Karyotypes of C. albicans fluconzole resistant mutants

白色念珠菌氟康唑抗性突变体的核型

• 批准号: 6739648
• 负责人: ELENA RUSTCHENKO
• 金额: $ 7.88万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6739648
• 关键词: Candida albicans; antifungal antibiotics; chromosomes; clinical research; drug resistance; fluconazole; gene expression; human tissue; karyotype; messenger RNA; microorganism culture; mutant

DESCRIPTION (provided by applicant): Through the work of many groups, fluconazole resistance in some, but not all clinical isolates has been associated with the gene target of the drug, ERG11, as well as genes for cellular pumps, CDR1, CDR2 and MDR1. The mechanisms implicating these genes in resistance are not fully understood, and it is uncertain that the entire repertoire of fluconazole genes have been identified. In our recent work, we showed for the first time that under laboratory condition 10 independent fluconazole resistant mutants that derived after a short exposure of one week to the drug, all lost a copy of chromosome 4. The mRNA level of the above mentioned genes in these mutants was not changed. A total of 7 independent fluconazole resistant mutants that derived after at least one month of exposure to the drug, all possessed the diminution of chromosome 4, and acquired a second specific change, an extra-copy of chromosome 3. Consistently, the expression of the gene CDR1, which is carried on the duplicated chromosome, but not of the other genes, was increased. Our results are remarkably similar to the published data describing series of C. albicans sequentially isolated clinical strains with a progressive increase of resistance over time, which did not initially show or did not have any mutations or overexpression of the genes associated with the resistance. We propose to test our hypothesis that the primary response of clinical isolates to fluconazole is specific changes in chromosomal copy number. The higher resistance levels of later clinical isolates can be viewed as secondary changes, which occur as a result of accumulation of gene mutations under prolonged selective pressure. We developed special procedure to isolate and handle C. albicans series of sequential genetically related strains from patients undergoing fluconazole treatment, thus protecting the isolates from stresses other than action of fluconazole, which can induce undesirable chromosomal instability. The reliable series of strains will be characterized for their electrophoretic karyotypes. If the change in copy number of a specific chromosome(s) is/are observed with clinical samples resistant to fluconazole, we are in the position to further study the still unknown genes responsible for this novel mechanism of resistance.

描述（由申请人提供）：通过许多小组的工作，一些但不是所有临床分离株的氟康唑耐药性与药物的基因靶点ERG 11以及细胞泵基因CDR 1、CDR 2和MDR 1相关。这些基因在耐药中的作用机制还不完全清楚，也不确定是否已鉴定出氟康唑基因的全部谱。在我们最近的工作中，我们首次发现，在实验室条件下，10个独立的氟康唑耐药突变体，在短时间暴露于药物一周后，都失去了4号染色体的拷贝。这些突变体中上述基因的mRNA水平没有变化。共7个独立的氟康唑耐药突变体，在至少一个月的药物暴露后，所有具有4号染色体的减少，并获得第二个特定的变化，额外的3号染色体的副本。因此，复制染色体上携带的基因CDR 1的表达增加，而其他基因的表达没有增加。我们的结果与已发表的描述C.白念珠菌相继分离出耐药性随时间逐渐增加的临床菌株，其最初不显示或不具有与耐药性相关的基因的任何突变或过表达。我们建议测试我们的假设，临床分离株的主要反应氟康唑是染色体拷贝数的具体变化。后期临床分离株的较高耐药水平可视为继发性变化，其是由于长期选择压力下基因突变积累所致。我们制定了特殊的程序来分离和处理C。白念珠菌系列的连续遗传相关菌株进行氟康唑治疗的患者，从而保护菌株从压力以外的氟康唑的作用，这可能会导致不希望的染色体不稳定性。将对可靠的菌株系列进行电泳核型表征。如果在临床样本中观察到对氟康唑耐药的特定染色体的拷贝数变化，我们将能够进一步研究负责这种新的耐药机制的未知基因。