Titrating Gene Function in the Human Fungal Pathogen Candida albicans through Poly-Adenosine Tract Insertion.
Titrating Gene Function in the Human Fungal Pathogen Candida albicans through Poly-Adenosine Tract Insertion.
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通过多聚腺苷束插入滴定人类真菌病原体白色念珠菌的基因功能。
DOI：
10.1128/msphere.00192-19
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发表时间：
2019
期刊：
影响因子：
4.8
通讯作者：
Palmer,GlenE
中科院分区：
文献类型：
--
作者：
Tournu,Helene;Butts,Arielle;Palmer,GlenE
最近的一项研究表明，将多聚腺苷(PolyA)链插入开放阅读框可以抑制编码蛋白在原核生物和真核生物中的表达。此外，抑制程度与Poly-A插入的长度成正比，因此可以提供一种可靠和可预测的方法来滴定特定蛋白质的表达。这项研究的目的是确定这种方法是否可以应用于调节人类常见的真菌病原体--白色念珠菌的蛋白质表达。在白色念珠菌羊毛甾醇脱甲基酶(Erg11p)的N端插入越来越多的编码赖氨酸的AAA密码子，表达逐渐减弱，但不会显著降低mRNA的水平。这表明Erg11p的表达在转录后水平减弱。还注意到插入的AAA密码子的数量与白色念珠菌对Erg11p抑制剂氟康唑的敏感性之间存在直接关联，表明Erg11p活性逐渐丧失。最后，我们构建了一系列在ARO1基因5‘端插入3-12个AAA密码子的白色念珠菌菌株，该基因编码一个五功能酶，催化芳香氨基酸生物合成途径的五个步骤。AAA密码子数量的增加逐渐降低了白色念珠菌在标准实验室培养基中的生长速度，表明ARO生物合成活性的逐渐丧失。这些数据清楚地证明了Poly-A插入法在检测白念珠菌中目标蛋白功能的表型结果方面的潜在效用。研究蛋白质在整个有机体水平上的功能重要性通常涉及改变其表达水平或其特定活性，并观察与生理或表型相关的结果。几种旨在部分或完全取消基因功能的方法已被广泛应用，包括从基因组中删除基因和使用促进条件表达的系统。然而，每一种方法都有显著的局限性，当需要确定动物模型中感染是否需要特定基因时，这些限制在病原微生物中尤其成问题。在这项研究中，我们试图确定另一种方法-在基因编码序列中插入PolyA重复序列-是否足以调节其在流行的人类真菌病原体白色念珠菌中的功能。我们的结果证实，这种方法使我们能够预测和逐步滴定蛋白质的表达水平，从而研究不同水平的基因/蛋白质功能的表型结果。
A recent study demonstrated that the insertion of poly-adenosine (poly-A) tracts into an open reading frame can suppress expression of the encoded protein in both prokaryotic and eukaryotic species. Furthermore, the degree of suppression is proportional to the length of the poly-A insertion, which can therefore provide a reliable and predictable means to titrate a specific protein’s expression. The goal of this study was to determine if this methodology can be applied to modulate the expression of proteins in the prevalent human fungal pathogen, Candida albicans. Insertion of increasing numbers of AAA codons encoding lysine at the N terminus of the C. albicans lanosterol demethylase (Erg11p) progressively diminished expression without significantly reducing the levels of mRNA. This suggests that Erg11p expression was attenuated at the posttranscriptional level. A direct correlation between the number of AAA codons inserted and C. albicans susceptibility to the Erg11p inhibitor fluconazole was also noted, indicating a progressive loss of Erg11p activity. Finally, we constructed a series of C. albicans strains with 3 to 12 AAA codons inserted at the 5′ end of theARO1gene, which encodes a pentafunctional enzyme catalyzing five sequential steps of the aromatic amino acid biosynthetic pathway. Increasing numbers of AAA codons progressively reduced the growth rate of C. albicans in standard laboratory medium, indicating a progressive loss of ARO biosynthetic activity. These data unequivocally demonstrate the potential utility of the poly-A insertion method to examine the phenotypic consequences of titrating target protein function in C. albicans.IMPORTANCEInvestigating a protein’s functional importance at the whole-organism level usually involves altering its expression level or its specific activity and observing the consequences with respect to physiology or phenotype. Several approaches designed to partially or completely abolish the function of a gene, including its deletion from the genome and the use of systems that facilitate conditional expression, have been widely applied. However, each has significant limitations that are especially problematic in pathogenic microbes when it is desirable to determine if a particular gene is required for infection in an animal model. In this study, we sought to determine if an alternative approach—the insertion of poly-A repeats within the coding sequence of the gene—is sufficient to modulate its function in the prevalent human fungal pathogen C. albicans. Our results confirm that this approach enables us to predictably and gradually titrate the expression level of a protein and thus to investigate the phenotypic consequences of various levels of gene/protein function.
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DOI：
--
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期刊：
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