fatty acids, a C18 α-hydroxy fatty acid is dominating in GlcCer from P. pastoris (Table II). In contrast, GlcCer from human (Gaucher's) spleen contain mainly nonhydroxy C24 and C22 very long chain fatty acids (Sigma; Matreya). Using the ELISA-based binding assay, RsAFP2 was found to interact in a dose-dependent manner with purified GlcCer from P. pastoris (Fig. 3A). No significant interaction of RsAFP2 with different concentrations of neither human or soybean GlcCer or with soybean monogalactosyldiacylglycerols could be detected, indicating that RsAFP2 interacts selectively with P. pastoris GlcCer. Because sphingolipids, including GlcCer, are associated with sterols in the plasma membrane to form rafts (31Xu X. Bittman R. Duportail G. Heissler D. Vilcheze C. London E. J. Biol. Chem. 2001; 276: 33540-33546Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 32Bagnat M. Keranen S. Shevchenko A. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (502) Google Scholar), we further investigated the effect of ergosterol, the main fungal sterol, on the interaction of RsAFP2 with fungal GlcCer. In a similar approach, we demonstrated recently the positive effect of ergosterol on the interaction of DmAMP1 with sphingolipids from S. cerevisiae (12Thevissen K. Francois I.E.J.A. Takemoto J.Y. Ferket K.K.A. Meert E.M.K. Cammue B.P.A. FEMS Microbiol. Lett. 2003; 226: 169-173Crossref PubMed Scopus (100) Google Scholar). Interaction of 50 nm RsAFP2 with different concentrations of ergosterol (ranging from 0.1 nmol to 5 nmol coated/well) was assessed. Under these conditions, no interaction of RsAFP2 with ergosterol could be observed (data not shown). Furthermore, interaction of 50 nm RsAFP2 with lipid mixtures consisting of 1 nmol of GlcCer and different amounts of ergosterol was assessed. However, no increase in RsAFP2 interaction with fungal GlcCer in the presence of various concentrations of ergosterol could be observed (data not shown). Link between the Antifungal Activity of RsAFP2 and Its Interaction with Fungal GlcCer—To address the link between the antifungal activity of RsAFP2 and its interaction with fungal GlcCer, the interaction of an RsAFP2 variant that is devoid of antifungal activity (RsAFP2(Y38G)) (21De Samblanx G.W. Goderis I.J. Thevissen K. Raemaekers R. Fant F. Borremans F. Acland D.P. Osborn R.W. Patel S. Broekaert W.F. J. Biol. Chem. 1997; 272: 1171-1179Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) with fungal GlcCer was assessed. Similarly to RsAFP2, RsAFP2(Y