The mycocidal, membrane-active complex of Cryptococcus humicola is a new type of cellobiose lipid with detergent features

AbstractThe chemical composition of the mycocidal complex (formerly known as microcin) secreted by Cryptococcus humicola was investigated by chemical, mass spectrometric and nuclear magnetic resonance methods. The results indicate that the mycocidal complex is composed of glycolipids with a highly acetylated (up to five acetyl groups) cellobiose backbone [β-D-Glcp-(1′→4)-β-D-Glcp] linked to the ω-hydroxyl group of α,ω-dihydroxy palmitate [16:0-α,ω-di-OH] with an unsubstituted carboxyl group. The acyl chain forming aglycon can be replaced by [18:0-(α,ω-di-OH)], [18:0-(α,ω-1,ω-tri-OH)], and [18:0-(α,ω-2,ω-tri-OH)]. The complex has a comparatively high surface activity; 0.5 mg/ml of it reduced the surface tension of 0.1 M NaHCO3 from 71 mN/m to 37 mN/m and interfacial tension against n-hexadecane from 39 mN/m to 10 mN/m. The critical micelle concentration of the complex at pH 4.0, determined by the fluorometric method with N-phenyl-1-naphthylamine as fluorescent probe and by the De Nouy ring method, was 2×10−5 M (taking the average molecular mass of the complex to be 750); it did not depend on the presence of 100 mM KCl and was an order of magnitude higher at pH 7.0. By fluorescence resonance energy transfer spectroscopy with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine as energy donor and N-(rhodamine B sulfonyl)-phosphatidylethanolamine as energy acceptor the complex was shown to intercalate into the liposomal lipid matrix. Primary lesions caused by the complex in planar lipid bilayers were revealed as short-living current fluctuations of a broad spectrum of amplitudes. The mycocidal effect of the complex is suggested to be associated with its detergent-like properties

Phosphate efflux as a test of plasma membrane leakage in Saccharomyces cerevisiae cells

Plasma membrane integrity is a key to cell viability. Currently, the main approach to assessing plasma membrane integrity is the detection of penetration of special dyes, such as trypan blue and propidium iodide, into the cells. However, this method needs expensive equipment: a fluorescent microscope or a flow cytometer. Besides, staining with propidium iodide occasionally gives false-positive results. Here, we suggest the phosphate (Pi) leakage assay as an approach to assess the increase in permeability of the plasma membrane of yeast cells. We studied the dependence of phosphate efflux and uptake into Saccharomyces cerevisiae cells on the composition of the incubation medium, time, and ambient pH. The difference in optimal conditions for these processes suggests that Pi efflux is not conducted by the Pi uptake system. The Pi efflux in water correlated with the proportion of cells stained with propidium iodide. This indicated that Pi efflux is associated with cytoplasmic membrane disruption in a portion of the yeast cell population. The assay of Pi efflux was used to evaluate membrane disruption in S. cerevisiae cells treated with some heavy metal ions and detergents.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

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

Polyphosphatase PPN1 of Saccharomyces cerevisiae: switching of exopolyphosphatase and endopolyphosphatase activities.

The polyphosphatase PPN1 of Saccharomyces cerevisiae shows an exopolyphosphatase activity splitting phosphate from chain end and an endopolyphosphatase activity fragmenting high molecular inorganic polyphosphates into shorter polymers. We revealed the compounds switching these activities of PPN1. Phosphate release and fragmentation of high molecular polyphosphate prevailed in the presence of Co2+ and Mg2+, respectively. Phosphate release and polyphosphate chain shortening in the presence of Co2+ were inhibited by ADP but not affected by ATP and argininе. The polyphosphate chain shortening in the presence of Mg2+ was activated by ADP and arginine but inhibited by ATP

Stress Resistance of Saccharomyces cerevisiae Strains Overexpressing Yeast Polyphosphatases

Inorganic polyphosphate (polyP) is an important factor in the stress resistance of microorganisms. The polyphosphate-overexpressing strains of yeast S. cerevisiae were used as a model for studying the inter-relationship between stress resistance and polyP level. We compared the polyP level and resistance to the oxidative, manganese, cadmium, and alkaline stresses in parent stain CRN and in strains overexpressing the four yeast polyphosphatases: Ppx1, Ppn1, Ppn2, and Ddp1. Strains overexpressing Ppx1, Ppn1, and Ppn2 have lower polyP content and the strain overexpressing Ddp1 has the same polyP content as the parent strain. The strains overexpressing Ppn1 and Ddp1 show higher resistance to peroxide and manganese. The strain overexpressing Ppx1 showed a decrease in peroxide resistance. The strain overexpressing Ppn2 was more resistant to alkaline and peroxide stresses. A similar increase in resistance to the manganese and peroxide stresses of strains overexpressing Ppn1 and Ddp1, which differ in polyP content, led to the conclusion that there is no direct relationship between polyP content and variations in this resistance. Thus, we speculate about the potential role of inositol pyrophosphates as signaling molecules in stress response

The effects of some compounds on exopolyphosphatase (A) and endopolyphosphatase (B, C) activities of purified PPN1 with PolyP₂₀₈ in the presence of 0.1 mM Co²⁺ or 0.25 mM Mg²⁺.

<p>The concentrations of effectors are (mM): P_i, PP_i, PolyP₃, ATP and ADP, 2.0; arginine, 100; spermidin and 1,3-diaminopropan, 50. The concentration of heparin was 0.01 mg/ml. Control, without effectors. A, white columns – exopolyphosphatase activity in the presence of Co²⁺, 100% corresponds to 290 U/ mg protein; black columns – exopolyphosphatase activity in the presence Mg²⁺, 100% corresponds to 22 U/mg protein. B, the endopolyphosphatase activity in the presence of Co²⁺; C, the endopolyphosphatase activity in the presence of Mg²⁺; - Me²⁺, without bivalent metal cation. The reaction time was 60 min. The PolyP PAGE was performed in 24% polyacrylamide gel with 7M urea; toluidine blue staining. PolyP₂₀₈, the substrate was incubated without the enzyme for 60 min. The experiments were repeated in triple and the average values and typical photographs are shown.</p

The effect of ATP on the endopolyphosphatase reaction of PPN1 in the presence of 0.25 mM MgSO₄.

<p>PolyP PAGE was performed in 24% polyacrylamide gel with 7M urea; toluidine blue staining. PolyP₂₀₈ - PolyP₂₀₈ was incubated without the enzyme at 30°C for 60 min. The numerals indicate ATP concentrations (mM). The PAGE experiment was repeated in triple and the photograph of typical experiment is shown.</p

The SDS-PAGE of purified protein (A) and time dependence of PolyP₂₀₈ hydrolysis by the purified PPN1 (B-E).

<p>The protein markers (A) were phosphorylase b (94 kD), albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), trypsin inhibitor (20.1 kD), α-lactalbumin (14.4 kD). B, P_i release, % of P in PolyP₂₀₈ used as substrate; (○) in the presence of 0.1 mM CoSO₄, (●) in the presence of 0.25 mM of MgSO₄. C-E, PolyP PAGE was performed in 24% polyacrylamide gel with 7M urea; toluidine blue staining. C, PolyP markers, commercial PolyP with the average chain lengths of 15, 25, 45 and 65 phosphate residues from Sigma (USA) and PolyP with an average chain length of 208 phosphate residues from Monsanto (USA). D, hydrolysis products of PolyP₂₀₈ in the presence of 0.1 mM CoSO₄. E, hydrolysis products of PolyP₂₀₈ in the presence 0.25 mM of MgSO₄. Control, PolyP₂₀₈ was incubated without the enzyme at 30°C for 120 min. The experiments were repeated in triple and the average values and typical photograph are shown.</p

Purification of recombinant PPN1 of <i>S. cerevisiae</i>.

<p>Purification of recombinant PPN1 of <i>S. cerevisiae</i>.</p

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