Maligie Marybeth A, Selitrennikoff Claude P
University of Colorado Health Sciences Center, Department of Cell and Developmental Biology, 12635 E. Montview Blvd., Suite 215, Aurora, Colorado 80010, USA.
Antimicrob Agents Chemother. 2005 Jul;49(7):2851-6. doi: 10.1128/AAC.49.7.2851-2856.2005.
(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.
（1,3）-β-D-葡聚糖合酶（EC 2.4.1.34，尿苷二磷酸葡萄糖：1,3-β-D-葡聚糖3-β-葡糖基转移酶）以尿苷二磷酸葡萄糖为底物，催化葡萄糖（[1,3]-β-连接）聚合，形成真菌细胞壁的主要碳水化合物成分。我们优化了新型隐球菌（1,3）-β-葡聚糖合酶活性的体外测定条件。在含有20%甘油、2 mM氟化钠、1 mM二硫苏糖醇、0.1 mM苯甲基磺酰氟、5 mM氯化镁、0.1%蛋白酶和磷酸酶抑制剂混合物以及60 μM GTPγS的50 mM Tris（pH 7.75）中裂解的细胞，在体外产生了最大比活性。我们针对（1,3）-β-葡聚糖合酶抑制剂卡泊芬净和西洛芬净测试了新型隐球菌（1,3）-β-葡聚糖合酶的体外活性，并确定（1,3）-β-葡聚糖合酶活性对这些棘白菌素非常敏感（表观K(i)分别为0.17±0.02 μM和22±5.7 μM）。结合新型隐球菌的高最低抑菌浓度（卡泊芬净最低抑菌浓度为16 μg/ml；西洛芬净最低抑菌浓度为64 μg/ml），我们的结果表明，新型隐球菌对卡泊芬净和西洛芬净耐药的机制与（1,3）-β-葡聚糖合酶耐药无关。
