Amino acid substitutions in the Candida albicans sterol Δ5,6-desaturase (Erg3p) confer azole resistance: characterization of two novel mutants with impaired virulence
Amino acid substitutions in the Candida albicans sterol Δ5,6-desaturase (Erg3p) confer azole resistance: characterization of two novel mutants with impaired virulence
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DOI：
10.1093/jac/dks186
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发表时间：
2012-09-01
影响因子：
5.2
通讯作者：
Le Pape, Patrice
中科院分区：
文献类型：
--
作者：
Morio, Florent;Pagniez, Fabrice;Le Pape, Patrice
为探讨2株白念珠菌（CAAL 2和CAAL 76）对氟康唑耐药的机制，采用微量肉汤稀释法（CLSI M27-A3）测定氟康唑和伏立康唑的MIC，用Etest法测定白念珠菌素B的MIC。通过RTPCR测定CDR 1、MDR 1和ERG 11的RNA表达水平。通过扩增和测序研究ERG 11和ERG 3中的突变。用气相色谱-质谱联用仪（GCMS）测定甾醇膜的膜分布。在侵袭性念珠菌病的小鼠模型中测定了体内毒力，两种分离株均显示唑类交叉耐药和对阿替霉素B敏感性降低，并且是新的（5，6）-去饱和酶（Erg 3 p）突变体。CAAL 2含有一个新的氨基酸取代（L193 R），而在CAAL 76中发现了一个13 bp的缺失，导致一个截短的Erg 3 p（366378）。如GCMS所示，这两种遗传变异都损害了Erg 3 p的功能（麦角甾醇含量低于10，麦角甾-7，22-二烯醇的积累高于40）。在体内，在侵袭性念珠菌病的小鼠模型中，CAAL 2和CAAL 76都表现出明显的毒力下降趋势，这似乎与菌丝生长能力降低有关。我们认为，这种减毒的体内毒力表型可能与菌丝生长潜力较低有关。总之，我们的研究结果强调了一个事实，即erg 3突变体必须考虑在未来的研究，旨在调查唑类抗真菌药物耐药性。
To determine the mechanisms responsible for fluconazole resistance in two Candida albicans isolates (CAAL2 and CAAL76) recovered from two hospitalized patients after fluconazole prophylaxis.MICs of fluconazole and voriconazole were determined by the broth microdilution method (CLSI M27-A3), and by Etest for amphotericin B. RNA expression levels of CDR1, MDR1 and ERG11 were determined by RTPCR. Mutations in ERG11 and ERG3 were investigated by amplification and sequencing. Sterol membrane profiles were determined by gas chromatographymass spectrometry (GCMS). In vivo virulence was determined in a murine model of invasive candidiasis.Both isolates displayed azole cross-resistance and reduced susceptibility to amphotericin B, and are novel (5,6)-desaturase (Erg3p) mutants. CAAL2 harbours a new amino acid substitution (L193R), whereas a 13 bp deletion leading to a truncated Erg3p (366378) was found in CAAL76. Both genetic alterations impaired Erg3p function as shown by GCMS in these isolates (ergosterol content below 10, and accumulation of ergosta-7,22-dienol above 40). In vivo, in a murine model of invasive candidiasis, both CAAL2 and CAAL76 exhibited a significant trend toward reduced virulence, which seems to be linked to a reduced capacity for hyphal growth.These findings demonstrate the critical role of residue 193 in Erg3p function and azole resistance. We suggest that this attenuated in vivo virulence phenotype could be linked to lower potential for hyphal growth. Taken together, our findings highlight the fact that erg3 mutants must be considered in future studies aiming at investigating azole antifungal drug resistance.