Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.
Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.
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DOI：
10.1371/journal.pone.0137869
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Steinbach WJ
中科院分区：
文献类型：
--
作者：
Falloon K;Juvvadi PR;Richards AD;Vargas-Muñiz JM;Renshaw H;Steinbach WJ
侵袭性曲霉病主要由烟曲霉引起，导致免疫抑制患者死亡人数不断增加。 FK506（他克莫司）等靶向钙调神经磷酸酶的免疫抑制剂已显示出抗真菌药物开发的前景。 FK506 结合蛋白 (FKBP) 在 FK506 (FKBP12-FK506) 存在的情况下与钙调磷酸酶形成复合物并抑制钙调磷酸酶活性。对真菌中 FKBP 的研究有限，之前还没有在烟曲霉中对 FKBP 进行过表征。我们在烟曲霉中鉴定了人类 FK506 结合伴侣 FKBP12 的四个直系同源基因，并将它们命名为 fkbp12-1、fkbp12-2、fkbp12-3 和 fkbp12-4。对四个基因的删除分析表明，Δfkbp12-1菌株对FK506具有抗性，表明FKBP12-1是FK506与钙调神经磷酸酶结合的关键介质。内源表达的 FKBP12-1-EGFP 融合蛋白在正常生长条件下定位于细胞质和细胞核，但在 FK506 处理后也定位于菌丝隔膜，揭示了其与钙调神经磷酸酶的相互作用。在 cnaA 缺失背景中存在 FK506 的情况下，FKBP12-1-EGFP 融合蛋白不会定位于隔膜，证实了其与钙调神经磷酸酶的相互作用。对大蜡螟曲霉病模型中所有缺失菌株的测试表明，这些蛋白质在毒力中并不发挥重要作用。虽然 Δfkbp12-2 和 Δfkbp12-3 菌株没有表现出任何可辨别的表型，但 Δfkbp12-4 菌株在正常生长条件下表现出轻微的生长缺陷，并在较高浓度的抗真菌卡泊芬净下抑制卡泊芬净介导的“反常生长效应”。总之，这些结果表明，虽然只有 FKBP12-1 是 FK506 的真正结合伴侣，导致烟曲霉中钙调神经磷酸酶的抑制，但 FKBP12-4 可能在基础生长和卡泊芬净介导的矛盾生长反应中发挥作用。利用烟曲霉 FKBP12-1 和人 FKBP12 之间的差异对于生成真菌特异性 FK506 类似物以抑制真菌钙调神经磷酸酶和治疗侵袭性真菌病至关重要。
Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn’t localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don’t play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn’t show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated “paradoxical growth effect” at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.