Schubert Sabrina, Rogers P David, Morschhäuser Joachim
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, Würzburg D-97070, Germany.
Antimicrob Agents Chemother. 2008 Dec;52(12):4274-80. doi: 10.1128/AAC.00740-08. Epub 2008 Sep 22.
Candida dubliniensis, a yeast that is closely related to Candida albicans, can rapidly develop resistance to the commonly used antifungal agent fluconazole in vitro and in vivo during antimycotic therapy. Fluconazole resistance in C. dubliniensis is usually caused by constitutive overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily. The zinc cluster transcription factor Mrr1p has recently been shown to control MDR1 expression in C. albicans in response to inducing stimuli, and gain-of-function mutations in the MRR1 gene result in constitutive upregulation of the MDR1 efflux pump. We identified a gene with a high degree of similarity to C. albicans MRR1 (CaMRR1) in the C. dubliniensis genome sequence. When C. dubliniensis MRR1 (CdMRR1) was expressed in C. albicans mrr1Delta mutants, it restored benomyl-inducible MDR1 expression, demonstrating that CdMRR1 is the ortholog of CaMRR1. To investigate whether MDR1 overexpression in C. dubliniensis is caused by mutations in MRR1, we sequenced the MRR1 alleles from a fluconazole-resistant, clinical C. dubliniensis isolate and a matched, fluconazole-susceptible isolate from the same patient as well as those from four in vitro-generated, fluconazole-resistant C. dubliniensis strains derived from two different C. dubliniensis isolates. We found that all five resistant strains contained single nucleotide substitutions or small in-frame deletions that resulted in amino acid changes in Mrr1p. Expression of these mutated alleles in C. albicans resulted in the constitutive activation of the MDR1 promoter and multidrug resistance. Therefore, mutations in MRR1 are the major cause of MDR1 upregulation in both C. albicans and C. dubliniensis, demonstrating that the transcription factor Mrr1p plays a central role in the development of drug resistance in these human fungal pathogens.
都柏林念珠菌是一种与白色念珠菌密切相关的酵母，在抗真菌治疗期间，它在体外和体内都能迅速对常用抗真菌药物氟康唑产生耐药性。都柏林念珠菌对氟康唑的耐药性通常是由MDR1基因的组成型过表达引起的，该基因编码主要易化子超家族的一种多药外排泵。锌簇转录因子Mrr1p最近被证明可响应诱导刺激来控制白色念珠菌中MDR1的表达，并且MRR1基因的功能获得性突变会导致MDR1外排泵的组成型上调。我们在都柏林念珠菌基因组序列中鉴定出一个与白色念珠菌MRR1（CaMRR1）高度相似的基因。当都柏林念珠菌MRR1（CdMRR1）在白色念珠菌mrr1Delta突变体中表达时，它恢复了苯菌灵诱导的MDR1表达，表明CdMRR1是CaMRR1的直系同源基因。为了研究都柏林念珠菌中MDR1的过表达是否由MRR1的突变引起，我们对来自一株氟康唑耐药的临床都柏林念珠菌分离株以及来自同一患者的一株匹配的氟康唑敏感分离株的MRR1等位基因进行了测序，同时也对来自两种不同都柏林念珠菌分离株的四个体外产生的氟康唑耐药都柏林念珠菌菌株的MRR1等位基因进行了测序。我们发现所有五个耐药菌株都含有单核苷酸替换或小的框内缺失，这些缺失导致Mrr1p中的氨基酸发生变化。这些突变等位基因在白色念珠菌中的表达导致MDR1启动子的组成型激活和多药耐药性。因此，MRR1的突变是白色念珠菌和都柏林念珠菌中MDR1上调的主要原因，这表明转录因子Mrr1p在这些人类真菌病原体的耐药性发展中起着核心作用。
