Sterol Biosynthesis and Azole Tolerance Is Governed by the Opposing Actions of SrbA and the CCAAT Binding Complex.
Sterol Biosynthesis and Azole Tolerance Is Governed by the Opposing Actions of SrbA and the CCAAT Binding Complex.
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DOI：
10.1371/journal.ppat.1005775
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发表时间：
2016-07
期刊：
影响因子：
6.7
通讯作者：
Bromley MJ
中科院分区：
文献类型：
--
作者：
Gsaller F;Hortschansky P;Furukawa T;Carr PD;Rash B;Capilla J;Müller C;Bracher F;Bowyer P;Haas H;Brakhage AA;Bromley MJ
唑类药物选择性靶向真菌甾醇生物合成，是我们的抗真菌治疗武器库的关键。然而，对这类药物的耐药性，特别是在主要的人类霉菌病原体烟曲霉中，正在出现并达到促使一些人认为它们将失去临床使用的现实可能性的水平。泛唑类耐药菌株的主要类别的特征在于在cyp 51 A的启动子内存在至少34个碱基的串联重复（TR 34），cyp 51 A是编码唑类药物靶固醇C14-脱甲基酶的基因。在这里，我们证明了TR 34中的重复序列被固醇调节元件结合蛋白（SREBP）SrbA和CCAAT结合复合物（CBC）结合。我们表明，CBC作为补充SrbA作为麦角固醇生物合成的负调节剂，并表明CBC活性的缺乏通过多个麦角固醇生物合成基因，包括那些编码HMG-CoA-合成酶，HMG-CoA-还原酶和甾醇C14-脱甲基酶的转录去抑制导致甾醇水平增加。与这些发现一致，CBC的失活增加了对靶向麦角固醇生物合成的不同类别药物的耐受性，包括唑类、烯丙胺（特比萘芬）和他汀类（辛伐他汀）。我们发现HapE（P88 L）中的临床相关突变显著损害CBC对其靶位点的结合亲和力。我们确定TR 34驱动cyp 51 A过表达的机制是由于SrbA的重复而不是CBC结合位点的重复，并表明34 mer的缺失导致cyp 51 A表达的缺乏和类似于cyp 51 A无效突变体的唑敏感性增加。最后，我们表明，菌株缺乏一个功能性CBC严重衰减的肺和全身模型曲霉菌病的致病性。 烟曲霉是世界范围内最重要的气传霉菌病原体和过敏原。据估计，每年有超过300万人患有侵入性或慢性感染，导致超过60万人死亡。很少有药物可用于治疗各种形式的曲霉病，我们主要依赖于抑制甾醇生物合成的唑类药物。对唑类药物的耐药性正在惊人地增长，主要是由具有两个主要遗传特征（TR 34/L98 H和TR 46/Y121 F/T289 A）的菌株驱动的。在这项研究中，我们确定了这组分离物中控制抗性的转录机制与2种转录调节因子SrbA和CBC的相反作用有关，并揭示了CBC在A.烟熏。我们建议靶向SrbA将为耐药菌株的治疗干预提供有效途径。
Azole drugs selectively target fungal sterol biosynthesis and are critical to our antifungal therapeutic arsenal. However, resistance to this class of drugs, particularly in the major human mould pathogen Aspergillus fumigatus, is emerging and reaching levels that have prompted some to suggest that there is a realistic probability that they will be lost for clinical use. The dominating class of pan-azole resistant isolates is characterized by the presence of a tandem repeat of at least 34 bases (TR34) within the promoter of cyp51A, the gene encoding the azole drug target sterol C14-demethylase. Here we demonstrate that the repeat sequence in TR34 is bound by both the sterol regulatory element binding protein (SREBP) SrbA, and the CCAAT binding complex (CBC). We show that the CBC acts complementary to SrbA as a negative regulator of ergosterol biosynthesis and show that lack of CBC activity results in increased sterol levels via transcriptional derepression of multiple ergosterol biosynthetic genes including those coding for HMG-CoA-synthase, HMG-CoA-reductase and sterol C14-demethylase. In agreement with these findings, inactivation of the CBC increased tolerance to different classes of drugs targeting ergosterol biosynthesis including the azoles, allylamines (terbinafine) and statins (simvastatin). We reveal that a clinically relevant mutation in HapE (P88L) significantly impairs the binding affinity of the CBC to its target site. We identify that the mechanism underpinning TR34 driven overexpression of cyp51A results from duplication of SrbA but not CBC binding sites and show that deletion of the 34 mer results in lack of cyp51A expression and increased azole susceptibility similar to a cyp51A null mutant. Finally we show that strains lacking a functional CBC are severely attenuated for pathogenicity in a pulmonary and systemic model of aspergillosis. Aspergillus fumigatus is the most important airborne mould pathogen and allergen worldwide. Estimates suggest that >3 million people have invasive or chronic infections that lead to >600,000 deaths every year. Very few drugs are available to treat the various forms of aspergillosis and we rely predominantly on the azole class of agents which inhibit sterol biosynthesis. Resistance to the azoles is growing alarmingly, primarily driven by strains with two principal genetic signatures (TR34/L98H and TR46/Y121F/T289A). In this study we identify that the transcriptional mechanism governing resistance in this group of isolates is linked to the opposing actions of 2 transcriptional regulators, SrbA and the CBC, and uncover a role for the CBC in sterol regulation and virulence in A. fumigatus. We propose targeting SrbA would provide an effective avenue for therapeutic intervention for resistant strains.