Genomic approach to identification of mutations affecting caspofungin susceptibility in Saccharomyces cerevisiae
Genomic approach to identification of mutations affecting caspofungin susceptibility in Saccharomyces cerevisiae
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DOI：
10.1128/aac.48.10.3871-3876.2004
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发表时间：
2004-10-01
影响因子：
4.9
通讯作者：
Osherov, N
中科院分区：
文献类型：
--
作者：
Markovich, S;Yekutiel, A;Osherov, N
抗真菌剂卡泊芬净（CAS）特异性干扰葡聚糖合成和细胞壁形成。为了进一步研究CAS影响的细胞过程，我们分析了酿酒酵母突变体集合（4，787个单独的敲除突变），以确定影响药物敏感性的新基因。对该集合进行CAS敏感性增加（CAS-IS）或CAS抗性增加（CAS-IR）筛选。通过肉汤微量稀释法测定MIC。20个基因的破坏导致CAS-IS（MIC降低4至8倍）。20个基因中有11个涉及细胞壁和膜功能，特别是蛋白激酶C（PKC）完整性途径（MID 2、FKS 1、SMI 1和BCK 1）、几丁质和甘露聚糖生物合成（CHS 3、CHS 4、CHS 7和MNN 10）以及麦角固醇生物合成（ERGS和ERG 6）。20个基因中的4个（TPO 1、YPS 65、VPS 25和CHC 1）参与液泡和运输功能，20个基因中的3个（CCR 4、POP 2和NPL 3）参与转录控制，20个基因中的2个功能未知。另外9个基因的破坏导致CAS-IR（MIC增加4倍）。这9个基因中的5个（SLG 1、ERG 3、VRP 1、CSG 2和CKA 2）参与细胞壁功能和信号转导，9个基因中的2个（YPS 67和SAC 2）参与液泡功能。为了评估CAS敏感性的特异性，检测了阿替霉素B、氟康唑、氟胞嘧啶和氟钙对菌株的MIC。20株CAS-IS菌株中有7株（FKS 1、SMI 1、BCK 1、CHS 4、ERGS、TPO 1和ILM 1被破坏）和9株CAS-IR菌株中有1株（SLGI被破坏）表现出对CAS的选择性敏感性。为了进一步探索PKC在CAS敏感性中的重要性，通过微量稀释测定法测试PKC抑制剂星形孢菌素与CAS组合对八种曲霉属临床分离株的活性。通过使用MIC和最低有效浓度终点，发现对所有8种分离株的协同或协同-相加活性。
The antifungal agent caspofungin (CAS) specifically interferes with glucan synthesis and cell wall formation. To further study the cellular processes affected by CAS, we analyzed a Saccharomyces cerevisiae mutant collection (4,787 individual knockout mutations) to identify new genes affecting susceptibility to the drug. This collection was screened for increased CAS sensitivity (CAS-IS) or increased CAS resistance (CAS-IR). MICs were determined by the broth microdilution method. Disruption of 20 genes led to CAS-IS (four- to eightfold reductions in the MIC). Eleven of the 20 genes are involved in cell wall and membrane function, notably in the protein kinase C (PKC) integrity pathway (MID2, FKS1, SMI1, and BCK1), chitin and mannan biosynthesis (CHS3, CHS4, CHS7, and MNN10), and ergosterol biosynthesis (ERGS and ERG6). Four of the 20 genes (TPO1, YPS65, VPS25, and CHC1) are involved in vacuole and transport functions, 3 of the 20 genes (CCR4, POP2, and NPL3) are involved in the control of transcription, and 2 of the 20 genes are of unknown function. Disruption of nine additional genes led to CAS-IR (a fourfold increase of MIC). Five of these nine genes (SLG1, ERG3, VRP1, CSG2, and CKA2) are involved in cell wall function and signal transduction, and two of the nine genes (YPS67 and SAC2) are involved in vacuole function. To assess the specificity of susceptibility to CAS, the MICs of amphotericin B, fluconazole, flucytosine, and calcofluor for the strains were tested. Seven of 20 CAS-IS strains (with disruption of FKS1, SMI1, BCK1, CHS4, ERGS, TPO1, and ILM1) and 1 of 9 CAS-IR strains (with disruption of SLGI) demonstrated selective susceptibility to CAS. To further explore the importance of PKC in CAS susceptibility, the activity of the PKC inhibitor staurosporine in combination with CAS was tested against eight Aspergillus clinical isolates by the microdilution assay. Synergistic or synergistic-to-additive activities were found against all eight isolates by use of both MIC and minimum effective concentration endpoints.