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Lipopeptides are antifungal agents that inhibit cell wall β-(1, 3)-glucan biosynthesis in fungal organisms. A mutant resistant to lipopeptides was generated by UV mutagenesis and characterized. The Candida albicans mutant (LP3-1) was stable and showed resistance specificity to a broad range of lipopeptides and certain glycolipid inhibitors. Other antifungal agents with diverse modes of action had a normal minimum inhibitory concentration profile for LP3-1 compared with the wild-type strain (CCH 442). In the in vitro β-(1, 3)-glucan synthase assay, both the lipopeptides and papulacandin-related agents had considerably higher 50% inhibitory concentration values in the LP3-1 strain than in the wild-type strain. In reconstitution assays, the resistance factor was associated with the integral membrane pellet rather than the peripheral GTP-binding protein. The LP3-1 strain had a membrane lipid profile similar to that of the parent strain and was virulent in a murine model of systemic candidiasis. Taken together, these results indicate that the resistance factor is associated with the integral membrane component of β-(1, 3)-glucan synthase. Lipopeptides are common antifungal agents encountered during screening of natural products. The LP3-1 strain was resistant to natural product extracts known to contain various lipopeptides. Thus, LP3-1 can be used in a dereplication assay.Key words: Candida albicans, β-(1, 3)-glucan synthase, lipopeptides, drug resistance.

Glucan synthesis was sensitive to several sulfhydryl reacting compounds: mercurials, reversible disulfides, and an alkylating sulfhydryl reagent (IC503–45 μM). Thiol groups associated with glucan synthesis were hydrophilic in nature, since both hydrophilic and hydrophobic reagents were active. Glucan synthase complex consists of at least two components: a peripheral GTP-binding protein that can be solubilized with detergents (supernatant) and the catalytic membrane-bound component (pellet). A rapid separation technique was developed to study sulfhydryl interactions with the complex. The GTP-binding protein was solubilized with 0.6% 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate from isolated microsomes of Candida albicans cells grown at either 10 or 30 °C. The residual membranous fraction contained the core catalytic moiety of glucan synthase. Both fractions were devoid of glucan synthase activity until they were reconstituted by mixing the two fractions together. In reconstitution experiments, the pellet lost almost 50% activity when preincubated with 2.5 μM N-ethylmaleimide and combined with an untreated supernatant whereas only 10% activity was lost when the supernatant was treated with N-ethylmaleimide. The catalytic active site of glucan synthase was not protected with UDP-Glc when preincubated with 10 μM N-ethylmaleimide but the GTP-binding fraction was partially protected with GTPγS.Key words: Candida albicans, (1,3)-β-glucan synthase, GTP-binding proteins, solubilization, sulfhydryl reagents.

The fuscandins, antifungal agents of the papulacandin class, are produced by a strain of Fusarium sambucinum. Fermentation yielded 60 mg/liter of fusacandin A and minor amounts of fusacandin B. As expected, the fusacandins inhibit (1,3)-beta-glucan synthesis. Fusacandin A is slightly less active than papulacandin B against Candida albicans and, like papulacandin, loses activity in the presence of serum.

Two new activators of (1,3)-β-glucan synthase fromSaccharomyces cerevisiae were identified, and a procedure for preparing enriched enzyme fractions by removal of peripheral membrane proteins and entrapped soluble proteins was developed. Microsomal enzyme activity, known to be enhanced by bovine serum albumin (BSA), was stimulated threefold by both β-lactoglobulin and Brij-35. Both apparently substituted for BSA, since no synergistic effects were observed with activators added in combination. Successive washings of microsomal fractions with the detergents Brij-35 and Tergitol NP-40 to remove peripheral and vesicle-entrapped proteins yielded particulate fractions five-fold enriched in glucan synthase activity. GTP, an important effector of glucan synthase, improved purification of the enzyme during detergent extractions. Various membrane fractions were photolabeled with 5[32P]N3UDP-Glc or 8N3[32P]GTP, and potential UDP-Glc and GTP-binding polypeptides were identified. However, further enrichment will be required to determine which of these might represent subunits of the yeast glucan synthase complex.

A whole-cell C. albicans screen was designed to identify novel inhibitors interacting with the synthesis, assembly and regulation of the fungal cell wall. C. albicans was grown in a paired broth assay in 96-well plates with natural product extracts or pure chemical compounds in the presence and absence of the osmotic stabilizer, sorbitol. Growth was visually examined over a 7-day period and scored into different growth categories. Positives from the sorbitol rescue were then examined under the microscope for morphological alterations and grouped into several morphological classes. Sorbitol protection and cell morphology were indicators of novel antifungal agents from natural product extracts and pure compounds.