Chitinolytic enzymes of Candida albicans
It has been envisaged that lytic enzymes may be crucial in determining the morphology and growth of fungi, and may therefore represent a target for antifungal agents. The chitinolytic system of Candida albicans was investigated using a range of 4-methylumbelliferyl glycosides as model substrates, and its potential as a target for antibiotics has been assessed. Maximum hydrolysis was obtained with substrates having monomer and tetramer chain lengths, being attributed to N-acetylglucosaminidase and endochitinase respectively. Activities were investigated in cell fractions, vacuoles, and also in whole cell preparations. The characteristics of both chitinase and N-acetylglucosaminidase were examined, including pH and temperature optima and Km values. Chitinase was semi-purified on Fast Protein Liquid chromatography system and activity could be located after native polyacrylamide gel electrophoresis. Analysis of chitinase activity during the growth of the yeast morphology of C.albicans revealed maximal activities during the logarithmic phase, suggesting a relationship of chitinase levels to active growth of the pathogen. It was found that the antibiotic allosamidin was a potent inhibitor of chitinase activity of C.albicans, but not of N-acetylglucosaminidase. Conversely, an analogue of N-acetylglucosamine was found to be a potent inhibitor of N-acetylglucosaminidase but not of chitinase. Treatment of yeast suspensions with allosamidin resulted in an increased chain length. No cell death, or discernible pattern of change in the radiolabelling was observed in the presence of either allosamidin or N-acetylglucosamine analogue. Similarly no consistent change in the optical density of cultures was observed in the presence of either inhibitor. Even in the presence of the membrane permeabilising agent amphotericin no effects were observed above those achieved with amphotericin alone. Comparative studies were carried out upon the chitinolytic activity of Kluyveromyces lactis toxin and bovine serum. The chitin synthetic system of Benjaminiella poitrasii was compared to that of C.albicans.