Miyazaki Taiga, Miyazaki Yoshitsugu, Izumikawa Koichi, Kakeya Hiroshi, Miyakoshi Shunichi, Bennett John E, Kohno Shigeru
Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan.
Antimicrob Agents Chemother. 2006 Feb;50(2):580-6. doi: 10.1128/AAC.50.2.580-586.2006.
Candida albicans ERG3 encodes a sterol C5,6-desaturase which is essential for synthesis of ergosterol. Defective sterol C5,6 desaturation has been considered to be one of the azole resistance mechanisms in this species. However, the clinical relevance of this resistance mechanism is still unclear. In this study, we created a C. albicans erg3/erg3 mutant by the "Ura-blaster" method and confirmed the expected azole resistance using standard in vitro testing and the presence of ergosta-7,22-dien-3beta-ol instead of ergosterol. For in vivo studies, a wild-type URA3 was placed back into its native locus in the erg3 homozygote to avoid positional effects on URA3 expression. Defective hyphal formation of the erg3 homozygote was observed not only in vitro but in kidney tissues. A marked attenuation of virulence was shown by the longer survival and the lower kidney burdens of mice inoculated with the reconstituted Ura+ erg3 homozygote relative to the control. To assess fluconazole efficacy in a murine model of disseminated candidiasis, inoculum sizes of the control and the erg3 homozygote were chosen which provided a similar organ burden. Under these conditions, fluconazole was highly effective in reducing the organ burden in both groups. This study demonstrates that an ERG3 mutation causing inactivation of sterol C5,6-desaturase cannot confer fluconazole resistance in vivo by itself regardless of resistance measured by standard in vitro testing. The finding questions the clinical significance of this resistance mechanism.
白色念珠菌ERG3编码一种甾醇C5,6-去饱和酶，该酶是麦角甾醇合成所必需的。甾醇C5,6去饱和缺陷被认为是该菌种对唑类耐药的机制之一。然而，这种耐药机制的临床相关性仍不清楚。在本研究中，我们通过“尿嘧啶轰击”方法创建了白色念珠菌erg3/erg3突变体，并使用标准体外试验以及存在麦角甾-7,22-二烯-3β-醇而非麦角甾醇来确认预期的唑类耐药性。对于体内研究，将野生型URA3放回erg3纯合子的天然位点，以避免对URA3表达的位置效应。不仅在体外而且在肾脏组织中都观察到erg3纯合子的菌丝形成缺陷。相对于对照组，接种重组的Ura + erg3纯合子的小鼠存活时间更长且肾脏负荷更低，表明毒力明显减弱。为了评估氟康唑在播散性念珠菌病小鼠模型中的疗效，选择了对照组和erg3纯合子的接种量，使其器官负荷相似。在这些条件下，氟康唑在降低两组的器官负荷方面都非常有效。本研究表明，导致甾醇C5,6-去饱和酶失活的ERG3突变本身不能在体内赋予氟康唑耐药性，无论通过标准体外试验测得的耐药性如何。这一发现质疑了这种耐药机制的临床意义。
