Defensins from insects and plants interact with fungal glucosylceramides

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.     

Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms

Cerebrosides are typical membrane lipids of many organisms. They occur in plants, fungi, animals, humans and some prokaryotes. Almost all of our knowledge on the physiological functions of cerebrosides results from experimental data obtained with mammalian cells. However, very little is known about the roles played by these lipids in plants and fungi. To initiate such investigations we have cloned and characterized a ceramide glucosyltransferase from the yeast Candida albicans. Functional expression of this gene in Saccharomyces cerevisiae led to the accumulation of new glycolipids which were not present in wild-type baker's yeast. They were identified by MS and NMR spectroscopy as beta-D-glucopyranosyl ceramides. The ceramide moieties of these cerebrosides comprised phytosphinganine and mainly long-chain (C(26)) alpha-hydroxy fatty acids in amide linkage. We also generated a ceramide glucosyltransferase-knock-out strain of C. albicans which was devoid of cerebrosides. The viability of this mutant showed that for this organism glucosyl ceramides are not essential for vegetative growth on complete or minimal media. In addition, we have cloned and functionally expressed one of the three putative glucosylceramide synthases from Caenorhabditis elegans, as well as a corresponding enzyme from Pichia pastoris.     

Existence of cerebroside in Saccharomyces kluyveri and its related species

Sphingolipids are ubiquitous compounds derived from ceramide that consist of a sphingoid long-chain base with a 2-amino group amide linked to fatty acid and are present in the membranes of many organisms. As a principal sphingolipid, Saccharomyces cerevisiae contains a free ceramide and its inositol-phosphorylated derivatives (acidic types) but not a neutral glycosylated ceramide, glucosylceramide (cerebroside), which usually appears in eukaryotic cells. When 31 strains accepted in the genera Saccharomyces, Torulaspora, Zygosaccharomyces, and Kluyveromyces were analyzed for sphingolipids, cerebrosides were found in S. kluyveri, Z. cidri, Z. fermentati, K. lactis, K. thermotolerans, and K. waltii. The cerebrosides of S. kluyveri and K. lactis included 9-methyl 4-trans, 8-trans-sphingadienine and its putative metabolic intermediates. A unique characteristic of S. kluyveri was the presence of a trihydroxy sphingoid base, which rarely occurs in fungal cerebrosides. A polymerase chain reaction with primers targeted to the glucosylceramide synthase gene of other microorganisms amplified the fragments of the expected size from S. kluyveri and K. lactis and further extended to the adjacent regions. The presumed protein of S. kluyveri had 54.4% similarity to that of K. lactis, higher than the glucosylceramide synthases from Candida albicans, Pichia pastoris, and other organisms. From these observations, the divergence of S. kluyveri from the lineage of K. lactis in their evolution is discussed.     

An inhibitor of glucosylceramide synthase inhibits the human enzyme, but not enzymes from other organisms

Specific inhibitors of glucosylceramide biosynthesis are used as drugs for the treatment of some human diseases correlated to glycosphingolipid metabolism. The target of the presently available inhibitors is the human glucosylceramide synthase (GCS), but effects on enzymes from other organisms have not been studied. We expressed cDNAs encoding GCS enzymes from lower animals, plants, fungi, and bacteria in the yeast P. pastoris. In vitro GCS assays with the GCS inhibitor D-threo-1-(3',4'-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol showed that this inhibitor did not affect non-human GCS enzymes.     

Identification of fungal sphingolipid C9-methyltransferases by phylogenetic profiling

Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24-71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and Delta4,8-desaturated ceramide as substrates.     

Two pathways of sphingolipid biosynthesis are separated in the yeast Pichia pastoris

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C(16)/C(18) acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis.     

Plant sphingolipids: structural diversity,biosynthesis, first genes and functions

In mammals and Saccharomyces cerevisiae, sphingolipids have been a subject of intensive research triggered by the interest in their structural diversity and in mammalian pathophysiology as well as in the availability of yeast mutants and suppressor strains. More recently, sphingolipids have attracted additional interest, because they are emerging as an important class of messenger molecules linked to many different cellular functions. In plants, sphingolipids show structural features differing from those found in animals and fungi, and much less is known about their biosynthesis and function. This review focuses on the sphingolipid modifications found in plants and on recent advances in the functional characterization of genes gaining new insight into plant sphingolipid biosynthesis. Recent studies indicate that plant sphingolipids may be also involved in signal transduction, membrane stability, host-pathogen interactions and stress responses.     

Intestinal absorption of dietary maize glucosylceramide in lymphatic duct cannulated rats

Sphingolipids are ubiquitous in all eukaryotic organisms. Various physiological functions of dietary sphingolipids, such as preventing colon cancer and improving the skin barrier function, have been recently reported. One of the common sphingolipids used as a foodstuff is glucosylceramide from plant sources, which is composed of sphingoid bases distinct from those of mammals. However, the fate of dietary sphingolipids derived from plants is still not understood. In this study, we investigated the absorption of maize glucosylceramide in the rat intestine using a lipid absorption assay of lymph from the thoracic duct. The free and complex forms of trans-4,cis-8-sphingadienine, the predominant sphingoid base of maize glucosylceramide, were found in the lymph after administration of maize glucosylceramide. This plant type of sphingoid base was detected in the ceramide fraction and N-palmitoyl-4,8-sphingadienine (C16:0-d18:2) and N-tricosanoyl-4,8-sphingadienine (C23:0-d18:2) were identified by LC-MS/MS. The cumulative recovery of 4t,8c-sphingadienine in the lymph was very low. These results indicate that dietary glucosylceramide originating from higher plants is slightly absorbed in the intestine and is incorporated into ceramide structures in the intestinal cells. However, it appears that the intact form of sphingoid bases is not reutilized well in the tissues.     

Interactions of Candida albicans with other Candida spp. and bacteria in the biofilms

AIMS: To study the interactions between Candida albicans and 12 other species of Candida and bacteria in biofilms. METHODS AND RESULTS: The number of cells within growing biofilms in a polystyrene tube model was measured after adding C. albicans to preformed biofilms of other micro-organisms and vice versa. It was also measured after simultaneous biofilm formation of C. albicans and other micro-organisms. The number of cells of C. albicans within the growing biofilms decreased significantly (P < 0.05) when the fungus was added to preformed biofilms of Candida spp. and bacteria except, with C. parapsilosis, Torulopsis glabrata and the glycocalyx producer Pseudomonas aeruginosa. When C. parapsilosis, Staphylococcus epidermidis (nonglycocalyx producer) or Serratia marcescens was added to preformed biofilms of C. albicans, the number of cells of these micro-organisms increased in the growing biofilms. CONCLUSIONS: Biofilms of C. albicans are capable of holding other micro-organisms and more likely to be heterogeneous with other bacteria and fungi in the environment and on medical devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of the heterogeneity of biofilm-associated organisms can influence treatment decisions, particularly in patients who do not respond to initial appropriate therapy.     

Plant Sphingolipids Today ‐ Are They Still Enigmatic?

Abstract: Sphingolipids are a diverse group of lipids found in all eukaryotes and some bacteria, consisting of a hydrophobic ceramide and a hydrophilic head group. We have summarised the contemporary understanding of the structure of plant sphingolipids with an emphasis on glucosylceramides and inositolphosphorylceramides. Plant glucosylceramides are important structural components of plasma and vacuole membranes. Inositolphosphorylceramides have been identified as moieties of the glycosylphosphorylinositol (GPI) anchors of plant proteins targeted to the plasma membrane. In the last few years, progress has been made in the cloning of plant genes coding for enzymes involved in sphingolipid metabolism. As found in yeast and mammals, the plant sphingolipid pathway is a potential generator of powerful cell signals. The role of plant sphingolipid metabolites in programmed cell death and calcium influx is discussed.     

Very long chain fatty acid and lipid signaling in the response of plants to pathogens

Recent findings indicate that lipid signaling is essential for plant resistance to pathogens. Besides oxylipins and unsaturated fatty acids known to play important signaling functions during plant-pathogen interactions, the very long chain fatty acid (VLCFA) biosynthesis pathway has been recently associated to plant defense through different aspects. VLCFAs are indeed required for the biosynthesis of the plant cuticle and the generation of sphingolipids. Elucidation of the roles of these lipids in biotic stress responses is the result of the use of genetic approaches together with the identification of the genes/proteins involved in their biosynthesis. This review focuses on recent observations which revealed the complex function of the cuticle and cuticle-derived signals, and the key role of sphingolipids as bioactive molecules involved in signal transduction and cell death regulation during plant-pathogen interactions.Key words: very long chain fatty acids (VLCFAs), plant-pathogen interactions, lipid signaling, sphingolipids, epicuticular waxes, lipid rafts, cuticle, plant defense     

Two-component signal transduction in human fungal pathogens

Signal transduction pathways provide mechanisms for adaptation to stress conditions. One of the most studied of these pathways is the HOG1 MAP kinase pathway that in Saccharomyces cerevisiae is used to adapt cells to osmostress. The HOG1 MAPK has also been studied in Candida albicans, and more recently observations on the Hog1p functions have been described in two other human pathogens, Aspergillus fumigatus and Cryptococcus neoformans. The important, but not surprising, concept is that this pathway is used for different yet similar functions in each of these fungi, given their need to adapt to different environmental signals. Current studies of C. albicans focus upon the identification of two-component signal proteins that, in both C. albicans and S. cerevisiae, regulate the HOG1 MAPK. In C. albicans, these proteins regulate cell wall biosynthesis (and, therefore, adherence to host cells), osmotic and oxidant adaptation, white-opaque switching, morphogenesis, and virulence of the organism.     

Adipocytes harbor a glucosylceramide biosynthesis pathway involved in iNKT cell activation

BackgroundNatural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis.ResultsInhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity.ConclusionGlucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes.SignificanceUnraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction.Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes.UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. β-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFNγ and IL4 secretion).Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue.Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes.     

Lysine biosynthesis in selected pathogenic fungi: characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans.

The alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena. Until now, this unique metabolic pathway has never been investigated in the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. Five of the eight enzymes (homocitrate synthase, homoisocitrate dehydrogenase, alpha-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase) of the alpha-aminoadipate pathway and glucose-6-phosphate dehydrogenase, a glycolytic enzyme used as a control, were demonstrated in wild-type cells of these organisms. All enzymes were present in Saccharomyces cerevisiae and the pathogenic organisms except C. neoformans 32608 serotype C, which exhibited no saccharopine reductase activity. The levels of enzyme activity varied considerably from strain to strain. Variation among organisms was also observed for the control enzyme. Among the pathogens, C. albicans exhibited much higher homocitrate synthase, homoisocitrate dehydrogenase, and alpha-aminoadipate reductase activities. Seven lysine auxotrophs of C. albicans and one of Candida tropicalis were characterized biochemically to determine the biochemical blocks and gene-enzyme relationships. Growth responses to alpha-aminoadipate- and lysine-supplemented media, accumulation of alpha-aminoadipate semialdehyde, and the lack of enzyme activity revealed that five of the mutants (WA104, WA153, WC7-1-3, WD1-31-2, and A5155) were blocked at the alpha-aminoadipate reductase step, two (STN57 and WD1-3-6) were blocked at the saccharopine dehydrogenase step, and the C. tropicalis mutant (X-16) was blocked at the saccharopine reductase step. The cloned LYS1 gene of C. albicans in the recombinant plasmid YpB1078 complemented saccharopine dehydrogenase (lys1) mutants of S. cerevisiae and C. albicans. The Lys1+ transformed strains exhibited significant saccharopine dehydrogenase activity in comparison with untransformed mutants. The cloned LYS1 gene has been localized on a 1.8-kb HindIII DNA insert of the recombinant plasmid YpB1041RG1. These results established the gene-enzyme relationship in the second half of the alpha-aminoadipate pathway. The presence of this unique pathway in the pathogenic fungi could be useful for their rapid detection and control.     

The antifungal activity of RsAFP2, a plant defensin from raphanus sativus, involves the induction of reactive oxygen species in Candida albicans

RsAFP2 (Raphanus sativus antifungal peptide 2), an antifungal plant defensin isolated from seed of R. sativus, interacts with glucosylceramides (GlcCer) in membranes of susceptible yeast and fungi and induces membrane permeabilization and fungal cell death. However, using carboxyfluorescein-containing small unilamellar vesicles containing purified GlcCer, we could not observe permeabilization as a consequence of insertion of RsAFP2 in such vesicles. Therefore, we focused on a putative RsAFP2-induced signaling cascade downstream of RsAFP2-binding to GlcCer in fungal membranes. We show that RsAFP2 induces reactive oxygen species (ROS) in Candida albicans wild type in a dose-dependent manner, but not at all in an RsAFP2-resistant DeltagcsC. albicans mutant that lacks the RsAFP2-binding site in its membranes. These findings indicate that upstream binding of RsAFP2 to GlcCer is needed for ROS production leading to yeast cell death. Moreover, the antioxidant ascorbic acid blocks RsAFP2-induced ROS generation, as well as RsAFP2 antifungal activity. These data point to the presence of an intracellular plant defensin-induced signaling cascade, which involves ROS generation and leads to fungal cell growth arrest.     

基于基因本体论的模式生物分子功能分布异同

基因本体论是关于基因和蛋白质知识的标准词汇,也是今后实现各种与基因相关的数据统一、数据转换、数据挖掘的基础。本文通过分子功能基因本体论比较了不同模式生物基因产物分子功能分布的异同。结果发现:在动物类、植物类以及真菌类模式生物中,大部分已知功能基因的分布比例是基本一致的,存在一定的同源性;但在动物中结合类基因数量较多而在植物与真菌中反而催化类基因数量较多,信号传导相关基因在动物中的分布数量多间接证明了动物在进化上的高等性,而植物中特有的大分子传递相关编码基因,可能与植物的养分、水分在机体种的传输相关。     

Processive lipid galactosyl/glucosyltransferases from Agrobacterium tumefaciens and Mesorhizobium loti display multiple specificities

The glycosyltransferase family 21 (GT21) includes both enzymes of eukaryotic and prokaryotic organisms. Many of the eukaryotic enzymes from animal, plant, and fungal origin have been characterized as uridine diphosphoglucose (UDP-Glc):ceramide glucosyltransferases (glucosylceramide synthases [Gcs], EC 2.4.1.80). As the acceptor molecule ceramide is not present in most bacteria, the enzymatic specificities and functions of the corresponding bacterial glycosyltransferases remain elusive. In this study, we investigated the homologous and heterologous expression of GT21 enzymes from Agrobacterium tumefaciens and Mesorhizobium loti in A. tumefaciens, Escherichia coli, and the yeast Pichia pastoris. Glycolipid analyses of the transgenic organisms revealed that the bacterial glycosyltransferases are involved in the synthesis of mono-, di- and even tri-glycosylated glycolipids. As products resulting from their activity, we identified 1,2-diacyl-3-(O-beta-D-galacto-pyranosyl)-sn-glycerol, 1,2-diacyl-3-(O-beta-D-gluco-pyranosyl)-sn-glycerol as well as higher glycosylated lipids such as 1,2-diacyl-3-[O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->6)-O-beta-D-gluco-pyranosyl]-sn-glycerol, and the deviatingly linked diglycosyldiacylglycerol 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->3)-O-beta-D-galacto-pyranosyl]-sn-glycerol. From a mixture of triglycosyldiacylglycerols, 1,2-diacyl-3-[O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol could be separated in a pure form. In vitro enzyme assays showed that the glycosyltransferase from A. tumefaciens favours uridine diphosphogalactose (UDP-Gal) over UDP-Glc. In conclusion, the bacterial GT21 enzymes differ from the eukaryotic ceramide glucosyltransferases by the successive transfer of up to three galactosyl and glucosyl moieties to diacylglycerol.     

Inositol phosphoryl transferases from human pathogenic fungi

The IPC1 gene from Saccharomyces cerevisiae, which encodes inositolphosphorylceramide (IPC) synthase, was first identified as a novel and essential gene encoding resistance to the natural product antifungal aureobasidin A (AUR1). The formation of IPC in fungi is essential for viability, suggesting inhibitors of IPC1p function would make ideal antifungal drug candidates. Homologs of the AUR1/IPC1 gene were identified from a number of human pathogenic fungi, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans. Comparison of these genes with other homologous genes from Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe reveals a conserved structural motif for inositolphosphoryl transferases which is similar to a motif recently described for lipid phosphatases, but with unique characteristics.