Novel Azole Resistance Mechanisms in Candida albicans

描述(由申请人提供)：关于在白色念珠菌中编码唑靶分子羊毛甾醇脱甲基酶(ERG11)及其转录调节因子(Upc2)的基因对唑类抗真菌耐药性的贡献，目前还缺乏相关知识。我们的长期目标是通过了解抗真菌耐药性的分子基础来改善念珠菌感染的治疗。这项建议的总体目标是了解白念珠菌中Upc2和ERG11介导的唑类耐药性的分子基础。我们的中心假设是，它的相互作用伙伴蛋白Upc2p和它的靶基因与Erg11p一起发挥着核心作用 在介导唑类抗真菌耐药性方面的作用。我们已经发现了激活Upc2p的突变，增加了ERG11的表达，并增加了对唑的耐药性。我们发现许多临床分离株都过度表达ERG11。在几个耐药菌株中，ERG11的过度表达并不是由于激活了Upc2的突变，这表明了尚未发现的新的耐药机制。本研究的目的1是利用配对的唑敏感和耐药菌株以及不匹配的耐药菌株，确定ERG11在白念珠菌对唑类耐药菌株中过度表达的机制。将通过测序和标准分子技术识别和表征Upc2中新的激活突变，而通过候选基因方法将识别ERG11过度表达的新机制。我们收集的许多菌株携带与耐药性相关的新的或特征的ERG11突变。虽然一些ERG11突变已经被证明与唑类耐药性有关或被证明影响了唑类耐药性，但它们对白色念珠菌这一表型的直接影响尚未被研究。目的2是通过构建与唑类耐药相关的ERG11突变的杂合子和纯合子的菌株，检测其对唑类抗真菌药物敏感性的影响，并表征其对唑类抗真菌药物与靶酶相互作用的生化影响，以确定特定的ERG11突变在白念珠菌对唑类耐药中的作用。此外，激活锌簇转录因子，如Upc2p，似乎涉及与这些转录因子直接相互作用的蛋白质 监管者。激活突变可能会影响Upc2p与其相互作用伙伴蛋白之间的相互作用。目的3是利用串联亲和纯化技术(TAP)鉴定白念珠菌中Upc2介导的唑类耐药所需的相互作用蛋白。最后，我们发现，与其他突变相比，Upc2中的一些激活突变显著增加了对唑类药物的耐药性，但对ERG11的表达也有类似的影响。因此，很可能是其他Upc2-靶基因导致了唑类耐药性。目的4确定除ERG11外的Upc2靶基因在白念珠菌Upc2介导的唑类耐药中的作用。这项拟议的研究具有重要意义，因为它将导致预测治疗失败、克服唑类耐药性和改进抗真菌治疗的新策略。我们的方法是创新的，因为它侧重于新颖的抵抗机制，并采用创造性的策略来实现拟议的具体目标。

DESCRIPTION (provided by applicant): There is a significant lack of knowledge concerning the contribution of the genes encoding the azole target lanosterol demethyase (ERG11) and its transcriptional regulator (UPC2) to azole antifungal resistance in C. albicans. Our long-term goal is to improve the treatment of Candida infections by understanding the molecular basis of antifungal resistance. The overall objective of this proposal is to understand the molecular basis of UPC2- and ERG11-mediated azole resistance in C. albicans. Our central hypothesis is that Upc2p, its interaction partner proteins, and its target genes play a central role along with Erg11p in mediating azole antifungal resistance. We have discovered mutations that activate Upc2p, increase expression of ERG11, and increase azole resistance. We have found that many clinical isolates overexpress ERG11. In several resistant isolates, ERG11 overexpression is not due to activating mutations in UPC2, suggesting undiscovered, novel resistance mechanisms. Aim 1 of this proposal is to identify mechanisms of ERG11 overexpression in azole resistant isolates of C. albicans by making use of matched pairs of azole susceptible and resistant isolates, as well as unmatched resistant isolates. Novel activating mutations in UPC2 will be identified and characterized through sequencing and standard molecular techniques whereas novel mechanisms of ERG11 overexpression will be identified through a candidate gene approach. Many isolates in our collection carry either novel or characterized ERG11 mutations associated with resistance. While some ERG11 mutations have been associated with or shown to influence azole resistance, their direct effect on this phenotype in C. albicans itself has not been investigated. Aim 2 is to determine the contribution of specific ERG11 mutations to azole resistance in C. albicans by constructing strains that are heterozygous and homozygous for ERG11 mutations that are associated with azole resistance, measuring their effect on susceptibility to azole antifungals, and characterizing their biochemical effects on the interactio between azole antifungals and their target enzyme. Moreover, activation of zinc cluster transcription factors, such as Upc2p, appears to involve proteins that interact directly with these regulators. Activating mutations may influence interactions between Upc2p and its interaction partner proteins. Aim 3 is to identify interaction partner proteins required for Upc2-mediated azole resistance in C. albicans using Tandem Affinity Purification (TAP). Finally, we have found that some activating mutations in UPC2 confer significant increases in azole resistance compared to others, yet have similar effects on ERG11 expression. It is therefore likely that other Upc2-targets contribute to azole resistance. Aim 4 is to determine the role of Upc2-target genes other than ERG11 in Upc2-mediated azole resistance in C. albicans. The proposed research is significant as it will lead to novel strategies for predicting treatment failure, overcoming azole resistance, and improving antifungal therapy. Our approach is innovative as it focuses on novel resistance mechanisms and employs creative strategies to achieve the proposed specific aims.