383The resistance of Candida albicans to amphotericin B methyl ester increases rapidly as cultures enter the stationary phase of growth; organisms harvested after several days in the stationary phase may have a resistance two or three orders of magnitude greater than that of exponentially growing organisms. This resistance is decreased by incubation of the organisms with enzymes whch attack components of the cell wall. Of the enzymes tested, (1+3)-P-~-glucanases are the most effective; incubation of 7 d batch cultures with exo-(l+3)-/?-D-glucanase at a concentration of 10 pg enzyme protein (mg dry wt organisms)-l for 24 h at 37 "C and pH 6.5 reduces the resistance of the organisms to a value approximating to that of exponentially growing organisms. Resistance is also decreased by treatment with chitinase, lipase, trypsin, a-mannosidase and (1+6)-/%~-glucanases but, on a specific activity basis, none of these enzymes is as effective as (1+3)-/3-~-glucanase. The action of (1+3)-P-~-glucanase is markedly enhanced by the addition during incubation of chitinase, trypsin or lipase. I N T R O D U C T I O NThe sensitivity to amphotericin B of Candida ulbicuns grown in batch culture at 37 "C varies markedly with the growth phase (Gale, 1974;Hammond et al., 1974). Judged by the concentration of amphotericin B methyl ester (AME) required to induce a standard rate of leakage of K+ under otherwise standard conditions, the sensitivity of C . albicans decreases from 0.1 to 0-2 pg AME ml-l for organisms in the exponential phase to greater than 30 pg AME ml-l for organisms in the late-stationary phase of growth. Protoplasts of stationary phase organisms have the same sensitivity to AME as those from exponentially growing organisms; the difference in sensitivity of the cells must therefore lie in alterations in the cell wall (Gale et al., 1975). Ultrastructure studies (Cassone et al., 1979) show that the walls of stationary phase organisms are thicker and do not show the pronounced electron-dense layering that can be seen in electron micrographs of walls of exponential phase organisms.The resistance of stationary phase organisms can be decreased by a reduction of oxygen tension or by treatment with SH-reducing agents, or irreversibly increased by treatment with SH-binding agents such as iodoacetamide or N-ethylmaleimide (Gale et al., 1975). Stationary phase cultures do not undergo a marked increase in resistance until endogenous Hdonors, especially glutamate in the metabolic pool, approach exhaustion (Gale et al., 1978). Stationary phase organisms contain more lipid than exponential phase organisms but the antagonism to AME displayed by lipid extracts from the two types of cell is not sufficiently different to explain the difference in their sensitivity (Gale et al., 1975).If phenotypic resistance of this nature is determined by the presence of specific compon-

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~~The phenotypic resistance to amphotericin methyl ester (AME) of stationary phase cultures of Candida albicans was decreased by alkaline pH values and by treatment with 2-mercaptoethanol or glucanase preparations, and was increased by acid pH values, increased aeration, treatment with N-ethylmaleimide, or the presence of inhibitors of protein synthesis such as trichodermin. The effects of such treatments on endogenous glucanase activity and on the incorporation of glucose residues into the 'glucan fraction' of the organism were studied. The changes in the endogenous levels of lytic activities on laminarin [as a measure of the total (1 +3)-P-~-glucanase] and on p-nitrophenyl-P-D-glucoside [reflecting the exo-( 1 +3)-P-~-glucanase] were followed in C. albicans cells under a variety of conditions. Treatments which increased AME sensitivity stimulated both total and exo-( 1 +3)-p-D-glucanase activities, while treatments which promoted resistance decreased the levels of both (1 +3)-p-~-glucanases. Changes in the 'glucan fraction' were followed by incubating suspensions of organisms in the presence of trace amounts of [U-'4C]glucose. The rate of incorporation of radioactivity fell during the first 2-3 d of stationary phase culture and then rose to high values by 7-8 d; AME resistance increased throughout this period. The rate of incorporation was markedly stimulated by prior treatment of the organisms with 2-mercaptoethanol or glucanase and inhibited by trichodermin or treatment with Nethylmaleimide.The addition in the concentration range 0.3-3 mM of the glucose analogues P-D-allose, 3-O-methyl-~-glucose, 2-deoxy-~-glucose or 5-thio-~-glucose to cultures 24 h after inoculation prevented any further increase in AME resistance for the next 2-3 d and resulted in a decrease in the level of resistance established at the time of addition. Radioactivity from 14C-or 3H-labelled analogues added, 24 h after inoculation, to stationary phase cultures was incorporated into the 'glucan fraction' of the organisms.The incorporation of glucose residues into the 'glucan fraction' is controlled by the activity of glucanases in producing glucose acceptor sites. The results reported confirm that there is a correlation between glucan metabolism, glucanase activity and resistance to AME, in that any factor leading to increased glucanase action also results in decreased resistance and vice versa, while incorporation of certain glucose analogues into the 'glucan fraction' delays the further increase in resistance. I N T R O D U C T I O NThe sensitivity of Candida albicans towards amphotericin methyl ester (AME) can be measured in terms of the amount of antibiotic required to induce the release of K+ at a standard rate from suspensions of the organism (Gale, 1974). Organisms grown in batch Present address:

Phenotypic resistance to both amphotericin B and miconazole develops in stationary phase cultures of Candida albicans and this resistance lies in changes in the cell wall. Study of the effects of growth conditions, treatment with SH-reactive agents and treatment with enzymes indicates that the nature of the changes leading to resistance must be different for the two drugs.

Treatment of cell suspensions of Saccharomyces fragilis with 0.01 M beta-mercaptoethanol or dithiothreitol released a variety of substances of high and low molecular weight. Twenty-two high-molecular-weight glycoproteins were separated by a combination of chromatography on DEAE cellulose and polyacrylamide gel electrophoresis in presence of sodium dodecylsulphate. The carbohydrate components consisted of at least 95% mannose and the protein components had threonine and serine as the major amino acids. Only very small amounts of phosphorus were associated with the high-molecular-weight components. The low-molecular-weight substances werr probably released from the internal cell pool and uracil and hypoxanthine were identified as components of this fraction. It is suggested that in addition to breaking disulphide bridges in the cell wall the thiols may also render the plasmalemma permeable to certain low-molecular-weight substances. Such effects are not lethal since the yeast can be trained to grow in presence of 0.01 M mercaptoethanol.