c.i.-fluorescent-brightening-agent-28 and nikkomycin

Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall β-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca(2+)-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsG mutant was hypersensitive to caspofungin, and all other ΔAfchs mutants tested remained capable of increasing their chitin content in response to treatment with CaCl2 and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchs mutants tested, with the exception of the ΔAfchsG mutant, which remained sensitive to caspofungin. In vitro exposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive echinocandin treatment in vivo by AfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections.

Topics: Aminoglycosides; Antifungal Agents; Aspergillus fumigatus; Benzenesulfonates; Calcineurin; Calcium Chloride; Calcium Signaling; Caspofungin; Cell Wall; Chitin; Chitin Synthase; Drug Synergism; Echinocandins; Enzyme Inhibitors; Fluorescent Dyes; Fungal Proteins; Gene Expression Regulation, Fungal; Hyphae; Lipopeptides; Microbial Sensitivity Tests; Mutation; Protein Kinase C

Analysis of Candida albicans cells using antibodies directed against Gas1p/Ggp1p, Saccharomyces cerevisiae homolog of Phr1p, revealed that Phr1p is a glycoprotein of about 88 kDa whose accumulation increases with the rise of external pH. This polypeptide is present both in the yeast form and during germ tube induction. In the Phr1- cells at pH 8 the solubility of glucans in alkali is greatly affected. In the parental strain the alkali-soluble/-insoluble glucan ratio shows a 50% decrease at pH 8 with respect to pH 4.5, whereas in the null mutant it is unchanged, indicating the lack of a polymer cross-linker activity induced by the rise of pH. The mutant has a sixfold increase in chitin level and is hypersensitive to calcofluor. Consistently with a role of chitin in strengthening the cell wall, Phr1- cells are more sensitive to nikkomycin Z than the parental strain.

Topics: Aminoglycosides; Anti-Bacterial Agents; Apoenzymes; Benzenesulfonates; Candida albicans; Chitin Synthase; Deoxyribodipyrimidine Photo-Lyase; Enzyme Inhibitors; Extracellular Matrix; Fluorescent Dyes; Fungal Proteins; Hydrogen-Ion Concentration; Membrane Glycoproteins; Molecular Weight; Morphogenesis; Mutation

Nikkomycin Z inhibits chitin synthase in vitro but does not exhibit antifungal activity against many pathogens. Assays of chitin synthase isozymes and growth assays with isozyme mutants were used to demonstrate that nikkomycin Z is a selective inhibitor of chitin synthase 3. The resistance of chitin synthase 2 to nikkomycin Z in vitro is likely responsible for the poor activity of this antibiotic against Saccharomyces cerevisiae.

Topics: Aminoglycosides; Anti-Bacterial Agents; Antifungal Agents; Benzenesulfonates; Chitin Synthase; Drug Resistance, Microbial; Fluorescent Dyes; Isoenzymes; Kinetics; Mutation; Pyrimidine Nucleosides; Saccharomyces cerevisiae

Flow cytometry was used to monitor chitin synthesis in regenerating protoplasts of the yeast Candida albicans. Comparisons of cells stained with Calcofluor White, a fluorochrome with known affinity for chitin, and cells incubated in the presence of N-[3H]-acetylglucosamine, the precursor substrate for chitin, showed a linear relationship between fluorescence and incorporation of label over time. Changes in both the fluorescence and light scatter of regenerating protoplasts treated with inhibitors of fungal chitin synthase were also quantitated by flow cytometry.

Topics: Aminoglycosides; Anti-Bacterial Agents; Antifungal Agents; Benzenesulfonates; Candida albicans; Chitin; Flow Cytometry; Fluorescent Dyes; Protoplasts