Novel Azole Resistance Mechanisms in Candida albicans

白色念珠菌的新唑耐药机制

• 批准号: 10313446
• 负责人: P. David Rogers
• 金额: $ 39.27万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313446
• 关键词: Novel; Azole; Resistance; Mechanisms; Candida

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.

在克服白色念珠菌中克服硫烷抗真菌抗性的进展的关键障碍是缺乏 完全了解其分子和遗传基础，因为已知的抗性机制DO 在许多临床分离株中没有完全解释抗性。我们的目标是进步 念珠菌感染通过鉴定可利用的新型唑的抗性机制，最终可以利用 克服这个问题。我们的中心假设是白色念珠菌临床分离株中的唑耐药是 多因素并涉及复杂的遗传变化，即1）改变Azole目标结合，2）激活转录 赋予阻力的程序，3）减少偶氮的吸收。我们的目标是1）描述 ERG11中与临床相关的突变，单独和组合其基因产物的活性，适应性， 2）确定新型Zn（2）Cys6转录因子（ZCFS）的临床意义 这会影响硫唑的敏感性，3）发现硫唑进口及其的决定因素及其 对临床分离株中硫唑耐药性的贡献。我们的初步数据表明不同的ERG11 突变会多样化影响固醇脱甲基酶活性，包括催化效率的改变，靶结合 动力学和反应速度。我们还观察到，C中的一组ZCF集的人工激活。 白色唱片增加了唑的耐药性。我们已经确定了抗唑的临床分离株 转录曲线与这些ZCF的激活一致，并且包含候选激活突变 在这些ZCF基因中。最后，我们已经证明，白色念珠菌通过能量占据氟康唑。 独立的促进扩散。我们已经观察到某些抗唑的抗分离株降低了 氟康唑吸收。在本提案的目标1中，我们将进行遗传学，微生物学和生化研究 为了剖析ERG11中单个和联合突变对固醇脱甲基酶敏感性的影响， 底物亲和力，唑结合，催化活性和适应性。在AIM 2中，我们将进行遗传和 微生物学研究以确定在抗性中编码新型ZCF的基因中是否发现突变 临床分离株会导致它们的激活并增加唑的耐药性。在AIM 3中，我们将确定 白色念珠菌临床分离株中硫唑抗真菌进口的机理及其对硫唑耐药性的贡献。 我们的方法具有创新性，因为我们将首次确定ERG11中的突变如何影响 酶活性，解剖突变的组合，并确定此类突变对适应性的影响 白色念珠菌。这项工作还探讨了唑耐药性的新机制。拟议的研究是 意义重大，因为它将提供最终克服偶氮抗性所需的理解 改进的试唑，干扰活化的ZCF以及增强唑的摄取。完全 了解取唑抗性的遗传基础，最终有可能发展基于非文化 快速，准确地检测临床分离株中唑的耐药性的策略。