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）：关于编码Azole靶标型鞭毛脱硫酶（ERG11）及其转录调节剂（UPC2）对阿唑抗真菌抗性的基因的贡献，缺乏明显的知识。我们的长期目标是通过了解抗真菌抗性的分子基础来改善念珠菌感染的治疗。该提案的总体目的是了解白色念珠菌中UPC2和ERG11介导的唑的抗性的分子基础。我们的核心假设是UPC2P，其相互作用伴侣蛋白及其靶基因与ERG11P一起起着核心作用 在介导唑抗真菌抗性时。我们发现了激活UPC2P，增加ERG11表达并增加唑耐药性的突变。我们发现许多临床分离株过表达ERG11。在几种抗性分离株中，ERG11的过表达并不是由于UPC2中激活突变，这表明未发现的新型抗性机制。该提案的目的1是通过使用匹配的Azole易感和抗性分离株以及无与伦比的抗性分离株来鉴定白色念珠菌抗甲唑的分离株ERG11过表达的机制。将通过测序和标准分子技术来鉴定和表征新的UPC2中的新型激活突变，而ERG11过表达的新型机制将通过候选基因方法鉴定。我们集合中的许多分离株都带有新颖或表征与抗性相关的ERG11突变。尽管某些ERG11突变已与或证明会影响硫唑的耐药性，但尚未研究它们对白色念珠菌本身中这种表型的直接影响。目的2是通过构建与偶氮抗性相关的ERG11突变的杂合和纯合的菌株来确定特定的ERG11突变对白色念珠菌抗唑的耐药性的贡献，从而测量了它们对Azole抗真菌性抗体的敏感性的影响，从而对其对Azole Attymals抗相互作用的影响进行了对抗体抗体的影响。此外，锌簇转录因子（例如UPC2P）的激活似乎涉及与这些蛋白质直接相互作用的蛋白质 监管机构。激活突变可能会影响UPC2P及其相互作用伴侣蛋白之间的相互作用。 AIM 3是使用串联亲和力纯化（TAP）确定UPC2介导的偶氮抗性所需的相互作用伴侣蛋白。最后，我们发现，与其他人相比，UPC2中的某些激活突变赋予了偶氮抗性的显着增加，但对ERG11表达的影响相似。因此，其他UPC2目标可能导致偶氮抗性。 AIM 4是确定ERG11以外的UPC2靶基因在upc2介导的白色念珠菌中的抗抗抗性中的作用。拟议的研究非常重要，因为它将导致预测治疗失败，克服唑耐药并改善抗真菌治疗的新型策略。我们的方法具有创新性，因为它专注于新颖的抵抗机制，并采用创造性策略来实现拟议的特定目标。 公共卫生相关性：拟议的研究与公共卫生有关，因为发现新颖的抗真菌抗性抗性的新机制将最终有助于制定预测治疗失败，克服硫唑耐药性并改善抗真菌疗法的新型策略。因此，这项研究与美国国家过敏和传染病研究所的那部分有关，该任务与支持基础和应用研究有关，以更好地理解，治疗，最终预防传染病，尤其是在抗菌抗性的重点方面。