Novel Azole Resistance Mechanisms in Candida albicans

白色念珠菌的新唑耐药机制

• 批准号: 8293723
• 负责人: P. David Rogers
• 金额: $ 37.45万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 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 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8293723
• 关键词: ABCB1 gene; Acquired Immunodeficiency Syndrome; Affect; Affinity Chromatography; Aneuploidy; Antifungal Agents; Antifungal Therapy; Antimicrobial Resistance; Applied Research; Area; Azole resistance; Azoles; Basic Science; Biochemical; CDR1 gene; Candida; Candida albicans; Candidate Disease Gene; Chromosomes, Human, Pair 5; Clinical; Collection; Communicable Diseases; Development; Disseminated candidiasis; Enzymes; Expenditure; Fungal Drug Resistance; Gene Targeting; Genes; Goals; Immunocompromised Host; Individual; Infection; Knowledge; Lanosterol; Lead; Measures; Mediating; Mission; Molecular; Mutation; National Institute of Allergy and Infectious Disease; Opportunistic Infections; Outcome; Patients; Pharmaceutical Preparations; Phenotype; Play; Predisposition; Process; Proteins; Public Health; Research; Resistance; Role; Sepsis; Sterol Biosynthesis Pathway; Sterols; TAC1 gene; Techniques; Testing; Transcription factor genes; Treatment Failure; United States; Work; Zinc Cluster; base; improved; innovation; mortality; novel; novel strategies; oropharyngeal thrush; overexpression; prevent; resistance mechanism; transcription factor; uptake

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.

描述（由申请人提供）：对于编码唑类靶点羊毛甾醇脱甲基酶（ERG11）及其转录调节因子（UPC2）的基因对白色念珠菌中唑类抗真菌耐药性的贡献，目前还缺乏了解。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。该提案的总体目标是了解白色念珠菌 UPC2 和 ERG11 介导的唑类耐药性的分子基础。我们的中心假设是 Upc2p、其相互作用伙伴蛋白及其靶基因与 Erg11p 一起发挥核心作用 介导唑类抗真菌耐药性。我们发现了激活 Upc2p、增加 ERG11 表达并增加唑类耐药性的突变。我们发现许多临床分离株过度表达 ERG11。在一些耐药菌株中，ERG11 过度表达并不是由于激活 UPC2 突变，这表明尚未发现的新耐药机制。该提案的目标 1 是通过使用匹配的唑敏感和耐药分离株以及不匹配的耐药分离株来确定白色念珠菌的唑耐药分离株中 ERG11 过度表达的机制。 UPC2 中的新激活突变将通过测序和标准分子技术进行鉴定和表征，而 ERG11 过度表达的新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 ERG11 突变。虽然一些 ERG11 突变与唑类耐药性相关或显示出影响，但它们对白色念珠菌本身表型的直接影响尚未得到研究。目标 2 是通过构建与唑类抗性相关的 ERG11 突变杂合和纯合菌株，测量其对唑类抗真菌药敏感性的影响，并表征其生化效应，确定特定 ERG11 突变对白色念珠菌中唑类抗性的贡献唑类抗真菌药与其靶酶之间的相互作用。此外，锌簇转录因子（例如 Upc2p）的激活似乎涉及与这些转录因子直接相互作用的蛋白质。 监管机构。激活突变可能会影响 Upc2p 及其相互作用伙伴蛋白之间的相互作用。目标 3 是使用串联亲和纯化 (TAP) 鉴定白色念珠菌中 Upc2 介导的唑类抗性所需的相互作用伴侣蛋白。最后，我们发现 UPC2 中的一些激活突变与其他突变相比，会显着增加唑类耐药性，但对 ERG11 表达也有类似的影响。因此，其他 Upc2 靶标可能会导致唑类耐药。目标 4 是确定除 ERG11 之外的 Upc2 靶基因在白色念珠菌中 Upc2 介导的唑类抗性中的作用。拟议的研究意义重大，因为它将带来预测治疗失败、克服唑类耐药性和改善抗真菌治疗的新策略。我们的方法是创新的，因为它专注于新颖的抵抗机制，并采用创造性的策略来实现拟议的具体目标。 公共健康相关性：拟议的研究与公共健康相关，因为唑类抗真菌耐药性新机制的发现最终将有助于开发预测治疗失败、克服唑类耐药性和改善抗真菌治疗的新策略。因此，这项研究与国家过敏和传染病研究所使命的一部分相关，即支持基础和应用研究，以更好地理解、治疗和最终预防传染病，特别是在抗菌素耐药性的重点领域。