ABSTRACT
Panomycocin, the killer toxin of Pichia anomala NCYC 434 (K5), is a 49 kDa monomeric glycoprotein with exo-beta-1,3-glucanase activity (patent pending). In this study we evaluated the in vitro activity of panomycocin against a panel of 109 human isolates of seven different pathogenic Candida spp. using microdilution and time-kill methods. Panomycocin was most active against C. tropicalis, C. pseudotropicalis and C. glabrata with MIC(90) values of 1 microg/ml. It displayed significant activity against C. albicans and C. parapsilosis with MIC(90) values of 4 and 2 microg/ml, respectively. For C. krusei, the MIC(90) value was 8 microg/ml. Panomycocin was fungicidal against all the tested Candida spp. The MFC values were only one or 2 dilutions higher than the MICs with the exception of C. krusei isolates with MFCs greater than or equal to 4xMIC. Results of this study indicated that panomycocin could be considered as a natural antifungal agent against Candida infections and has significant potential for further investigation.
Subject(s)
Antifungal Agents/pharmacology , Candida/drug effects , Glycoside Hydrolases/pharmacology , Mycotoxins/pharmacology , Pichia/chemistry , Antifungal Agents/isolation & purification , Drug Resistance, Fungal , Glycoside Hydrolases/isolation & purification , Humans , Microbial Sensitivity Tests , Mycotoxins/isolation & purificationABSTRACT
Killer proteins that are produced and secreted into the environment by certain yeast strains are considered as promising antifungal agents. In this study, in vitro activity of Pichia anomala NCYC 434 (K5) killer protein, panomycocin, which is a 49 kDa glycoprotein with an exo-beta-1,3-glucanase activity was tested against 41 isolates of dermatophytes. Minimum inhibitory concentrations (MICs) were determined by a broth microdilution method based on the reference document M38-A of Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS). For panomycocin MIC determinations two end point criteria MIC-2 (prominent growth inhibition) and MIC-0 (complete growth inhibition) were recorded. All the tested isolates (Microsporum spp. and Trichophyton spp.) were found susceptible to panomycocin. The MIC-2 values ranged from 0.25 to 2 microg ml(-1) and MIC-0 values ranged from 1 to 8 microg ml(-1). These results showed that panomycocin is active in vitro against fungal strains that cause superficial infections and highlighted its probable use as a topical antifungal agent.
Subject(s)
Antifungal Agents/pharmacology , Arthrodermataceae/drug effects , Glucan 1,3-beta-Glucosidase/pharmacology , Pichia/enzymology , Antifungal Agents/isolation & purification , Antifungal Agents/metabolism , Arthrodermataceae/classification , Glucan 1,3-beta-Glucosidase/isolation & purification , Glucan 1,3-beta-Glucosidase/metabolism , Microbial Sensitivity Tests/standards , Microsporum/classification , Microsporum/drug effects , Trichophyton/classification , Trichophyton/drug effectsABSTRACT
K5-type yeast killer toxin secreted by P. anomala NCYC 434 cells has a broad killing spectrum. Competitive inhibiton of killer activity showed that glucans, mainly the beta-1,3 glucan, represent the primary toxin binding site within the cell wall of sensitive cells. Its hydrolytic activity on laminarin in an exo-like fashion revealed that the toxin exerts its killing effect by exo-beta-1,3-glucanase activity. Its specific activity on laminarin was 120 U/mg, and the Michaelis constants K(m) and V(max) for laminarin hydrolysis were 0.25 mg/ml and 370 micromol/min/mg. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Production of the toxin by the cells was induced only when they were grown in culture media rich in beta-glucan sources, and the addition of glucose increased the specific production rate. The enzymic activity of the toxin was fully inhibited by Hg(+2), but increased with some other metal ions, most of all by Pb(+2).
Subject(s)
Glucan 1,3-beta-Glucosidase/metabolism , Glucans/metabolism , Mycotoxins/pharmacology , Binding Sites , Binding, Competitive , Kinetics , Mycotoxins/metabolism , Polysaccharides/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/drug effects , YeastsABSTRACT
K5-type yeast killer protein in the culture supernatant of Pichia anomala NCYC 434 cells was concentrated by ultrafiltration and purified to homogeneity by ion-exchange chromatography with a POROS HQ/M column followed by gel filtration with a TSK G2000SW column. The protein migrated as a single band on discontinuous gradient SDS-PAGE and had a molecular mass of 49,000 Da. The pI value of the K5-type killer protein was measured at pH 3.7 by high voltage vertical gel electrofocusing. The result of an enzyme immuno assay revealed that it was a glycosylated protein. Its internal amino acid sequencing yielded the sequences LNDFWQQGYHNL, IPIGYWAFQLLDNDPY, and YGGSDYGDVVIGIELL, which are 100% identical to exo-beta-1,3-glucanase (accession no. AJ222862) of Pichia anomala (strain K). The purified protein was highly stable at pH values between 3 and 5.5 and temperatures up to 37 degrees C.
