Mechanisms of drug resistance in Candida albicans

白色念珠菌的耐药机制

• 批准号: 7247242
• 负责人: Brian Wong
• 金额: $ 36.03万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-15 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7247242
• 关键词: ABCB1 gene; Acquired Immunodeficiency Syndrome; Antifungal Agents; Azole resistance; Azoles; Biochemical; Biological; CDR1 gene; Candida albicans; Carrier Proteins; Cell membrane; Cells; Clinical; Code; Collection; Condition; Disease; Drug Efflux; Drug resistance; Fluconazole; Genetic; Goals; Membrane; Mutation; Open Reading Frames; Patients; Plasmids; Polymerase Chain Reaction; Property; Protein Overexpression; Proteins; Pump; Recombinant Proteins; Recombinants; Recurrence; Secretory Vesicles; Structural Genes; Substrate Specificity; Testing; Vesicle; efflux pump; inhibitor/antagonist; mutant; oropharyngeal thrush; physical property; prevent

DESCRIPTION (provided by applicant): A major cause of resistance to azole antifungal drugs in Candida albicans is overproduction of the plasma membrane drug efflux pumps Cdr1p, Cdr2p and/or Mdr1p, but little is known about these pumps' physical and catalytic properties. It also is not known if mutations in these pumps' structural genes (CDR1, CDR2 and MDR1) cause azole resistance in clinical C. albicans strains. This project will use genetic, cell biological and biochemical approaches to pursue two specific aims. Aim 1 is to determine if Cdr1p, Cdr2p and Mdr1p actively transport azoles across the plasma membrane and to define these proteins' properties. CDR1, CDR2 or MDR1 will be expressed in a conditional C. albicans sec4 mutant, and secretory vesicles from these cells will be tested for the ability to transport [3H]-fluconazole across their membranes. The vesicles will also be used to examine each recombinant protein's physical properties, catalytic constants, substrate specificities and inhibitor profile. Aim 2 is to determine if mutations in the CDR1, CDR2 and/or MDR1 coding sequences cause azole resistance in C. albicans. Azole-susceptible and azole-resistant C. albicans strains from different groups of patients will be tested for [3H]-fluconazole accumulation and for CDR1, CDR2 and MDR1 overexpression. The CDR1, CDR2 and MDR1 ORFs from [3H]-fluconazole-underaccumulating strains that do not overexpress CDR1, CDR2 or MDR1 will be cloned and expressed in a C. albicans cdr1 cdr2 mdr1 null mutant. Any ORF that confers higher levels of azole resistance in the cdr1 cdr2 mdr1 mutant than does its wild-type counterpart will be sequenced, its protein product will be analyzed using isolated C. albicans secretory vesicles, and its effects on azole resistance will be verified in wild-type C. albicans transformants. The longterm goal of these studies is to develop new strategies and approaches for overcoming and/or preventing the emerging problem of azole resistance in C. albicans.

描述（由申请人提供）：白色念珠菌中抗烷基抗真菌药物的耐药性的主要原因是质膜膜药外泵的过量生产CDR1P，CDR2P和/或MDR1P，但对这些泵的物理和​​催化性质知之甚少。这些泵的结构基因中的突变（CDR1，CDR2和MDR1）是否在临床白色念珠菌菌株中引起硫代抗性。该项目将使用遗传，细胞生物学和生化方法来追求两个具体目标。 AIM 1是确定CDR1P，CDR2P和MDR1P是否在质膜上积极运输叠唑并定义这些蛋白质的特性。 CDR1，CDR2或MDR1将在有条件的白色念珠菌Sec4突变体中表达，这些细胞的分泌囊泡将经过测试，以便在其膜上运输[3H]氟康唑的能力。囊泡还将用于检查每种重组蛋白的物理特性，催化常数，底物特异性和抑制剂谱。 AIM 2是确定CDR1，CDR2和/或MDR1编码序列中的突变是否在白色念珠菌中引起Azole抗性。来自不同患者组的硫唑敏感和抗唑的白色念珠菌菌株将进行[3H] - 氟康唑积累以及CDR1，CDR2和MDR1过表达的测试。来自[3H] - 氟康唑纳维菌菌株的CDR1，CDR2和MDR1 ORF，不会过表达CDR1，CDR2或MDR1的菌株，并将在C. albicans CDR1 CDR1 CDR1 CDR2 MDR1 NULL突变体中进行克隆并表达。将对CDR1 CDR2 MDR1突变体含有较高水平的唑耐药性的任何ORF比其野生型对应物进行测序，将使用孤立的白色念珠菌分泌囊泡对其蛋白质产物进行测序，并且其对Azole耐药性的影响将在Wildpy C. albicans Transfants中得到验证。这些研究的长期目标是制定新的策略和方法来克服和/或防止白色念珠菌中新兴的硫唑耐药性问题。