Novel Azole Resistance Mechanisms in Candida albicans

描述（由申请人提供）：关于编码唑靶羊毛甾醇脱甲基酶（ERG 11）及其转录调节因子（UPC 2）的基因对C.白色念珠菌我们的长期目标是通过了解抗真菌药物耐药的分子基础来改善念珠菌感染的治疗。本提案的总体目标是了解UPC 2和ERG 11介导的C.白色念珠菌我们的中心假设是Upc 2 p、其相互作用伴侣蛋白及其靶基因与Erg 11 p一起发挥沿着核心作用 介导唑类抗真菌药耐药性。我们发现了激活Upc 2 p、增加ERG 11表达和增加唑类耐药的突变。我们发现许多临床分离株过表达ERG 11。在一些耐药菌株中，ERG 11过表达不是由于UPC 2中的激活突变，这表明未发现的新耐药机制。本研究的第一个目的是确定ERG 11在唑类耐药念珠菌中的过表达机制。白色念珠菌的唑敏感和耐药菌株的配对，以及不匹配的耐药菌株。UPC 2中的新型激活突变将通过测序和标准分子技术进行鉴定和表征，而ERG 11过表达的新型机制将通过候选基因方法进行鉴定。我们收集的许多分离株携带与耐药性相关的新型或特征性ERG 11突变。虽然一些ERG 11突变与唑类耐药相关或影响唑类耐药，但它们对C.白色念珠菌本身尚未调查。目的2是确定特异性ERG 11突变对C.通过构建与唑类耐药相关的ERG 11突变的杂合和纯合菌株，测量其对唑类抗真菌药敏感性的影响，并表征其对唑类抗真菌药与其靶酶之间相互作用的生化作用，此外，锌簇转录因子如Upc 2 p的激活似乎涉及与这些转录因子直接相互作用的蛋白质。 监管部门激活突变可能会影响Upc 2 p与其相互作用伴侣蛋白之间的相互作用。目的3是鉴定UPc 2介导的唑类耐药所需的相互作用伴侣蛋白。使用串联亲和纯化（TAP）纯化白色念珠菌。最后，我们发现UPC 2中的一些激活突变与其他突变相比显著增加了唑类耐药，但对ERG 11表达具有相似的影响。因此，其他Upc 2-靶标可能有助于唑类耐药性。目的4：研究UPc 2靶基因在UPc 2介导的唑类耐药中的作用。白色念珠菌这项研究意义重大，因为它将为预测治疗失败、克服唑类耐药和改善抗真菌治疗提供新的策略。我们的方法是创新的，因为它专注于新的耐药机制，并采用创造性的策略来实现所提出的具体目标。 公共卫生相关性：拟议的研究与公共卫生有关，因为唑类抗真菌药物耐药的新机制的发现将最终有助于预测治疗失败，克服唑类耐药和改善抗真菌治疗的新策略的发展。因此，这项研究与国家过敏和传染病研究所的使命有关，该使命涉及支持基础和应用研究，以更好地了解，治疗和最终预防传染病，特别是关于抗菌素耐药性的重点领域。

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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the discovery of novel mechanisms of azole antifungal resistance will ultimately contribute to the development of novel strategies for predicting treatment failure, overcoming azole resistance, and improving antifungal therapy. This research is therefore relevant to that part of the National Institute of Allergy and Infectious Diseases' mission that pertains to supporting basic and applied research to better understand, treat, and ultimately prevent infectious diseases, particularly with regard to the emphasis area of antimicrobial resistance.