Novel Azole Resistance Mechanisms in Candida albicans

在克服白色念珠菌对唑类抗真菌耐药性方面取得进展的一个关键障碍是缺乏 完全了解它的分子和遗传基础，因为已知的抗性机制 不能完全解释在许多临床分离株中观察到的耐药性。我们的目标是推进治疗 通过鉴定新的唑类耐药机制，最终可以利用这些机制来感染念珠菌 克服这个问题。我们的中心假设是临床分离的白色念珠菌对唑的耐药性是 多因素的，涉及复杂的遗传变化，1)改变唑靶结合，2)激活转录 传播抗药性的方案，以及3)减少唑类摄取。我们的目标是1)描述 ERG11的临床相关突变，单独和组合，对其基因产物的活性，适合度， 2)确定新的锌(2)Cys6转录因子(ZCFs)的临床意义 3)发现减少唑类进口的决定因素及其影响因素 临床分离株对唑类耐药的贡献。我们的初步数据显示不同的ERG11 突变以不同的方式影响固醇去甲基酶的活性，包括催化效率、靶结合的改变 动力学和反应速度。我们还观察到，在C. 白色念珠菌增加了对唑类的耐药性。我们已经鉴定出对唑类药物耐药的临床菌株 与这些ZCFs的激活一致且包含候选激活突变的转录图谱 在这些ZCF基因中。最后，我们证明了白色念珠菌通过能量摄取氟康唑- 独立促进扩散。我们观察到一些对唑类耐药菌株的耐药性降低。 氟康唑摄取。在这项提案的目标1中，我们将进行遗传、微生物学和生化研究。 为了分析ERG11单一和组合突变对甾醇脱甲基酶易感性的影响， 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编码新的ZCFs的基因中发现突变 临床分离株导致其活性和对唑类耐药性的增加。在目标3中，我们将确定 白念珠菌临床分离株的唑类抗真菌导入机制及其对唑类耐药性的贡献。 我们的方法是创新的，因为我们将首次准确地确定ERG11突变如何影响 酶活性，剖析突变组合，并确定这些突变对 白色念珠菌。这项工作还探索了唑类耐药性的新机制。拟议的研究是 因为它将提供所需的理解，以通过 改进的唑类化合物的开发，对活化的ZCFs的干扰，以及提高唑类的摄取。通过充分的 了解唑类耐药性的遗传基础，最终将有可能发展出基于非培养的 快速、准确地检测临床分离株中的唑类耐药性的策略。

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.