Cloning and functional characterization of the WdSTUA and WdPACC genes of Wangiella dermatitidis

Abstract

To study the function of WdStuAp and WdPacCp in Wangiella dermatitidis, a black, polymorphic fungal pathogen of humans with yeast phase predominance, WdSTUA and WdPACC were cloned, sequenced, disrupted and expressed. WdStuAp was most similar to the APSES proteins of Aspergillus species and its APSES DNA-binding domain was located in its N-terminal half. Deletion of WdSTUA in W. dermatitidis induced convoluted instead of normal smooth colony surface growth on the rich, yeast maintenance agar medium, YPDA, at 37°C. Additionally, deletion of WdSTUA repressed aerial hyphal growth, conidiation and invasive hyphal growth on the nitrogen poor, hyphae-inducing agar medium, PDA, at 25°C. Ectopic expression of WdSTU Arepressed the convoluted colony surface growth on YPDA at 37°C, and also strongly repressed hyphal growth on PDA at 25°C and 37°C. Expression of WdSTUA in S. cerevisiae induced pseudohyphal growth on the nitrogen poor medium. WdPacCp was also most similar to the PacCp proteins of Aspergillus species. Three zinc finger DNA-binding motifs were at the N-terminus, and the C-terminus had the signaling protease cleavage site. WdPACC was more expressed at neutral-alkaline pH than at acidic pH. Truncation of the coding sequence for about 40 residues upstream of the conserved processing protease cleavage site of WdPacCp affected growth on YPDA, increased sensitivity to Na⁺ stress, decreased growth level at neutral-alkaline pH, and repressed hyphal growth on PDA at 25°C. Truncation of the coding sequence for the conserved signaling protease box of WdPacCp impaired growth and reduced RNA expression of class II chitin synthase gene WdCHS1 at acidic pH, and activated hyphal growth on PDA. My results suggested that WdStuAp and WdPacCp play important roles in yeast-hyphal transitions in W. dermatitidis, and that WdPacCp is particularly important for W. dermatitidis to adapt to different ambient pH conditions.