Cryptococcus neoformans resistance to echinocandins:: (1,3)β-glucan synthase activity is sensitive to echinocandins
Cryptococcus neoformans resistance to echinocandins:: (1,3)β-glucan synthase activity is sensitive to echinocandins
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DOI：
10.1128/aac.49.7.2851-2856.2005
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发表时间：
2005-07-01
影响因子：
4.9
通讯作者：
Selitrennikoff, CP
中科院分区：
文献类型：
--
作者：
Maligie, MA;Selitrennikoff, CP
（1，3）β-D-葡聚糖合酶（EC 2.4.1.34. UDP-葡萄糖：1，3-β-D-葡聚糖3-β-葡糖基转移酶）使用UDP-葡萄糖作为底物并催化葡萄糖（[1，3]-β-键）聚合以形成真菌细胞壁的主要碳水化合物组分。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。细胞在50 mM Tris（pH 7.75）中裂解，含有20%甘油、2 mM NaF、1 mM二硫苏糖醇、0.1 mM苯甲基磺酰氟、5 mM MgCl 2、0.1%蛋白酶和磷酸酶抑制剂混合物和60 μ M GTP γ S，在体外产生最大比活性。我们在体外测试了C。新形式（1，3）β-葡聚糖合酶活性对（1，3）β-葡聚糖合酶抑制剂卡泊芬净和西洛芬净的抑制作用，并且已经确定（1，3）β-葡聚糖合酶活性对这些棘白菌素非常敏感（表观Ki分别为0.17 ± 0.02 μ M和22 ± 5.7 μ M）。与C的高MIC一起。新型念珠菌（卡泊芬净MIC，16 μ g/ml;西洛芬净MIC，64 μ g/ml），我们的结果表明C.新生儿通过与（1，3）β-葡聚糖合酶抗性无关的机制对卡泊芬净和西洛芬净具有抗性。
(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 MgCl2, 0.1% protease and phosphatase inhibitor cocktails, and 60 mu M GTP gamma S 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 mu M and 22 +/- 5.7 mu M, respectively) to these echinocandins. Taken together with high MICs for C. neoformans (caspofungin MIC, 16 mu g/ml; cilofungin MIC, 64 mu g/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.