Novel Azole Resistance Mechanisms in Candida albicans

在克服白念珠菌唑类抗真菌药耐药性方面取得进展的一个关键障碍是缺乏一种抗真菌药物， 完整的了解其分子和遗传基础，因为已知的耐药机制， 不能完全解释在许多临床分离株中观察到的耐药性。我们的目标是推进 通过鉴定新的唑类耐药机制， 克服这个问题。我们的中心假设是临床分离的C.以白色 多因素，涉及复杂的遗传变化，1）改变唑靶结合，2）激活转录 赋予抗性的程序，和3）减少唑类吸收。我们的目标是：1）描述 ERG 11中的临床相关突变，单独和组合，对其基因产物的活性，适应性， 和唑类药物敏感性，2）确定新的Zn（2）Cys 6转录因子（ZCF）的临床意义 影响唑类药物敏感性的因素，以及3）发现减少唑类药物输入的决定因素及其 在临床分离株中对唑类耐药的贡献。我们的初步数据表明，不同的ERG 11 突变影响甾醇脱甲基酶活性，包括催化效率的改变，靶点结合 动力学和反应速度。我们还观察到，人工激活一组不同的ZCF在C。 白念珠菌增加唑类耐药。我们已经确定了唑类耐药的临床分离株， 转录谱与这些ZCF的激活一致，并且含有候选激活突变 这些ZCF基因。最后，我们证明了C.白色念珠菌通过能量吸收氟康唑- 独立促进扩散。我们已经观察到一些唑类耐药菌株表现出降低的 氟康唑摄取在本提案的目标1中，我们将进行遗传学、微生物学和生物化学研究 为了剖析ERG 11中的单个和组合突变对甾醇脱甲基酶敏感性的影响， 底物亲和力、唑结合、催化活性和适合性。在目标2中，我们将进行遗传和 微生物学研究，以确定是否以及如何在耐药的编码新ZCF的基因中发现突变， 临床分离株导致其活化和唑类耐药性增加。在目标3中，我们将确定 唑类抗真菌药的输入机制及其对临床分离的念珠菌唑类耐药的贡献。白色念珠菌。 我们的方法是创新的，因为我们将第一次精确地确定ERG 11突变如何影响 酶的活性，解剖突变的组合，并确定这些突变对适应性的影响， C.白色念珠菌这项工作还探讨了唑类耐药的新机制。拟议的研究是 重要的是，它将提供最终克服唑类耐药性所需的理解， 开发改进的唑类药物，干扰活化的ZCF，并增强唑类药物的摄取。通过完全 了解唑类耐药性的遗传基础，最终将有可能开发出非培养型的 快速准确检测临床分离株中唑类耐药的策略。

A critical barrier to progress in overcoming azole antifungal resistance in Candida albicans is the lack of a complete understanding of its molecular and genetic basis because the known mechanisms of resistance do not fully explain resistance observed among many clinical isolates. Our goal is to advance the treatment of Candida infections by identifying novel azole resistance mechanisms that can be exploited to ultimately overcome this problem. Our central hypothesis is that azole resistance in clinical isolates of C. albicans is multifactorial and involves complex genetic changes that 1) alter azole target binding, 2) activate transcriptional programs that impart resistance, and 3) reduce azole uptake. Our objectives are to 1) delineate the effects of clinically relevant mutations in ERG11, alone and in combination, on the activity of its gene product, fitness, and azole susceptibility, 2) determine the clinical significance of novel Zn(2)Cys6 transcription factors (ZCFs) that influence azole susceptibility, and 3) to discover the determinants of reduced azole import and their contribution to azole resistance in clinical isolates. Our preliminary data suggest that different ERG11 mutations diversely affect sterol demethylase activity, including alterations of catalytic efficiency, target binding kinetics, and reaction velocity. We have also observed that artificial activation of a distinct set of ZCFs in C. albicans increases azole resistance. We have identified azole-resistant clinical isolates that exhibit transcriptional profiles consistent with activation of these ZCFs and that contain candidate activating mutations in these ZCF genes. Finally, we have demonstrated that C. albicans takes up fluconazole by energy- independent facilitated diffusion. We have observed that some azole resistant isolates exhibit reduced fluconazole uptake. In Aim 1 of this proposal we will undertake genetic, microbiologic, and biochemical studies to dissect the effects of single and combinatorial mutations in ERG11 on sterol demethylase susceptibility, substrate affinity, azole binding, catalytic activity, and fitness. In Aim 2 we will undertake genetic and microbiologic studies to determine if and how mutations found in the genes encoding novel ZCFs in resistant clinical isolates result in their activation and increased azole resistance. In Aim 3 we will determine the mechanism of azole antifungal import and its contribution to azole resistance in clinical isolates of C. albicans. Our approach is innovative as we will determine for the first time precisely how mutations in ERG11 influence enzyme activity, dissect combinations of mutations, and determine the impact of such mutations on fitness of C. albicans. This work also explores novel mechanisms of azole resistance. The proposed research is significant as it will provide the understanding needed to ultimately overcome azole resistance through the development of improved azoles, interference with activated ZCFs, and enhancement of azole uptake. By fully understanding the genetic basis of azole resistance it will be possible to eventually develop non-culture based strategies to rapidly and accurately detect azole resistance in clinical isolates.