Functions of 14-3-3 protein Bmh1 in cell growth and hyphal development of Candida albicans

Abstract:

[Objective] We studied the roles of 14-3-3 protein Bmh1 of Candida albicans in its cell growth and hyphal development by using a Tet-off promoter. [Methods] Based on the URA3⁺ strain SN152 of C. albicans, we deleted one BMH1 allele and replaced the promoter of the other allele with the Tet-off promoter, generating a strain in which Bmh1 expression was controlled by Doxycycline. Then we explored the phenotypes of cell growth and hyphal development via spot analysis and morphological observations. Further, we preliminarily studied the position of Bmh1 in the network of hyphal development regulation via overexpression of Bmh1 in the mutants of ras1, flo8, efg1, cph1 and tec1, which were very important hyphal development regulators. Finally, we constructed some Bmh1 mutants with different C-terminals and detected their effects on cell growth and hyphal development of Candida albicans. [Results] Doxycycline-induced knockdown of Bmh1 inhibited cell growth severely. Highly-expressed Bmh1 improved hyphal development significantly without Doxycycline induction. This improvement bypassed the effects of ras1, efg1, cph1 and tec1 gene deletions and was blocked by flo8 deficiency. All Bmh1 mutants with C-terminal deletion or heterologous C-terminals grew normally with Doxycycline induction and the hyphal development was not improved obviously. [Conclusion] We validated that the 14-3-3 protein Bmh1 of Candida albicans is essential for cell growth, proving the tight controlling of the Tet-off promoter on Bmh1 expression. Bmh1 functions as a positive regulator of hyphal development and plays roles in the downstream of Ras1, Efg1, Cph1 and Tec1, and in the upstream of Flo8. The conserved domain of Bmh1 is required for cell growth while the C-terminal is not.