Mutational Analysis of Fks1: Intrinsic Echinocandin Resistance

棘球菌素(ECs)是一种新的抗真菌药物，包括卡泊芬净、米卡芬净和 阿尼杜拉方金。ECS代表了抗真菌化疗的一个深远的重要发展，因为 它们通常是杀菌的，无毒的，并且对获得抗药性的菌株保持活性 抗真菌药。这些优势源于EC独特的作用机制，包括抑制 真菌的p-L、3-葡聚糖合成酶，因此细胞壁的合成。棘球绦虫易感性降低(RES) 可以通过编码FKS1的突变在正常敏感的真菌中发展，如白色念珠菌 主要的葡聚糖合成酶，但通常这种突变很少发生。然而，有一些人没有 常见但往往更致命的真菌病原体表现出不同程度的内在Re。镰刀菌， 单孢菌属、隐球菌属、毛孢子菌属和S接合菌属具有高水平的 而曲霉菌和近平假丝酵母菌表现出低水平的内禀Res。 固有的RES严重限制了EC的使用，因为抗真菌治疗通常是经验性的和 在高危患者中，越来越依赖于抗FungaJ预防。为了解决这一限制，我们 必须了解内在RE的基础。我们的中心假设是内在RE，如在 突变获得的RES，是由FKS蛋白序列介导的。对这一假说的支持；IS一直是 由我们最近发表的检测FC内在耐药性的研究提供。妄想症(GarciaEffron 等人..200秒。Me 52：2305)和Fusariwn Solani(Katiyar和&i1ind.2009年。媒体中的MC)。我们 建议用酿酒酵母作为模型和替代宿主来研究这一问题。 通过以下目的：(1)突变确定酿酒酵母中的EC靶标。入门 使用我们最近扩展的自发RES突变数据库(手稿正在准备中)。这 AIM将依赖于一种新的高效的基于每个位点的定向突变(PSM)方法， 多才多艺。并对以前的“L”伪迹免疫。微光的不同影响约&lt； 米卡芬净、阿尼杜拉和阿尼杜林敏感性用于推断EC结合部位。这些数据将 与FHT拓扑学相关联，FTM PSM用于精化这些模型。(2)检查bflsis是否 所选真菌病原菌(ScedQsrum，CRYP(QCQccus.)和曲霉属) 异源FKS在酿酒酵母中的表达。与AIM 1数据相结合的序列比对将 身份候选重新调解区域。这些将被用来在酿酒酵母中产生福L杂交种。 使用PSM方法，然后。通过对EC的敏感性测试。最后，将使用PSM来测试 预测特定FKS残基的作用；在固有RE中。这些研究将加速该领域的发展 第二代ECs提供更广泛的保护，免受真菌病原体的侵袭。

The echinocandins (ECs) are a new antifungal group that includes caspofungin, micafungin, and anidulafungin. ECs represent a profoundly important development in antifungal chemotherapy because they are generally fungicidal, non-toxic, and remain active versus strains that acquire resistance to other antifungals. These advantages stem from the unique mechanism ofEC action involving inhibition of fungal p-l ,3-glucan synthase and hence cell wall synthesis. Reduced echinocandin susceptibility (RES) can develop in normally susceptible fungi such iIS Candida albicans by mutation of FKSl encoding the major glucan synthase, but fOrtw'l ately such mutations occur only rarely. However, a number ofless common but often more lethal fungal pathogens demonstrate intrinsic RES of varying levels. FusarIUm, Scedosporium, Cryptococcus, Trichosporon, and zygomycete s~ies are characterized by high-level intrinsic RES, while Aspergillus specics and Candida parapsilosis exhibit low-level intrinsic RES. Intrinsic RES represents a serious limitation to EC use, since antifungal treatment is often empirical and antifungaJ prophylaxis is increasingly relied upon in high risk patients. To address this limitation, we must understand the basis for intrinsic RES. Our central hypothesis is that intrinsic RES, as in mutationally acquired RES, is mediated by Fks protein sequences. Support for this hypothe;is has been provided by Our recently published studies examining intrinsic resistance ofC. paraosilosis (GarciaEffron et al .. 200S. Me 52:2305) and Fusariwn solani (Katiyar and &i1ind. 2009. MC in press). We propose to fyrfug tcst this by using the yeast Saccharomyces cerevisiae as model and surrogate host, through the following Aims: (I) Mutatiorlally defIne the EC target in S. cerevisiae FksJ. Begirlning with our recently expanded database of spontaneous RES mutations (manuscripts in preparation). this Aim will rely on a new method for peR-based site-direcled mutagenesis (pSM) that is efficient, versatile... and immune to ex"l)ression artifacts. Differential efferu of mlltation~ on ca.<;JlOfUrWn.. micafungin, and anidulat\ltlgln susceptibility will be used to deduce the EC binding site. These data will be correlated with Fht topology, ftM PSM used to refine these models. (2) Examine the bflsis for intrinsic RES of selected fungal pathogens (ScedQsrorium , Cryp(QCQccus. and Aspergillus species) by hetaologous FKS expression in S. cerevisiae. Sequence aligrunents coupled with Aim 1 data will identity candidate RES-mediating regions. These will be used to generate fu l hybrids in S. ceevi.siae using the PSM method, followed. by tests ofEC susceptibility. Finally, PSM will be used to test the predicted roles of specific Fks residue; in intrinsic RES. These studies will accelerate the development of second generation ECs that provide a broader spectrum of protection from fungal pathogens.