Karyotypes of C. albicans fluconazole resistant mutants

描述（由申请人提供）：通过许多小组的工作，一些（但不是所有的）临床分离株的氟康唑耐药与药物的基因靶点ERG11以及细胞泵基因CDR1、CDR2和MDR1有关。这些基因在耐药性中的作用机制尚不完全清楚，也不确定是否已鉴定出氟康唑的全部基因库。在我们最近的工作中，我们首次证明了在实验室条件下，10个独立的氟康唑抗性突变体在短时间暴露于药物一周后产生，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。暴露于氟康唑至少一个月后产生的7个独立的氟康唑耐药突变体均具有4号染色体的减少，并获得第二个特异性变化，即3号染色体的额外拷贝。与此一致的是，在复制染色体上携带的基因CDR1的表达增加了，而其他基因则没有。我们的结果与已发表的数据非常相似，这些数据描述了一系列连续分离的白色念珠菌临床菌株，随着时间的推移，耐药性逐渐增加，最初没有显示或没有任何与耐药性相关的基因突变或过表达。我们建议验证我们的假设，即临床分离株对氟康唑的主要反应是染色体拷贝数的特异性变化。后期临床分离株较高的耐药水平可被视为继发性变化，这是在长期选择压力下基因突变积累的结果。我们开发了特殊的程序来分离和处理从接受氟康唑治疗的患者中序列遗传相关的白色念珠菌系列菌株，从而保护分离株免受氟康唑作用以外的压力，这些压力可能导致不良的染色体不稳定。可靠的菌株系列将以其电泳核型为特征。如果临床对氟康唑耐药的样本中观察到特定染色体拷贝数的变化，我们就可以进一步研究这种新的耐药机制的未知基因。

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.