Mutational Analysis of Fks1: Intrinsic Echinocandin Resistance

棘白菌素类是一种新的抗真菌药物，包括卡泊芬净、米卡芬净和卡泊芬净。 阿尼芬净EC代表了抗真菌化疗的一个非常重要的发展， 它们通常是杀真菌的、无毒的，并且对获得对其它细菌的抗性的菌株保持活性。 抗真菌药这些优点源于EC作用的独特机制，包括抑制 真菌β-1，3-葡聚糖合酶和因此的细胞壁合成。棘白菌素敏感性降低（RES） 可以通过编码FKS 1的突变在正常敏感的真菌如iIS白色念珠菌中发展， 主要葡聚糖合酶的突变，但最重要的是这种突变很少发生。然而， 常见但通常更致命的真菌病原体表现出不同水平的内在RES。FusarIum， Scedosporium、Cryptococcus、Trichosporon和Acetamycyte的特征是高水平 固有RES，而曲霉属和近平滑念珠菌表现出低水平的固有RES。 内源性RES代表了EC使用的严重限制，因为抗真菌治疗通常是经验性的， 在高危患者中越来越依赖抗真菌药物预防。为了解决这个问题，我们 我们的中心假设是，内在RES，如在 突变获得性RES由Fks蛋白序列介导。对这一假说的支持; 由我们最近发表的研究提供，研究了C.副黏菌病（GarciaEffron 等。两百多Me 52：2305）and Fusariwn solani（Katiyar and &i1ind. 2009. MC in press）。我们 建议用酿酒酵母作为模型和替代宿主来进一步研究这一点， （1）突变性地确定S.酿酒酵母贝格尔宁 我们最近扩展的自发RES突变数据库（手稿正在准备中）。这 Aim将依赖于一种有效的基于peR的定点诱变（pSM）的新方法， 多才多艺并且对表情艺术品免疫。微分方程的求解。<;JlOfUrWn.. 米卡芬净和阿尼杜拉敏感性将用于推断EC结合位点。这些数据将 与Fht拓扑结构相关，ftM PSM用于改进这些模型。(2)检查bflsis， 选择的真菌病原体（ScedQsrorium，Cryp（QCQccus.和曲霉属物种）， 在S.啤酒。与Aim 1数据结合的序列比对将 身份候选RES介导区域。这些将用于在S. ceevi.siae 使用PSM方法，然后。通过EC敏感性试验。最后，PSM将用于测试 这些研究将加速Fks残基在内源性RES中的作用， 第二代EC可提供更广泛的真菌病原体保护。

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.