The Extracellular Vesicles Containing Inorganic Polyphosphate of Candida Yeast upon Growth on Hexadecane.

The cell wall of Candida yeast grown on presence of hexadecane as a sole carbon source undergoes structural and functional changes including the formation of specific supramolecular complexes-canals. The canals contain specific polysaccharides and enzymes that provide primary oxidization of alkanes. In addition, inorganic polyphosphate (polyP) was identified in Candida maltosa canals. The aim of the work was a comparative study of the features of cell walls and extracellular structures in yeast C. maltosa, C. albicans and C. tropicalis with special attention to inorganic polyphosphates as possible part of these structures when grown on the widely used xenobiotic hexadecane (diesel fuel). Fluorescence microscopy with DAPI has shown an unusual localization of polyP on the cell surface and in the exovesicles in the three yeast species, when growing on hexadecane. Electron-scanning microscopy showed that the exovesicles were associated with the cell wall and also presented in the external environment probably as biofilm components. Treatment of hexadecane-grown cells with purified Ppx1 polyphosphatase led to the release of phosphate into the incubation medium and the disappearance of polyP in vesicles and cell wall observed using microscopic methods. The results indicate the important role of polyP in the formation of extracellular structures in the Candida yeast when consuming hexadecane and are important for the design of xenobiotic destructors based on yeast or mixed cultures.

Effect of Deletions of the Genes Encoding Pho3p and Bgl2p on Polyphosphate Level, Stress Adaptation, and Attachments of These Proteins to Saccharomyces cerevisiae Cell Wall.

Inorganic polyphosphates (polyP), according to literature data, are involved in the regulatory processes of molecular complex of the Saccharomyces cerevisiae cell wall (CW). The aim of the work was to reveal relationship between polyP, acid phosphatase Pho3p, and the major CW protein, glucanosyltransglycosylase Bgl2p, which is the main glucan-remodelling enzyme with amyloid properties. It has been shown that the yeast cells with deletion of the PHO3 gene contain more high molecular alkali-soluble polyP and are also more resistant to exposure to alkali and manganese ions compared to the wild type strain. This suggests that Pho3p is responsible for hydrolysis of the high molecular polyP on the surface of yeast cells, and these polyP belong to the stress resistance factors. The S. cerevisiae strain with deletion of the BGL2 gene is similar to the Δpho3 strain both in the level of high molecular alkali-soluble polyP and in the increased resistance to alkali and manganese. Comparative analysis of the CW proteins demonstrated correlation between the extractability of the acid phosphatase and Bgl2p, and also revealed a change in the mode of Bgl2p attachment to the CW of the strain lacking Pho3p. It has been suggested that Bgl2p and Pho3p are able to form a metabolon or its parts that connects biogenesis of the main structural polymer of the CW, glucan, and catabolism of an important regulatory polymer, polyphosphates.

Multiple effects of the PHO91 gene knockout in Ogataea parapolymorpha.

Pho91 is a vacuolar phosphate transporter that exports phosphate from the vacuolar lumen to the cytosol in yeast cells. In this study, we have demonstrated the pleiotropic effects of the PHO91 gene knockout in the methylotrophic yeast Ogataea parapolymorpha (Hansenula polymorpha, Ogataea angusta). The content of both acid-soluble and acid-insoluble inorganic polyphosphate (polyP) in the ∆pho91 cells was slightly higher compared to the strain with wild-type PHO91, when the cells were cultivated on glucose. The pho91-Δ mutations both in O. parapolymorpha and in Saccharomyces cerevisiae diminished resistance to cadmium and increased resistance to manganese and peroxide stresses. The cells of the mutant strain of O. parapolymorpha were unable to consume methanol due to the lack of methanol oxidase activity. We speculate that these effects are associated with the inability of mutant cells to mobilize phosphate from the vacuolar pool and/or defects in the signaling pathways involving phosphate, polyP, and inositol polyphosphates.

PHM6 and PHM7 genes are essential for phosphate surplus in the cells of Saccharomyces cerevisiae.

The cells of Saccharomyces cerevisiae are capable for phosphate surplus: the increased uptake of phosphate (Pi) and accumulation of inorganic polyphosphate (polyP) occur when the cells after Pi limitation were cultivated in a medium supplemented with Pi. We demonstrated that single knockout mutations in the PHO84, PHO87, and PHO89 genes encoding plasma membrane phosphate transporters suppressed the Pi uptake and polyP accumulation under phosphate surplus at nitrogen starvation. The knockout strains in the PHM6 and PHM7 genes encoding unannotated PHO-proteins showed decreased polyP accumulation under Pi surplus both at nitrogen starvation and in complete YPD medium. This is due to the suppression of Pi uptake in the cells of these mutant strains. We speculate that Pi transporters of plasma membrane, and Phm6 and Phm7 proteins function in concert providing increased Pi uptake at phosphate surplus conditions.

The γ-Core Motif Peptides of AMPs from Grasses Display Inhibitory Activity against Human and Plant Pathogens.

Antimicrobial peptides (AMPs) constitute an essential part of the plant immune system. They are regarded as alternatives to conventional antibiotics and pesticides. In this study, we have identified the γ-core motifs, which are associated with antimicrobial activity, in 18 AMPs from grasses and assayed their antimicrobial properties against nine pathogens, including yeasts affecting humans, as well as plant pathogenic bacteria and fungi. All the tested peptides displayed antimicrobial properties. We discovered a number of short AMP-derived peptides with high antimicrobial activity both against human and plant pathogens. For the first time, antimicrobial activity was revealed in the peptides designed from the 4-Cys-containing defensin-like peptides, whose role in plant immunity has remained unknown, as well as the knottin-like peptide and the C-terminal prodomain of the thionin, which points to the direct involvement of these peptides in defense mechanisms. Studies of the mode of action of the eight most active γ-core motif peptides on yeast cells using staining with propidium iodide showed that all of them induced membrane permeabilization leading to cell lysis. In addition to identification of the antimicrobial determinants in plant AMPs, this work provides short candidate peptide molecules for the development of novel drugs effective against opportunistic fungal infections and biopesticides to control plant pathogens.

Synthetic Oligopeptides Mimicking γ-Core Regions of Cysteine-Rich Peptides of Solanum lycopersicum Possess Antimicrobial Activity against Human and Plant Pathogens.

Plant cysteine-rich peptides (CRPs) represent a diverse group of molecules involved in different aspects of plant physiology. Antimicrobial peptides, which directly suppress the growth of pathogens, are regarded as promising templates for the development of next-generation pharmaceuticals and ecologically friendly plant disease control agents. Their oligopeptide fragments are even more promising because of their low production costs. The goal of this work was to explore the antimicrobial activity of nine short peptides derived from the γ-core-containing regions of tomato CRPs against important plant and human pathogens. We discovered antimicrobial activity in peptides derived from the defensin-like peptides, snakins, and MEG, which demonstrates the direct involvement of these CRPs in defense reactions in tomato. The CRP-derived short peptides appeared particularly active against the gram-positive bacterium Clavibacter michiganensis, which causes bacterial wilt-opening up new possibilities for their use in agriculture to control this dangerous disease. Furthermore, high inhibitory potency of short oligopeptides was demonstrated against the yeast Cryptococcus neoformans, which causes serious diseases in humans, making these peptide molecules promising candidates for the development of next-generation pharmaceuticals. Studies of the mode of action of the two most active peptides indicate fungal membrane permeabilization as a mechanism of antimicrobial action.

VTC4 Polyphosphate Polymerase Knockout Increases Stress Resistance of Saccharomyces cerevisiae Cells.

Inorganic polyphosphate (polyP) is an important factor of alkaline, heavy metal, and oxidative stress resistance in microbial cells. In yeast, polyP is synthesized by Vtc4, a subunit of the vacuole transporter chaperone complex. Here, we report reduced but reliably detectable amounts of acid-soluble and acid-insoluble polyPs in the Δvtc4 strain of Saccharomyces cerevisiae, reaching 10% and 20% of the respective levels of the wild-type strain. The Δvtc4 strain has decreased resistance to alkaline stress but, unexpectedly, increased resistance to oxidation and heavy metal excess. We suggest that increased resistance is achieved through elevated expression of DDR2, which is implicated in stress response, and reduced expression of PHO84 encoding a phosphate and divalent metal transporter. The decreased Mg2+-dependent phosphate accumulation in Δvtc4 cells is consistent with reduced expression of PHO84. We discuss a possible role that polyP level plays in cellular signaling of stress response mobilization in yeast.

Changes in cell wall structure and protein set in Candida maltosa grown on hexadecane.

The yeast Candida maltosa is a model organism for studying adaptive changes in the structure and function of the cell wall when consuming water-insoluble nutrient sources. The cells of C. maltosa that utilize hydrocarbons contain supramolecular structures, so-called "canals" in the cell wall. Differences in protein profiles of culture liquids and cell wall extracts of C. maltosa grown on glucose and hexadecane were analyzed. Three proteins specific of cells grown on hexadecane were revealed using mass spectrometry: glycosyl hydrolase EPD2 in the culture liquid; a protein belonging to the cytochrome C family in the 0.5 mol/L NaCl extract; and PPIA_CANAL protein known as chaperone, in the 0.1% SDS extract. The possible role of these proteins in cell wall structures responsible for adaptation to hexadecane utilization is discussed.

Mannan and phosphomannan from Kuraishia capsulata yeast.

Linear mannan and branched phosphomannan were identified as exopolysaccharides produced by Kuraishia capsulata yeast. Their structures were determined using nuclear magnetic resonance spectroscopy. The repeating unit of mannan was found to be a trisaccharide →6)-α-Manp-(1→2)-α-Manp-(1→2)-α-Manp-(1→, while the phosphomannan was shown to be built of ß-Manp-(1→2)-α-Manp-(1 disaccharide blocks linked by phosphodiester bonds via C-1 and C-6 of the reducing unit. The production of both polysaccharides was shown to depend on the phosphate concentration in the culture medium. In the absence of phosphate, only mannan was obtained, while an excess of KH2PO4 led to the exclusive production of phosphomannan. Chemical depolymerisation of phosphomannan led to the formation of disaccharide ß-Manp-(1→2)-(6-P)-Manp, representing the repeating unit of the hydrolysed polysaccharide. The treatment of the disaccharide with alkaline phosphatase resulted in the formation of disaccharide ß-Manp-(1→2)-Manp. The latest products can be transformed into glycosyl donors applicable further in the synthesis of oligosaccharides related to Candida cell wall polysaccharides.

The cadmium tolerance in Saccharomyces cerevisiae depends on inorganic polyphosphate.

The sensitivity to cadmium (Cd(II)), an important environmental pollutant, was studied in the cells of Saccharomyces cerevisiae strains with genetically altered polyphosphate metabolism. The strains overproducing polyphosphatases PPX1 or PPN1 were more sensitive to Cd(II) than the parent strain. The half maximal inhibitory concentrations were 0.02 and 0.05 mM for the transformants and the parent strain, respectively. Transformant strains cultivated in the presence of Cd(II) show a decrease in the content of short-chained cytosolic acid soluble polyphosphate. The role of this polyphosphate fraction in detoxification of heavy metal ions is discussed.

The antibiotic and membrane-damaging activities of cellobiose lipids and sophorose lipids.

Antibiotic activity was compared for Cryptococcus humicola cellobiose lipids, the mixture of 2,3,4-О-triacetyl-ß-D-glucopyranosyl-(1→4)-(6-О-acetyl-ß-D-glucopyranosyl-(1→16)-2,16-dihydroxyhexodecanoic acid and 2,3,4-О-triacetyl-ß-D-glucopyranosyl-(1→4)-(6-О-acetyl-ß-D-glucopyranosyl-(1→16)-2,17,18-trihydroxyoctotodecanoic acid, and the commercial sophorose lipid mixture of a mono-acetylated acidic sophorose lipid and a di-acetylated acidic sophorose lipid, both containing the C18:1 fatty acid residue. The MIC values of cellobiose lipids were 0.005 and 0.04 mg/mL for Filobasidiella neoformans and Candida tropicalis, respectively. The MIC values of sophorose lipids were 1 and 15 mg/mL for F. neoformans and C. tropicalis, respectively. MIC values for some bacteria were in the range of 10-30 mg/mL for both glycolipid preparations. Both sophorose lipids and cellobiose lipids displayed a membrane-damaging activity against F. neoformans. The treatment with these glycolipids reduces the content of ATP in the cells of test cultures and results in their staining with ethidium bromide.

Triterpenoid saponins from the roots of Acanthophyllum gypsophiloides Regel.

Two new triterpenoid saponins 1 and 2 were isolated from the methanol extract of the roots of Acanthophyllum gypsophiloides Regel. These saponins have quillaic acid or gypsogenin moieties as an aglycon, and both bear similar sets of two oligosaccharide chains, which are 3-O-linked to the triterpenoid part trisaccharide α-L-Arap-(1→3)-[α-D-Galp-(1→2)]-ß-D-GlcpA and pentasaccharide ß-D-Xylp-(1→3)-ß-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[ß-D-Quip-(1→4)]-ß-D-Fucp connected through an ester linkage to C-28. The structures of the obtained saponins were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy. A study of acute toxicity, hemolytic, anti-inflammatory, immunoadjuvant and antifungal activity was carried out. Both saponins 1 and 2 were shown to exhibit immunoadjuvant properties within the vaccine composition with keyhole limpet hemocyanin-based immunogen. The availability of saponins 1 and 2 as individual pure compounds from the extract of the roots of A. gypsophiloides makes it a prospective source of immunoactive agents.

The antifungal effect of cellobiose lipid on the cells of Saccharomyces cerevisiae depends on carbon source.

The cellobiose lipid of Cryptococcus humicola, 16-(tetra-O-acetyl-ß-cellobiosyloxy)-2-hydroxyhexadecanoic acid, is a natural fungicide. Sensitivity of the cells of Saccharomyces cerevisiae to the fungicide depends on a carbon source. Cellobiose lipid concentrations inducing the leakage of potassium ions and ATP were similar for the cells grown in the medium with glucose and ethanol. However, the cells grown on glucose and ethanol died at 0.05 mg ml(-1) and 0.2 mg ml(-1) cellobiose lipid, respectively. Inorganic polyphosphate (PolyP) synthesis was 65% of the control with 0.05 mg ml(-1) cellobiose lipid during cultivation on ethanol. PolyP synthesis was not observed during the cultivation on glucose at the same cellobiose lipid concentration. The content of longer-chain polyP was higher during cultivation on ethanol. We speculate the long-chained polyP participate in the viability restoring of ethanol-grown cells after treatment with the cellobiose lipid.

Extracellular cellobiose lipid from yeast and their analogues: structures and fungicidal activities.

Basidiomycetous yeasts Cryptococcus humicola and Pseudozyma fusiformata secrete cellobiose lipids into the culture broth. In the case of Cr. humicola, 16-(tetra-O-acetyl-beta-cellobiosyloxy)-2-hydroxyhexadecanoic acid was defined as major product and 16-(tetra-O-acetyl-beta-cellobiosyloxy)-2,15-dihydrohexadecanoic acid was defined as minor product, while Ps. fusiformata secreted mainly 16-[6-O-acetyl-2'-O-(3-hydroxyhexanoyl)-beta-cellobiosyloxy)-2,15-dihydroxyhexadecanoic acid. These compounds exhibit similar fungicidal activities against different yeasts including pathogenic Cryptococcus and Candida species. The cells of Filobasidiella neoformans causing systemic cryptococcosis completely died after 30-min incubation with 0.02 mg mL(-1) of cellobiose lipids. The same effect on ascomycetous yeast, including pathogenic Candida species, is achieved at 0.1-0.3 mg mL(-1) of cellobiose lipids depending on the test culture used. Cellobiose lipid of Ps. fusiformata inhibits the growth of phytopathogenic fungi Sclerotinia sclerotiorum and Phomopsis helianthi more efficiently than cellobiose lipids from Cr. humicola. Fully O-deacylated analogue, namely 16-(beta-cellobiosyloxy)-2-hydroxyhexadecanoic acid, and totally synthetic compound, 16-(beta-cellobiosyloxy)-hexadecanoic acid, do not inhibit the growth of F. neoformans and Saccharomyces cerevisiae, while 16-(beta-cellobiosyloxy)-2,15-dihydroxyhexadecanoic acid inhibits the growth of both test cultures but at higher concentrations than cellobiose lipids of Cr. humicola and Ps. fusiformata. The amide of 16-(beta-cellobiosyloxy)-2,15-dihydroxyhexadecanoic acid possessed no fungicide activity. Thus, the structures of both the carbohydrate part and fatty acid aglycon moiety are important for the fungicidal activity of cellobiose lipids.

Ustilagic acid secretion by Pseudozyma fusiformata strains.

Eight strains of Pseudozyma fusiformata were examined for antifungal activity. All of them had the same spectrum of action and were active against many species of yeasts, yeast-like and filamentous fungi. They secreted glycolipids, which were purified from the culture liquid by column and thin-layer chromatography. According to nuclear magnetic resonance and mass-spectroscopy experiments all strains produced ustilagic acid, a cellobioside-containing 2,15,16-trihydroxypalmitic acid as aglycon, 3-hydroxycaproic acid and acetic acid as O-acylic substituents.

Characterization of an antifungal glycolipid secreted by the yeast Sympodiomycopsis paphiopedili.

An antifungal glycolipid was purified from the culture liquid of the ustilaginomycetous yeast Sympodiomycopsis paphiopedili by column and thin-layer chromatography. According to nuclear magnetic resonance and mass-spectroscopy experiments it was a cellobioside containing 2,15,16-trihydroxypalmitic acid as an aglycon. The minimal effective concentrations leading to ATP leakage and growth inhibition were 45 and 160 microg ml(-1) for Cryptococcus terreus and Candida albicans, respectively.

ATP leakage from yeast cells treated by extracellular glycolipids of Pseudozyma fusiformata.

The ustilaginaceous yeast Pseudozyma fusiformata secreted glycolipids which were lethal to many yeasts and fungi more active at pH of about 4.0, and in the temperature range of 20-30 degrees C. Purified glycolipids enhanced non-specific permeability of the cytoplasmic membrane in sensitive cells, which resulted in ATP leakage and susceptibility of the cells to staining with bromocresol purple. Cells of Saccharomyces cerevisiae lost the ability to acidify the medium. Basidiomycetous yeasts were more sensitive to the glycolipids than ascomycetous ones. The minimal effective glycolipid concentration was 0.13 and 0.26 mg ml(-1) for Cryptococcus terreus and Filobasidiella neoformans, while for Candida albicans and Saccharomyces cerevisiae it was 1.0 and 1.6 mg ml(-1).