Institute of Medicine and School of Medicine, Chung Shan Medical University, Taichung City 40201, Taiwan, R.O.C.
Department of Biomedical Sciences, Chung Shan Medical University, Taichung City 40201, Taiwan, R.O.C.
Int J Mol Med. 2018 Dec;42(6):3193-3208. doi: 10.3892/ijmm.2018.3930. Epub 2018 Oct 12.
Candida albicans (C. albicans) CDC4 (CaCDC4), encoding the F‑box protein for the substrate specificity of the Skp1‑cullin‑F‑box E3 ubiquitin ligase complex, suppresses the yeast‑to‑filament transition in C. albicans. In our previous study, Thr1 was identified as a CaCdc4‑associated protein using affinity purification. THR1 encodes a homoserine kinase, which is involved in the threonine biosynthesis pathway. The present study generated a strain with repressible CaCDC4 expression and continuous THR1 expression. Colony and cell morphology analyses, as well as immunoblotting, revealed that the Thr1 protein was detectable under conditions in which the expression of CaCDC4 was repressed and that the filaments resulting from the repressed expression of CaCDC4 were suppressed by the constitutive expression of THR1 in C. albicans. Additionally, by using the CaSAT1‑flipper method, the present study produced null mutants of THR1, GCN4, and CaCDC4. The phenotypic consequences were evaluated by growth curves, spotting assays, microscopic analysis, reverse transcription‑polymerase chain reaction and XTT‑based biofilm formation ability. The results revealed that fewer cells lacking THR1 entered the stationary phase but had no apparent morphological alteration. It was observed that the expression of THR1 was upregulated concurrently with GCN4 during nutrient depletion and that cells lacking GCN4 rescued the lethality of cells in the absence of THR1 in conditions accumulating homoserine in the threonine biosynthesis pathway. Of note, it was found that cells with either CaCDC4 or THR1 loss were sensitive to oxidative stress and osmotic stress, with those with THR1 loss being more sensitive. In addition, it was observed that cells with loss of either CaCDC4 or THR1 exhibited the ability to increase biofilm formation, with those lacking CaCDC4 exhibiting a greater extent of enhancement. It was concluded that CaCDC4 is important in the coordination of morphogenesis, nutrient sensing, and the stress response through THR1 in C. albicans.
白色念珠菌（C. albicans）CDC4（CaCDC4），编码 Skp1- 细胞周期蛋白 F-box E3 泛素连接酶复合物的底物特异性的 F-box 蛋白，抑制白色念珠菌中的酵母 - 丝状过渡。在我们之前的研究中，使用亲和纯化鉴定 Thr1 是与 CaCdc4 相关的蛋白。THR1 编码同型丝氨酸激酶，参与苏氨酸生物合成途径。本研究生成了一种可抑制 CaCDC4 表达和持续表达 THR1 的菌株。菌落和细胞形态分析以及免疫印迹显示，在抑制 CaCDC4 表达的条件下可检测到 Thr1 蛋白，并且通过在白色念珠菌中组成型表达 THR1 可抑制由 CaCDC4 抑制表达引起的丝状细胞。此外，通过使用 CaSAT1- 翻转方法，本研究产生了 THR1、GCN4 和 CaCDC4 的 null 突变体。通过生长曲线、点样测定、显微镜分析、逆转录-聚合酶链反应和 XTT 基于生物膜形成能力评估表型后果。结果表明，缺乏 THR1 的细胞进入静止期的细胞数量较少，但形态无明显改变。观察到在营养物质耗尽时，THR1 的表达与 GCN4 同时上调，并且在苏氨酸生物合成途径中积累同型丝氨酸的条件下，缺乏 GCN4 的细胞可挽救缺乏 THR1 的细胞的致死性。值得注意的是，发现 CaCDC4 或 THR1 缺失的细胞对氧化应激和渗透应激敏感，而缺乏 THR1 的细胞更为敏感。此外，观察到 CaCDC4 或 THR1 缺失的细胞能够增加生物膜形成，而缺乏 CaCDC4 的细胞增强程度更大。综上所述，在白色念珠菌中，CaCDC4 通过 THR1 对形态发生、营养感应和应激反应的协调具有重要作用。
