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首页> 外文期刊>Infection and immunity >Molecular Cloning and Characterization ofWdPKS1, a Gene Involved in Dihydroxynaphthalene Melanin Biosynthesis and Virulence in Wangiella(Exophiala) dermatitidis
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Molecular Cloning and Characterization ofWdPKS1, a Gene Involved in Dihydroxynaphthalene Melanin Biosynthesis and Virulence in Wangiella(Exophiala) dermatitidis

机译：WdPKS1的分子克隆和表征，WdPKS1，一个基因参与了皮肤旺吉氏菌（Exophiala）皮肤病中二羟基萘黑色素的生物合成和毒力测定。

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1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resultingwdpks1Δ disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intactWdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a β-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1Δ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in awdpks1Δ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.
机译：1,8-二羟基萘（1,8-DHN）是一种真菌聚酮化合物，当它与人类毛囊菌病的病原体 Wangiella dermatitidis 的细胞壁中聚合成1,8-DHN黑色素时，会产生毒性。 。要开始对导致1,8-DHN黑色素生物合成的初始合成步骤进行遗传分析，请使用基于已知会产生第一个环化的真菌聚酮化合物合酶（Pks）保守区域的引物，从基因组DNA中扩增772-bp PCR产物。 1,8-DHN-黑色素途径中间体1,3,6,8-四羟基萘。然后将克隆的PCR产物用作靶向序列，以破坏 W中假定的聚酮化合物合酶基因 WdPKS1 。皮肤炎。产生的 wdpks1 Δ破坏基因除白化病表型外没有其他形态缺陷，并且以与黑色野生型亲本相同的速率生长。使用标记拯救方法，然后成功地从两个质粒中回收了完整的 WdPKS1 基因。还通过互补 W的白化突变体中的 mel3 突变独立地分离了 WdPKS1 基因。使用粘粒文库的皮肤病。序列分析证实， WdPKS1 在一个由三个短内含子隔开的三个外显子组成的单一开放阅读框中编码了一个假定的聚酮化合物合酶（WdPks1p）。该结论得到了推导的氨基酸序列中β-酮酰基合酶，乙酰丙二酰基转移酶，两个酰基载体蛋白和硫酯酶的高度保守的Pks结构域的支持。使用嗜中性粒细胞杀伤试验和小鼠急性感染模型进行的研究证实，与野生型亲本相比，所有 wdpks1 Δ菌株对杀伤的抵抗力和毒性均较低。在 wdpks1 Δ菌株中1,8-DHN黑色素生物合成的重建重新确立了其对嗜中性粒细胞杀伤的抵抗力和引起致命小鼠感染的能力。

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