Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Fungal Genet Biol. 2013 Aug;57:85-91. doi: 10.1016/j.fgb.2013.06.004. Epub 2013 Jun 21.
Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Although A. fumigatus can be treated with many of the available antifungal drugs, including azole compounds, drug resistant isolates are being recovered at an increasing rate. In other fungal pathogens such as the Candida species, ATP-binding cassette (ABC) transporter proteins play important roles in development of clinically-significant azole resistance phenotypes. Central among these ABC transporter proteins are homologues of the Saccharomyces cerevisiae Pdr5 multidrug transporter. In this work, we test the two A. fumigatus genes encoding proteins sharing the highest degree of sequence similarity to S. cerevisiae Pdr5 for their ability to be function in a heterologous pdr5Δ strain of S. cerevisiae. Expression of full-length cDNAs for these two Afu proteins failed to suppress the drug sensitive phenotype of a pdr5Δ strain and no evidence could be obtained for their expression as green fluorescent protein (GFP) fusions. To improve the expression of one of these Afu ABC transporters (XP_755847), we changed the sequence of the cDNA to use codons corresponding to the major tRNA species in S. cerevisiae. This codon-optimized (CO Afu abcA) cDNA was efficiently expressed in pdr5Δ cells and able to be detected as a GFP fusion protein. The CO Afu abcA did not correct the drug sensitivity of the pdr5Δ strain and exhibited a high degree of perinuclear fluorescence suggesting that this fusion protein was localized to the S. cerevisiae ER. Interestingly, when these experiments were repeated at 37 °C, the CO Afu abcA was able to complement the drug sensitive phenotype of pdr5Δ cells and exhibited less intracellular fluorescence. Additionally, we found that the CO Afu abcA was able to reduce resistance to drugs like phytosphingosine that act via causing mislocalization of amino acid permeases in fungi. These data suggest that the Afu abcA protein can carry out two different functions of Pdr5: drug transport and regulation of protein internalization from the plasma membrane.
烟曲霉是人类主要的丝状真菌病原体。尽管烟曲霉可以用许多现有的抗真菌药物治疗，包括唑类化合物，但具有耐药性的分离株的回收率正在以越来越快的速度增加。在其他真菌病原体，如念珠菌属物种中，三磷酸腺苷结合盒（ABC）转运蛋白在临床上显著的唑类耐药表型的发展中发挥重要作用。在这些 ABC 转运蛋白中，最重要的是酿酒酵母 Pdr5 多药转运蛋白的同源物。在这项工作中，我们测试了编码与酿酒酵母 Pdr5 具有最高序列相似性的蛋白质的两个烟曲霉基因，以测试它们在酿酒酵母 pdr5Δ 菌株中的功能。这两个 Afu 蛋白的全长 cDNA 的表达未能抑制 pdr5Δ 菌株的药物敏感表型，也无法获得其作为绿色荧光蛋白（GFP）融合蛋白表达的证据。为了提高其中一个 Afu ABC 转运蛋白（XP_755847）的表达，我们改变了 cDNA 的序列，使用与酿酒酵母中主要 tRNA 种类相对应的密码子。这种密码子优化的（CO Afu abcA）cDNA 在 pdr5Δ 细胞中得到高效表达，并能被检测为 GFP 融合蛋白。CO Afu abcA 不能纠正 pdr5Δ 菌株的药物敏感性，并表现出高度的核周荧光，表明该融合蛋白定位于酿酒酵母内质网。有趣的是，当这些实验在 37°C 下重复时，CO Afu abcA 能够补充 pdr5Δ 细胞的药物敏感表型，并表现出较少的细胞内荧光。此外，我们发现 CO Afu abcA 能够降低植物鞘氨醇等药物的耐药性，植物鞘氨醇等药物通过导致真菌中氨基酸渗透酶的定位错误而起作用。这些数据表明，Afu abcA 蛋白可以执行 Pdr5 的两种不同功能：药物转运和调节从质膜内化的蛋白质。
