The Aspergillus fumigatus Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility.
The Aspergillus fumigatus Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility.
复制标题
烟曲霉损伤抗性蛋白家族协调调节麦角甾醇生物合成和唑敏感性
作者：
Song J;Zhai P;Zhang Y;Zhang C;Sang H;Han G;Keller NP;Lu L
麦角甾醇是真菌质膜的主要特异性成分，因此，催化麦角甾醇合成的细胞色素P450酶（Erg蛋白）已被选为唑类抗真菌药物的有价值靶点。然而，机会致病菌烟曲霉（Aspergillus fumigatus）在世界范围内对唑类药物产生了耐药性，主要是通过细胞色素P450酶Cyp51 （Erg11）的突变。在这项研究中，我们证明了一个细胞色素b5样血红素结合损伤抵抗蛋白（Dap）家族，由DapA， DapB和DapC组成，协调调节细胞色素P450酶Erg5和Erg11的功能，并相反地影响对唑类药物的敏感性。这三个基因的表达均以唑浓度依赖性的方式诱导，对唑类药物敏感性的降低需要DapA稳定细胞色素P450蛋白活性。相反，过表达DapB和DapC导致Erg5和Erg11功能障碍，导致甾醇中间体异常积累，进一步增强ΔdapA菌株对唑类药物的敏感性。外源血红素拯救和血红素结合位点诱变实验结果表明，DapA的血红素结合有助于降低唑敏感性，而DapB和c能够通过消耗血红素来降低Erg5和Erg11的活性。体内实验数据表明，在侵袭性肺曲霉病免疫功能低下的小鼠模型中，灭活的DapA与活化的DapB联合产生一种烟曲霉突变体，这种突变体很容易用唑治疗。与在酿酒酵母和pombe裂糖酵母中发现的单一Dap蛋白相比，我们认为这个复杂的Dap家族调节系统是在真菌进化过程中作为一种适应环境刺激调节麦角甾醇合成的适应性手段而出现的。了解真菌病原体麦角甾醇的生物合成途径有助于设计新的抗真菌药物，并有助于抗真菌耐药机制的研究。在这项研究中，我们证明了三种细胞色素b5样Dap蛋白协同调节细胞色素P450蛋白催化的唑抗性和麦角甾醇生物合成。我们对真菌病原体中Dap调控系统的新见解可能具有广泛的治疗影响，而不仅仅是它们对经典的唑类抗真菌药物的有用性。此外，我们对Dap通过血红素结合活性调控细胞色素P450蛋白功能的分子机制的阐明可能超出真菌领域，适用于Dap蛋白调控哺乳动物固醇合成。了解麦角甾醇在真菌病原体中的生物合成途径有助于设计新的抗真菌药物，并有助于抗真菌耐药机制的研究。在这项研究中，我们证明了三种细胞色素b5样Dap蛋白协同调节细胞色素P450蛋白催化的唑抗性和麦角甾醇生物合成。我们对真菌病原体中Dap调控系统的新见解可能具有广泛的治疗影响，而不仅仅是它们对经典的唑类抗真菌药物的有用性。此外，我们对Dap通过血红素结合活性调控细胞色素P450蛋白功能的分子机制的阐明可能超出真菌领域，适用于Dap蛋白调控哺乳动物固醇合成。
ABSTRACT Ergosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochrome b5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapA strains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme. In vivo data demonstrate that inactivated DapA combined with activated DapB yields an A. fumigatus mutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found in Saccharomyces cerevisiae and Schizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli. IMPORTANCE Knowledge of the ergosterol biosynthesis route in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of antifungal-drug resistance mechanisms. In this study, we demonstrate that three cytochrome b5-like Dap proteins coordinately regulate the azole resistance and ergosterol biosynthesis catalyzed by cytochrome P450 proteins. Our new insights into the Dap regulatory system in fungal pathogens may have broad therapeutic ramifications beyond their usefulness for classic azole antifungals. Moreover, our elucidation of the molecular mechanism of Dap regulation of cytochrome P450 protein functionality through heme-binding activity may extend beyond the Kingdom Fungi with applicability toward Dap protein regulation of mammalian sterol synthesis. Knowledge of the ergosterol biosynthesis route in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of antifungal-drug resistance mechanisms. In this study, we demonstrate that three cytochrome b5-like Dap proteins coordinately regulate the azole resistance and ergosterol biosynthesis catalyzed by cytochrome P450 proteins. Our new insights into the Dap regulatory system in fungal pathogens may have broad therapeutic ramifications beyond their usefulness for classic azole antifungals. Moreover, our elucidation of the molecular mechanism of Dap regulation of cytochrome P450 protein functionality through heme-binding activity may extend beyond the Kingdom Fungi with applicability toward Dap protein regulation of mammalian sterol synthesis.