Evaluation of Lysine Biosynthesis as an Antifungal Drug Target: Biochemical Characterization of Aspergillus fumigatus Homocitrate Synthase and Virulence Studies
Evaluation of Lysine Biosynthesis as an Antifungal Drug Target: Biochemical Characterization of Aspergillus fumigatus Homocitrate Synthase and Virulence Studies
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DOI：
10.1128/ec.00020-10
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发表时间：
2010-06-01
期刊：
影响因子：
--
通讯作者：
Brock, Matthias
中科院分区：
文献类型：
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作者：
Schoebel, Felicitas;Jacobsen, Ilse D.;Brock, Matthias
烟曲霉是严重侵袭性曲霉病的主要致病菌。为了对抗这种危及生命的感染，只有有限数量的抗真菌药物可用。真菌α-氨基己二酸途径是赖氨酸生物合成所必需的，已被认为是潜在的抗真菌药物靶点。在这里，我们重新分析了这一途径在建立侵袭性曲霉病小鼠模型中的作用。我们选择了第一个途径特异性酶，高柠檬酸合酶（HcsA），生化特性和研究其在毒力中的作用。A.烟曲霉HcsA对底物乙酰辅酶A（acetyl-CoA）和α-酮戊二酸具有特异性，并且其活性不依赖于任何金属离子。与其他高柠檬酸脱氢酶的情况相反，酶活性几乎不受赖氨酸的影响，并且在赖氨酸补充的条件下基因表达增加。一个hcsA缺失突变体赖氨酸营养缺陷型，不能发芽的未水解的蛋白质作为唯一的营养源。然而，添加部分纯化的A.烟曲霉蛋白酶恢复了生长，证实了游离赖氨酸对补充营养缺陷型的重要性。与其他真菌物种的赖氨酸营养缺陷型突变体相反，该突变体在血液和血清上生长，表明存在高亲和力的赖氨酸摄取系统。在协议中，虽然突变体的毒力被强烈衰减，在小鼠模型的支气管肺曲霉菌病，毒力部分恢复赖氨酸补充通过饮用水。此外，与减毒肺部感染的情况相反，突变体在静脉注射时保留了全部毒力。因此，我们得出的结论是，抑制真菌赖氨酸生物合成，至少对于传播性侵袭性曲菌病来说，似乎并不能为新的抗真菌药物提供合适的靶点。
Aspergillus fumigatus is the main cause of severe invasive aspergillosis. To combat this life-threatening infection, only limited numbers of antifungals are available. The fungal alpha-aminoadipate pathway, which is essential for lysine biosynthesis, has been suggested as a potential antifungal drug target. Here we reanalyzed the role of this pathway for establishment of invasive aspergillosis in murine models. We selected the first pathway-specific enzyme, homocitrate synthase (HcsA), for biochemical characterization and for study of its role in virulence. A. fumigatus HcsA was specific for the substrates acetyl-coenzyme A (acetyl-CoA) and alpha-ketoglutarate, and its activity was independent of any metal ions. In contrast to the case for other homocitrate synthases, enzymatic activity was hardly affected by lysine and gene expression increased under conditions of lysine supplementation. An hcsA deletion mutant was lysine auxotrophic and unable to germinate on unhydrolyzed proteins given as a sole nutrient source. However, the addition of partially purified A. fumigatus proteases restored growth, confirming the importance of free lysine to complement auxotrophy. In contrast to lysine-auxotrophic mutants from other fungal species, the mutant grew on blood and serum, indicating the existence of high-affinity lysine uptake systems. In agreement, although the virulence of the mutant was strongly attenuated in murine models of bronchopulmonary aspergillosis, virulence was partially restored by lysine supplementation via the drinking water. Additionally, in contrast to the case for attenuated pulmonary infections, the mutant retained full virulence when injected intravenously. Therefore, we concluded that inhibition of fungal lysine biosynthesis, at least for disseminating invasive aspergillosis, does not appear to provide a suitable target for new antifungals.