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.     

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2‐hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast‐to‐hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24‐ceramides in membranes of RsAFP2‐treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.     

Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi.

Glucosylceramides are membrane lipids in most eukaryotic organisms and in a few bacteria. The physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in plants, fungi, and bacteria. In an attempt to establish appropriate experimental systems to study glucosylceramide functions in these organisms, we performed a systematic functional analysis of a glycosyltransferase gene family with members of animal, plant, fungal, and bacterial origin. Deletion of such putative glycosyltransferase genes in Candida albicans and Pichia pastoris resulted in the complete loss of glucosylceramides. When the corresponding knock-out strains were used as host cells for homologous or heterologous expression of candidate glycosyltransferase genes, five novel glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase) genes were identified from the plant Gossypium arboreum (cotton), the nematode Caenorhabditis elegans, and the fungi Magnaporthe grisea, Candida albicans, and P. pastoris. The glycosyltransferase gene expressions led to the biosynthesis of different molecular species of glucosylceramides that contained either C18 or very long chain fatty acids. The latter are usually channeled exclusively into inositol-containing sphingolipids known from Saccharomyces cerevisiae and other yeasts. Implications for the biosynthesis, transport, and function of sphingolipids will be discussed.     

Isolation and characterization of Neurospora crassa mutants resistant to antifungal plant defensins

Twenty-five Neurospora crassa mutants obtained by chemical mutagenesis were screened for increased resistance to various antifungal plant defensins. Plant defensin-resistant N. crassa mutants were further tested for their cross-resistance towards other families of structurally different antimicrobial peptides. Two N. crassa mutants, termed MUT16 and MUT24, displaying resistance towards all plant defensins tested but not to structurally different antimicrobial peptides were selected for further characterization. MUT16 and MUT24 were more resistant towards plant defensin-induced membrane permeabilization as compared to the N. crassa wild-type. Based on the previously demonstrated key role of fungal sphingolipids in the mechanism of growth inhibition by plant defensins, membrane sphingolipids of MUT16 and MUT24 were analysed. Membranes of these mutants contained structurally different glucosylceramides, novel glycosylinositolphosphorylceramides, and an altered level of steryl glucosides. Evidence is provided to link these clear differences in sphingolipid profiles of N. crassa mutants with their resistance towards different plant defensins.     

Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastoris

Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris , which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae . Using P. pastoris psd1 Δ and psd2 Δ mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP)–ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris , which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes.     

Comparative signature-tagged mutagenesis identifies Pseudomonas factors conferring resistance to the pulmonary collectin SP-A

The pulmonary collectin, surfactant protein A (SP-A), is a broad spectrum opsonin with microbicidal membrane permeabilization properties that plays a role in the innate immune response of the lung. However, the factors that govern SP-A's microbial specificity and the mechanisms by which it mediates membrane permeabilization and opsonization are not fully understood. In an effort to identify bacterial factors that confer susceptibility or resistance to SP-A, we used comparative signature-tagged mutagenesis to screen a library of 1,680 Pseudomonas aeruginosa mutants for evidence of differential pulmonary clearance in SP-A-sufficient (SP-A) and SP-A-deficient (SP-A) mice. Two SP-A-sensitive P. aeruginosa mutants harboring transposon insertions in genes required for salicylate biosynthesis (pch) and phosphoenolpyruvate-protein-phosphotransferase (ptsP) were recovered. The mutants were indistinguishable from the parental wild-type PA01 with regard to opsonization by SP-A, but they exhibited increased susceptibility to SP-A-mediated membrane permeabilization. These results suggest that bacterial gene functions that are required to maintain membrane integrity play crucial roles in resistance of P. aeruginosa to the permeabilizing effects of SP-A.     

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.     

A peptide based on the pore-forming domain of pro-apoptotic poliovirus 2B viroporin targets mitochondria

Non-structural poliovirus 2B protein induces plasma membrane permeabilization and has been recently implicated in triggering apoptosis via the mitochondrial pathway. Here we describe that the pore-forming P3 peptide, based on the 2B amphipathic domain, translocates through the plasma membrane of culture cells and targets mitochondria. Cell permeabilization by P3 versions of different lengths, together with peptide uptake analyses supported an internalization mechanism dependent on P3 capacity to interact physically with lipid bilayers and establish permeating pores therein. Internalized P3 was found associated with mitochondria, but contrary to the parental 2B protein, the short peptide did not affect the morphology or cell distribution of these organelles, nor induced apoptosis. We conclude that P3 constitutes a mitochondriotropic sequence, which is however devoid of 2B pro-apoptotic activity.     

Glutathione and thioredoxin peroxidases mediate susceptibility of yeast mitochondria to Ca(2+)-induced damage

The effect of thioredoxin peroxidases on the protection of Ca(2+)-induced inner mitochondrial membrane permeabilization was studied in the yeast Saccharomyces cerevisiae using null mutants for these genes. Since deletion of a gene can promote several other effects besides the absence of the respective protein, characterizations of the redox state of the mutant strains were performed. Whole cellular extracts from all the mutants presented lower capacity to decompose H(2)O(2) and lower GSH/GSSG ratios, as expected for strains deficient for peroxide-removing enzymes. Interestingly, when glutathione contents in mitochondrial pools were analyzed, all mutants presented lower GSH/GSSG ratios than wild-type cells, with the exception of DeltacTPxI strain (cells in which cytosolic thioredoxin peroxidase I gene was disrupted) that presented higher GSH/GSSG ratio. Low GSH/GSSG ratios in mitochondria increased the susceptibility of yeast to damage induced by Ca(2+) as determined by membrane potential and oxygen consumption experiments. However, H(2)O(2) removal activity appears also to be important for mitochondria protection against permeabilization because exogenously added catalase strongly inhibited loss of mitochondrial potential. Moreover, exogenously added recombinant peroxiredoxins prevented inner mitochondrial membrane permeabilization. GSH/GSSG ratios decreased after Ca(2+) addition, suggesting that reactive oxygen species (ROS) probably mediate this process. Taken together our results indicate that both mitochondrial glutathione pools and peroxide-removing enzymes are key components for the protection of yeast mitochondria against Ca(2+)-induced damage.     

The flagellum of Pseudomonas aeruginosa is required for resistance to clearance by surfactant protein A

Surfactant protein A (SP-A) is an important lung innate immune protein that kills microbial pathogens by opsonization and membrane permeabilization. We investigated the basis of SP-A-mediated pulmonary clearance of Pseudomonas aeruginosa using genetically-engineered SP-A mice and a library of signature-tagged P. aeruginosa mutants. A mutant with an insertion into flgE, the gene that encodes flagellar hook protein, was preferentially cleared by the SP-A(+/+) mice, but survived in the SP-A(-/-) mice. Opsonization by SP-A did not play a role in flgE clearance. However, exposure to SP-A directly permeabilized and killed the flgE mutant, but not the wild-type parental strain. P. aeruginosa strains with mutation in other flagellar genes, as well as mucoid, nonmotile isolates from cystic fibrosis patients, were also permeabilized by SP-A. Provision of the wild-type fliC gene restored the resistance to SP-A-mediated membrane permeabilization in the fliC-deficient bacteria. In addition, non-mucoid, motile revertants of CF isolates reacquired resistance to SP-A-mediated membrane permeability. Resistance to SP-A was dependent on the presence of an intact flagellar structure, and independent of flagellar-dependent motility. We provide evidence that flagellar-deficient mutants harbor inadequate amounts of LPS required to resist membrane permeabilization by SP-A and cellular lysis by detergent targeting bacterial outer membranes. Thus, the flagellum of P. aeruginosa plays an indirect but important role resisting SP-A-mediated clearance and membrane permeabilization.     

Sterol glucosyltransferases have different functional roles in Pichia pastoris and Yarrowia lipolytica

Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia lipolytica defective in the orthologue of UGT51 (encoding sterol glucosyltransferase) were isolated and compared. These mutants do not contain the specific ergosterol derivate, ergosterol glucoside. We observed that the P. pastoris UGT51 gene is required for pexophagy, the process by which peroxisomes containing methanol-metabolizing enzymes are selectively shipped to and degraded in the vacuole upon shifting methanol-grown cells of this yeast to glucose or ethanol. PpUGT51 is also required for other vacuole related processes. In contrast, the Y. lipolytica UGT51 gene is required for utilization of decane, but not for pexophagy. Thus, sterol glucosyltransferases play different functional roles in P. pastoris and Y. lipolytica.     

Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin.

The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.     

Control of glucosylceramide production and morphogenesis by the Bar1 ceramide synthase in Fusarium graminearum

The contribution of plasma membrane proteins to the virulence of plant pathogenic fungi is poorly understood. Accordingly, the objective of this study was to characterize the acyl-CoA dependent ceramide synthase Bar1 (previously implicated in plasma membrane organization) in the wheat pathogen Fusarium graminearum. The role of Bar1 in mediating cell membrane organization was confirmed as ΔBAR1 mutants failed to display a distinct sterol-rich domain at the hyphal tip. The ΔBAR1 mutants were non-pathogenic when inoculated onto wheat heads, and their in vitro growth also was severely perturbed. ΔBAR1 mutants were incapable of producing perithecia (sexual fruiting structures) and only produced macroconidia (asexual spores) in the presence of NaCl. Sphingolipid analyses indicated that Bar1 is specifically necessary for the production of glucosylceramides in both F. graminearum and Aspergillus nidulans. Interestingly, glucosylceramides appear to mediate sensitivity to heat stable antifungal factor (HSAF), as, in addition to ΔBAR1 mutants, a glucosylceramide synthase deficient mutant of Yarrowia lipolytica is also resistant to HSAF.     

Characterization of sugar transport in 2-deoxy-d-glucose resistant mutants of yeast

Summary A number of 2-deoxy-d-glucose (2-DOG) resistant mutants exhibiting resistance to glucose repression were isolated from variousSaccharomyces yeast strains. Most of the mutants isolated were observed to have improved maltose uptake ability in the presence of glucose. Fermentation studies indicated that maltose was taken up at a faster rate and glucose taken up at a slower rate in the mutant strains compared to the parental strains, when these sugars were fermented together. When these sugars were fermented separately, only the 2-DOG resistant mutant obtained fromSaccharomyces cerevisiae strain 1190 exhibited alterations in glucose and maltose uptake compared to the parental strain. Kinetic analysis of sugar transport employing radiolabelled glucose and maltose indicated that both glucose and maltose were transported with higher rates in the mutant strain. These results suggested that the high affinity glucose transport system was regulated by glucose repression in the parental strain but was derepressed in the mutant.     

P1 bacteriophage and tellurite sensitivity in Klebsiella pneumoniae and Escherichia coli

Klebsiella pneumoniae and Escherichia coli respond inversely toward P1 bacteriophage or TeO3(-2). Klebsiella pneumoniae is resistant to both antagonists and E. coli is sensitive. However, P1 cmts lysogens (P1 cmts resistant) of K. pneumoniae became sensitive to tellurite and when cured from P1 cmts regained resistance. Escherichia coli spontaneous mutants selected for resistance to either P1 or TeO3(-2) were collaterally resistant to the other. As well, TeO3(-3) enhanced the adsorption of P1 vir to both E. coli and K. pneumoniae. Several outer membrane proteins were enhanced in the K. pneumoniae lysogens and were reduced in E. coli lysogens; one of which was the same molecular weight (77 000) in both bacteria. When partially purified it enhanced the plaque efficiency of P1 vir. Lipopolysaccharide (LPS) from E. coli C600 inactivated P1 vir, but neither the P1 lysogens nor LPS derived from the lysogens inactivated P1 vir. Escherichia coli P1 lysogens produced only heptose-deficient LPS. It is suggested that both LPS and outer membrane protein(s) comprise the P1 receptor. TeO3(-2) may interact with one or both components.     

Identification of a new family of genes involved in beta-1,2-mannosylation of glycans in Pichia pastoris and Candida albicans

Structural studies of cell wall components of the pathogenic yeast Candida albicans have demonstrated the presence of beta-1,2-linked oligomannosides in phosphopeptidomannan and phospholipomannan. During C. albicans infection, beta-1,2-oligomannosides play an important role in host/pathogen interactions by acting as adhesins and by interfering with the host immune response. Despite the importance of beta-1,2-oligomannosides, the genes responsible for their synthesis have not been identified. The main reason is that the reference species Saccharomyces cerevisiae does not synthesize beta-linked mannoses. On the other hand, the presence of beta-1,2-oligomannosides has been reported in the cell wall of the more genetically tractable C. albicans relative, P. pastoris. Here we present the identification, cloning, and characterization of a novel family of fungal genes involved in beta-mannose transfer. Employing in silico analysis, we identified a family of four related new genes in P. pastoris and subsequently nine homologs in C. albicans. Biochemical, immunological, and structural analyses following deletion of four genes in P. pastoris and deletion of four genes acting specifically on C. albicans mannan demonstrated the involvement of these new genes in beta-1,2-oligomannoside synthesis. Phenotypic characterization of the strains deleted in beta-mannosyltransferase genes (BMTs) allowed us to describe the stepwise activity of Bmtps and acceptor specificity. For C. albicans, despite structural similarities between mannan and phospholipomannan, phospholipomannan beta-mannosylation was not affected by any of the CaBMT1-4 deletions. Surprisingly, depletion in mannan major beta-1,2-oligomannoside epitopes had little impact on cell wall surface beta-1,2-oligomannoside antigenic expression.     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

Catalases and thioredoxin peroxidase protect Saccharomyces cerevisiae against Ca(2+)-induced mitochondrial membrane permeabilization and cell death

The involvement of reactive oxygen species in Ca(2+)-induced mitochondrial membrane permeabilization and cell viability was studied using yeast cells in which the thioredoxin peroxidase (TPx) gene was disrupted and/or catalase was inhibited by 3-amino-1,2, 4-triazole (ATZ) treatment. Wild-type Saccharomyces cerevisiae cells were very resistant to Ca(2+) and inorganic phosphate or t-butyl hydroperoxide-induced mitochondrial membrane permeabilization, but suffered an immediate decrease in mitochondrial membrane potential when treated with Ca(2+) and the dithiol binding reagent phenylarsine oxide. In contrast, S. cerevisiae spheroblasts lacking the TPx gene and/or treated with ATZ suffered a decrease in mitochondrial membrane potential, generated higher amounts of hydrogen peroxide and had decreased viability under these conditions. In all cases, the decrease in mitochondrial membrane potential could be inhibited by ethylene glycol-bis(beta-aminoethyl ether) N,N, N',N'-tetraacetic acid, dithiothreitol or ADP, but not by cyclosporin A. We conclude that TPx and catalase act together, maintaining cell viability and protecting S. cerevisiae mitochondria against Ca(2+)-promoted membrane permeabilization, which presents similar characteristics to mammalian permeability transition.     

Identification and susceptibility against fluconazole and albaconazole of 100 yeasts' strains isolated from vaginal discharge

Vulvovaginal candidiasis is a condition that affects a great number of fertile women. It is considered the second cause of genital infection after vaginosis due to GAM complex. Candida albicans is the most frequent isolated species from vaginal discharge. However, sometimes more than one yeast species could be found in the same clinical sample that are more resistant to antifungal drugs. Nowadays, it is necessary to identify properly up to species level the isolated microorganism and to determine the antifungal susceptibility profile. One hundred strains obtained from vaginal discharge of 94 patients suffering acute vulvovaginal candidiasis were studied. The identification of the isolates showed: C. albicans 86%, Candida glabrata 6%, Candida inconspicua 3%, Candida krusei 2% and Candida intermedia, Candida holmii and Trichosporon asahii one case each. Minimal inhibitory concentrations (MIC) of all the yeasts against fluconazole and albaconazole were performed. C. glabrata, C. krusei and C. inconspicua were the most resistant against fluconazole, on the other hand albicans was susceptible to this drug. All the isolates presented MIC against albaconazole much lower than fluconazole.