Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase

Abstract

The lipopeptide antifungal agents, echinocandins, papulacandins, and pneumocandins, kill Candida albicans by inhibiting glucan synthesis. For this fungus, there is a good correlation of in vitro enzyme inhibition with in vitro assays of MICs. Semisynthetic lipopeptides such as cilofungin, LY303366,989,560 have activity in vivo against Aspergillus infections but appear to be inactive in broth dilution in vitro tests (MICs, > 128 ,ug/ml). To understand how compounds which lack activity in vitro can have good in vivo activity, we monitored the effect of pneumocandins on the morphology ofAspergillusf migatus and A. flavus strains by light microscopy and electron microscopy and related the changes in growth to inhibition of glucan synthesis.Pneumocandin Bo caused profound changes in hyphal growth; light micrographs showed abnormally swollen germ tubes, highly branched hyphal tips, and many cells with distended balloon shapes. Aspergillus electron micrographs confirmed that lipopeptides produce changes in cell walls; drug-treated germlings showed very stubby growth with thick walls and a conspicuous dark outer layer which was much thicker in the subapical regions. The rest of the hyphal tip ultrastructure was unaffected by the drug, indicating considerable specificity for the primary target. The drug-induced growth alteration produced very compact clumps in broth dilution wells, making it possible to score the morphological effect macroscopically. The morphological changes could be assayed quantitatively by using conventional broth microdilution susceptibility assay conditions. We defined the endpoint as the lowest concentration required to produce the morphological effect and called it the minimum effective concentration to distinguish it from the no-growth endpoints used in MIC determinations.The minimum effective concentration assay was related to inhibition of glucan synthase activity in vitro and may provide a starting point for development of susceptibility testing methods for lipopeptides.Echinocandins, pneumocandins, and papulacandins are antifungal agents which inhibit the synthesis of 1,3-p-D-glucan (22,39,43,44). ,B-Glucans are vital cell wall polymers in clinically important pathogenic fungi, including Candida and Aspergillus spp. (8). The proportion of this polysaccharide in the walls of different organisms varies, but at least 10% of the dry weight of the walls of these organisms is in the form of 1,3-p-D-glucan (4). This polysaccharide is synthesized by a membrane-associated activity which has been partially characterized in yeasts such as Candida albicans and Saccharomyces cerevisiae (31,40,46). The enzyme uses UDP-glucose as a substrate and catalyzes the polymerization of a linear polymer of 1,3-p-D-glucan reported to be 60 U (40) or as much as 700 U (35) long. The Km for UDP-glucose is approximately 2 mM, and the reaction is stimulated by nucleoside triphosphates, particularly GTP (31, 42). For both C. albicans and S. cerevisiae, the in vitro product, which is synthesized without a requirement for ...

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A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase

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