Cryptococcus neoformans resistance to echinocandins: (1,3)beta-glucan synthase activity is sensitive to echinocandins.
(1,3)Beta-D-glucan synthase (EC 2.4.1.34. UDP-glucose: 1,3-beta-D-glucan 3-beta-glucosyltransferase) uses UDP-glucose as substrate and catalyzes the polymerization of glucose ([1,3]-beta-linkages) to form the major carbohydrate component of the fungal cell wall. We have optimized in vitro assay conditions for (1,3)beta-glucan synthase activity from Cryptococcus neoformans. Cells lysed in 50 mM Tris, pH 7.75, containing 20% glycerol, 2 mM NaF, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl(2), 0.1% protease and phosphatase inhibitor cocktails, and 60 microM GTPgammaS produced maximum specific activity in vitro. We tested in vitro C. neoformans (1,3)beta-glucan synthase activity against the (1,3)beta-glucan synthase inhibitors, caspofungin and cilofungin, and have determined that (1,3)beta-glucan synthase activity is very sensitive (apparent K(i) of 0.17 +/- 0.02 microM and 22 +/- 5.7 microM, respectively) to these echinocandins. Taken together with high MICs for C. neoformans (caspofungin MIC, 16 microg/ml; cilofungin MIC, 64 microg/ml), our results indicate that C. neoformans is resistant to caspofungin and cilofungin by a mechanism(s) unrelated to (1,3)beta-glucan synthase resistance.
University of Colorado Health Sciences Center, Department of Cell and Developmental Biology, 12635 E. Montview Blvd., Suite 215, Aurora, Colorado 80010, USA.
Marybeth A
MA
Maligie
Claude P
CP
Selitrennikoff
D000935:Antifungal Agents / Q000494:pharmacology*; D000077336:Caspofungin; D003455:Cryptococcus neoformans / Q000187:drug effects* / Q000201:enzymology; D025141:Drug Resistance, Fungal; D054714:Echinocandins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D055666:Lipopeptides; D008826:Microbial Sensitivity Tests; D010456:Peptides, Cyclic / Q000494:pharmacology*
D000935:Antifungal Agents; D054714:Echinocandins; D005656:Fungal Proteins; D055666:Lipopeptides; D010456:Peptides, Cyclic; C042101:cilofungin; D005964:Glucosyltransferases; C040232:glucan synthase; D000077336:Caspofungin