Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors
Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors
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DOI：
10.1128/aac.41.11.2471
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发表时间：
1997-11-01
影响因子：
4.9
通讯作者：
Kurtz, MB
中科院分区：
文献类型：
--
作者：
Douglas, CM;DIppolito, JA;Kurtz, MB
肺炎粘附素和棘球菌素是目前在临床开发中的杀菌抗生素，可以抑制几种人类真菌病原体的1，3-β-D-葡聚糖合成酶(GS)。我们已经从二倍体生物体白色念珠菌中鉴定出一个基因，它编码这些抑制剂的一个靶标。该基因有一个2.1kb的片段，命名为CaFKS1，与酿酒酵母的FKS1和FKS2基因有显著的同源性，这两个基因编码部分功能冗余的GS亚基。为了评估CaFks1p在棘球菌素敏感性中的作用，我们在自发的白念珠菌耐药突变株CAI4R1、NR2、NR3和NR4上分别干扰了CaFKS1的一个同源物。这些突变体之前已经在含有肺炎粘附素L-733,560的琼脂平板上进行了筛选。在液体微量稀释法和体外GS测定中，该转化系对L-733,560的抑制既有抗性(Ech(R))，也有完全敏感的(Ech(S))。通过Southern印迹分析，利用CaFKS1基因的限制性内切酶切点多态性来区分这两个等位基因(命名为CaFKS1h和CaFKS1b)。对于菌株CAI4R1和NR2，CaFKS1b等位基因在每个Ech(R)转化子中都被破坏；对于菌株NR4，CaFKS1h在每个Ech(R)转化子中被破坏。我们的结论是：(I)CAI4R1、NR2和NR4菌株是CaFKS1上的显性或半显性肺炎粘附素耐药突变的杂合子，(Ii)CaFKS1等位基因中的任何一个都可能发生耐药突变，以及(Iii)CaFks1p是棘球绦虫的靶标。对于菌株NR3的转化子，我们分析的所有克隆都是均匀的Ech(R)，在基因组Southern blotts上只检测到CaFKS1h等位基因，无论是中断的还是野生型的。我们认为，CaFKS1基因座的基因转换可能产生了两个Cafks1h等位基因，每个等位基因都包含一个Ech(R)突变。在播散性念珠菌病的小鼠模型中，对来自突变体的转化子进行了对肺炎粘菌素治疗的敏感性分析。抗性等位基因杂合子的菌株(白念珠菌CAI4R1、NR2和NR4)对治疗表现为中等抗性，而没有功能Ech(S)等位基因的菌株(即NR3菌株及其衍生菌株CAI4R1与Ech(S)等位基因整合的中断质粒整合)在体内表现出较强的棘球菌素抗性。最后，我们不能通过两步整合干扰来灭活CaFKS1上的两个等位基因，这表明CaFks1p可能是白色念珠菌的一种必需蛋白。
Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFks1p in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech(r)) or fully sensitive (Ech(s)) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech(s) allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.