Effects of amphotericin B, nystatin, and other polyene antibiotics on chitin synthase.

Abstract

The effects of amphotericin B (AmB), nystatin, filipin, and pimaricin were tested on purified chitin synthase (EC 2.4.1.16) (chitosomes from yeast cells of Mucor rouxii). AmB and nystatin inhibited the enzyme at concentrations 210 #g/ml, filiin was weakly inhibitory, and pimaricin had no effect. The inhibition of chitin synthase by AmB appears to be noncompetitive, with a K; value of about 0.13 mM. The effect of nystatin was more complex and included a sharp stimulation of chitin synthase activity at "'50 jug/ml. Our Chitosomes are microvesicular organelles that can synthesize chitin fibrils in vitro (1-4). Chitosomes have been isolated from representatives of all major groups of the chitinous fungi (5). Digitonin, a well known sterol-complexing agent (6, 7), has profound effects on chitosomes. Depending on concentration, this saponin can stimulate or inhibit the chitin synthase activity (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-,B-acetamidodeoxy-D-glucosyltransferase, EC 2.4.1.16) of chitosomes from Mucor rouxii and cause their 'dissociation into subunits (8)."Solubilization" (9, 10) and inhibition (9) of chitin synthase by digitonin have also been noted for other fungi.The interaction of digitonin with chitosomal chitin synthase prompted us to examine the effect of polyene antibiotics on chitosomes because these agents have, like digitonin, a marked sterol-binding capacity (11-15). MATERIALS AND METHODSGrowth of the yeast form of Mucor rouxii, IM-80 (American Type Culture Collection 24905), and preparation of purified chitosomes were done as described (5). Chitosomes were kept at 4°C and used within 24 hr.Chitin Synthase Assay. The standard incubation mixture contained, in a total volume of 125 ,ul, enzyme preparation (50 p1), polyene solution (25 ,ul), and the following final concentrations of reagents: Rennilase (a crude acid protease; 1 mg/ ml), UDP-['4CJGlcNAc (0.4 mM; 22,500 dpm), GIcNAc (20 mM), and ATP (0.2 mM). All solutions were prepared in 0.05 M KH2POjNaOH buffer containing 0.01 M MgCl2, pH 6.5 (hereafter referred to as "buffer"). The duration of the preincubation of chitin synthase with Rennilase and polyene prior to the addition of the substrate was 20 min.The reaction time was 1 hr and the temperature was 220C. The radioactive chitin formed was determined by a filtration method (5, 16). The reaction rate (in the absence of polyene) was constant for at least 90 min, even with the most active preparations.Growth Inhibition Tests. Sporangiospores of M. rouxii, prepared as described (17), were inoculated into 125-ml Erlenmeyer flasks with 25 ml of YPG medium (18) to a final density of 104 spores per ml. The antibiotics were dissolved in either dimethyl sulfoxide or N,N-dimethylformamide, and 250 Al of such solutions was added to each culture. These two solvents caused slight growth inhibition of M. rouxii. Growth inhibition (percent) was calculated relative to control cultures with dimethyl sulfoxide or dimethylformamide. The flasks were incubated aerobically on a shaker at 30°C in the da...

Change of cell wall chitin content in amphotericin B resistant Kluyveromyces strains

“…This notion is supported by previous results [22], which show the non-competitive inhibition of puri¢ed chitin synthetase by a high concentration of AmB.…”

Effect of Candida morphology on amphotericin B susceptibility

“…Secondly, during germ tube formation certain synthetic activities are concentrated at restricted regions of the cell (10 is possible that AmB causes cell death by localizing to these regions and preferentially inhibiting the formation of new cell wall. Rast and Bartnicki-Garcia demonstrated that AmB inhibits chitin synthase at concentrations around 1 pLg/ml (8). This formulation would not require an increase in total drug uptake and would be supported by the observation that germ tube lengthening is inhibited by AmB but that new second germ tubes can develop.…”

Aspergillus and Aspergillosis

“…Amphotericin B at concentrations in excess of the minimum growth inhibitory concentrations, inhibits chitin synthase in chitosomal preparations from filamentous fungi (Rast and Bartnicki-Gareis, 1981). This may result from the polyene affecting the lipid environment of the enzyme and hence its activity.…”