Hanaoka Nozomu, Takano Yukie, Shibuya Kazutoshi, Fugo Hajime, Uehara Yoshimasa, Niimi Masakazu
Department of Bioactive Molecules, National Institute of Infectious Diseases, Toyama, Tokyo, Japan.
Eukaryot Cell. 2008 Oct;7(10):1640-8. doi: 10.1128/EC.00129-08. Epub 2008 Aug 15.
Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Delta, yvh1Delta, sit4Delta, and ptc1Delta) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Delta, yvh1Delta, and sit4Delta mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Delta revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.
蛋白磷酸酶对于许多细胞过程的调节至关重要。利用URA3和ARG4标记基因，通过连续等位基因替换构建了白色念珠菌21种假定蛋白磷酸酶的缺失突变体。使用一种简单的白色念珠菌感染家蚕模型来筛选突变体库。相对于对照菌株和亲本菌株，四种缺失突变体（cmp1Δ、yvh1Δ、sit4Δ和ptc1Δ）菌株在家蚕模型中显示出毒力减弱。其中三种突变体，即cmp1Δ、yvh1Δ和sit4Δ突变体，先前已被鉴定为在传统小鼠模型中影响毒力，这表明家蚕模型筛选的有效性。白色念珠菌假定蛋白磷酸酶基因PTC1与酿酒酵母2C型蛋白磷酸酶PTC1具有52%的同一性，其破坏在家蚕模型中显著降低了毒力。该突变体在播散性念珠菌病小鼠模型中也无致病性。使用在组成型ACT1启动子控制下包含任一PTC1等位基因的盒式结构，将白色念珠菌的任一PTC1等位基因重新导入缺失菌株，可在两种感染模型中恢复毒力。ptc1Δ的特性分析揭示了其他表型特征，包括体外和体内菌丝生长减少以及细胞外蛋白水解活性降低。我们得出结论，PTC1可能在白色念珠菌的致病性中起作用。
