MORPHOLOGICAL EFFECTS OF LIPOPEPTIDES AGAINST ASPERGILLUS-FUMIGATUS CORRELATE WITH ACTIVITIES AGAINST (1,3)-BETA-D-GLUCAN SYNTHASE

The lipopeptide antifungal agents, echinocandins, papulacandins, and p neumocandins, kill Candida albicans by inhibiting glucan synthesis. Fo r this fungus, there is a good correlation of in vitro enzyme inhibiti on with in vitro assays of MICs. Semisynthetic lipopeptides such as ci lofungin, LY303366, L-693,989, and L-733,560 have activity in vivo aga inst Aspergillus infections but appear to be inactive in broth dilutio n in vitro tests (MICs, >128 mu g/ml). To understand how compounds whi ch lack activity in vitro can have good in vivo activity, we monitored the effect of pneumocandins on the morphology of Aspergillus fumigatu s and A. flavus strains by light microscopy and electron microscopy an d related the changes in growth to inhibition of glucan synthesis. Pne umocandin B-0 caused profound changes in hyphal growth; light microgra phs showed abnormally swollen germ tubes, highly branched hyphal tips, and many cells with distended balloon shapes. Aspergillus electron mi crographs confirmed that lipopeptides produce changes in cell walls; d rug-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 t he drug, indicating considerable specificity for the primary target. T he drug-induced growth alteration produced very compact clumps in brot h dilution wells, making it possible to score the morphological effect macroscopically. The morphological changes could be assayed quantitat ively by using conventional broth microdilution susceptibility assay c onditions. We defined the endpoint as the lowest concentration require d to produce the morphological effect and called it the minimum effect ive concentration to distinguish it from the no-growth endpoints used in MIC determinations. The minimum effective concentration assay was r elated to inhibition of glucan synthase activity in vitro and may prov ide a starting point for development of susceptibility testing methods for lipopeptides.