papulacandin-b and cilofungin

Analysis of the in vitro sensitivity of clinical isolates of different yeast species to amphotericine B and two inhibitors of cell wall beta-glucane synthesis: the lipopeptide cilofungin and the liposaccharide papulacandin B, as well as the possible interactions among these antifungal types.. The in vitro sensibility of C. albicans strains and other yeast species of clinical origin was studied, using the macrodilution method in RPMI broth as recommended by NCCLS. In six C. albicans and two T. glabrata strains the interaction between amphotericine B and one of the beta-glucan inhibitors was also assessed, by combining one antifungal agent in a two-fold dilution series with subinhibitory concentrations (1/4MIC) of the second antifungal.. Of all three antifungals, amphothericine B proved to be the more active one, with a MIC90 of 1.25 micrograms/ml for C. albicans. MIC90 of cilofungin and papulacandine B is 5 micrograms/ml for both antifungals. The spectrum of action for the different yeast species is broader for papulacandin B than for cilofungin. On the other hand, no synergism has been observed between the two glucane inhibitors and amphotericine B.

Topics: Aminoglycosides; Amphotericin B; Anti-Bacterial Agents; Antifungal Agents; Ascomycota; beta-Glucans; Drug Synergism; Echinocandins; Glucans; Microbial Sensitivity Tests; Peptides, Cyclic; Yeasts

A whole-cell C. albicans screen was designed to identify novel inhibitors interacting with the synthesis, assembly and regulation of the fungal cell wall. C. albicans was grown in a paired broth assay in 96-well plates with natural product extracts or pure chemical compounds in the presence and absence of the osmotic stabilizer, sorbitol. Growth was visually examined over a 7-day period and scored into different growth categories. Positives from the sorbitol rescue were then examined under the microscope for morphological alterations and grouped into several morphological classes. Sorbitol protection and cell morphology were indicators of novel antifungal agents from natural product extracts and pure compounds.

Topics: Aminoglycosides; Anti-Bacterial Agents; Antifungal Agents; Benzenesulfonates; beta-Glucans; Candida albicans; Cell Wall; Chitin; Echinocandins; Fungal Proteins; Glucans; Peptides; Peptides, Cyclic; Protein Kinase C; Sorbitol

The synthesis and metabolism of yeast cell wall glucan were studied using a Saccharomyces cerevisiae construct in which radiolabelled galactose is metabolized to UDP-glucose and preferentially incorporated into glucan. Greater than 85% of the incorporated radiolabel was found within insoluble cell wall material. Our study also demonstrated that radiolabelled wall glucan is released from cells growing exponentially, and that the released radiolabel is reutilizable low molecular mass material. Size exclusion chromatography and enzymic analysis indicate that laminaribiose comprises approximately 50% of the released fraction. This is consistent with in vitro findings that laminaribiose is a by-product of a newly identified glucosyltransferase (R. P. Hartland, G. W. Emerson & P. A. Sullivan, 1991, Proc R Soc Lond B 246, 155-160) associated with fungal cell walls. Our results also suggest that pre-existing glucan undergoes less metabolic processes than newly synthesized material as evidenced by a decrease in released radiolabel over time. Pulse double labelling of glucan and total cellular protein indicate that glucan metabolism and protein synthesis (ps) are not tightly coupled although they do parallel each other during exponential growth. Inhibitors of glucan synthesis (gs) decrease the glucan to protein ratio. Measurement of ps allows normalization for non-specific decreases in the rate of cell wall synthesis due to general cessation of growth. Cilofungin and papulacandin B, two putative inhibitors of gs, inhibited galactose incorporation into glucan and thus showed a decrease in the glucan to protein ratio, although ps was affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aminoglycosides; Anti-Bacterial Agents; Antifungal Agents; Cell Wall; Echinocandins; Fungal Proteins; Galactose; Genes, Fungal; Glucans; Glycogen; Kinetics; Peptides, Cyclic; Saccharomyces cerevisiae; Uridine Diphosphate Glucose

A systematic evaluation of the in vitro (1,3)-beta-glucan synthase assay parameters was performed using microsomes prepared from Candida albicans from either yeast or mycelial phase cells. Enzyme activities of both yeast and mycelial phase microsomes depended on the presence of guanosine-5'-O-(3-thiophosphate) and either bovine serum albumin or a detergent [W-1 (polyoxyethylene ether detergent) or Brij-35 (polyoxyethylene ether, 23 lauryl ether)]. Brij-35 was included in standard assays as it was compatible with the permeabilized whole-cell assay. Microsomes derived from both the yeast and mycelial phases generally yielded similar glucan synthase activities under a range of different assay conditions. Brij-35 significantly stabilized the enzyme, yielding a half-life of 5.6 d at 4 degrees C, compared with 0.9 d without detergent. The addition of detergent during mechanical breakage of yeast cells dramatically improved glucan synthase stability and activity. Enzyme catalysis was linear for at least 75 min with 100 micrograms protein from microsomes of yeast cells grown to mid-exponential phase, with an apparent Km for UDP-glucose of 1.1 mM. The pH and temperature optima were 7.75 and 30 degrees C, respectively. Glucan synthase activity was highest in cells derived from early mid-exponential phase and declined to a basal level by stationary phase. A permeabilization-based in situ assay for glucan synthase was developed. Cells were permeabilized with 2% (v/v) solution of toluene/methanol (1:1) and assayed for glucan synthase activity using standard reaction mixtures. Reactions were linear for 30 min and were inhibited by known inhibitors of glucan synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aminoglycosides; Anti-Bacterial Agents; Antifungal Agents; Candida albicans; Cell Membrane Permeability; Detergents; Echinocandins; Enzyme Stability; Fungal Proteins; Glucosyltransferases; Guanosine Triphosphate; Kinetics; Membrane Proteins; Microsomes; Peptides; Peptides, Cyclic; Polidocanol; Polyethylene Glycols; Schizosaccharomyces pombe Proteins

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.

Topics: Aminoglycosides; Anti-Bacterial Agents; Antifungal Agents; Centrifugation, Density Gradient; Echinocandins; Fungal Proteins; Glucosyltransferases; Kinetics; Membrane Proteins; Neurospora crassa; Peptides; Peptides, Cyclic; Protoplasts; Schizosaccharomyces pombe Proteins; Sorbose; Uridine Diphosphate