Genetic Dissection of Azole Resistance Mechanisms in Candida albicans and Their Validation in a Mouse Model of Disseminated Infection
Genetic Dissection of Azole Resistance Mechanisms in Candida albicans and Their Validation in a Mouse Model of Disseminated Infection
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DOI：
10.1128/aac.01645-09
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发表时间：
2010-04-01
影响因子：
4.9
通讯作者：
Sanglard, Dominique
中科院分区：
文献类型：
--
作者：
MacCallum, Donna M.;Coste, Alix;Sanglard, Dominique
唑类抗真菌药耐药的主要机制包括多药转运蛋白的上调和靶酶的修饰，细胞色素P450（Erg 11）参与麦角甾醇的14 α-去甲基化。这些机制往往结合在唑耐药的白色念珠菌分离的患者。然而，各个机制对C.由于对这种二倍体物种进行遗传操作的技术困难，因此很难确定白念珠菌对特异性唑类的抗性。最近的进展使遗传操作变得更加容易，因此，我们对耐唑临床分离株的耐药机制进行了遗传解剖。该分离株（DSY 296）上调多药转运蛋白基因CDR 1和CDR 2，并在两个ERG 11等位基因中获得G464 S置换。在DSY 296中，灭活TAC 1（一种含有功能获得性突变的转录因子），然后用野生型等位基因顺序替换ERG 11突变等位基因，使唑类敏感性恢复至亲本唑类敏感分离株（DSY 294）的水平。这些连续的遗传操作不仅证明了这两种抗性机制是DSY 296中抗性发展的原因，而且还表明通过MIC测定体外测量的抗性定量水平是任何菌株中遗传抗性机制数量的函数。还在一种新的侵袭性C.小鼠白色念珠菌感染。氟康唑对DSY 296的50%有效剂量（艾德（50）s）最高，并随着各耐药机制的相继消除而成比例地降低。然而，虽然ED 50的倍数差异与MIC的倍数差异成比例，但其幅度低于体外测量的幅度，并取决于特定的耐药机制。
Principal mechanisms of resistance to azole antifungals include the upregulation of multidrug transporters and the modification of the target enzyme, a cytochrome P450 (Erg11) involved in the 14 alpha-demethylation of ergosterol. These mechanisms are often combined in azole-resistant Candida albicans isolates recovered from patients. However, the precise contributions of individual mechanisms to C. albicans resistance to specific azoles have been difficult to establish because of the technical difficulties in the genetic manipulation of this diploid species. Recent advances have made genetic manipulations easier, and we therefore undertook the genetic dissection of resistance mechanisms in an azole-resistant clinical isolate. This isolate (DSY296) upregulates the multidrug transporter genes CDR1 and CDR2 and has acquired a G464S substitution in both ERG11 alleles. In DSY296, inactivation of TAC1, a transcription factor containing a gain-of-function mutation, followed by sequential replacement of ERG11 mutant alleles with wild-type alleles, restored azole susceptibility to the levels measured for a parent azole-susceptible isolate (DSY294). These sequential genetic manipulations not only demonstrated that these two resistance mechanisms were those responsible for the development of resistance in DSY296 but also indicated that the quantitative level of resistance as measured in vitro by MIC determinations was a function of the number of genetic resistance mechanisms operating in any strain. The engineered strains were also tested for their responses to fluconazole treatment in a novel 3-day model of invasive C. albicans infection of mice. Fifty percent effective doses (ED(50)s) of fluconazole were highest for DSY296 and decreased proportionally with the sequential removal of each resistance mechanism. However, while the fold differences in ED50 were proportional to the fold differences in MICs, their magnitude was lower than that measured in vitro and depended on the specific resistance mechanism operating.