Functional characterization of Candida glabrata ORF, CAGL0M02233g for its role in stress tolerance and virulence

Present investigation is aimed to analyze the role of an uncharacterized ORF of Candida glabrata (CBS138), CAGL0M02233g (an ortholog of RAD53, a key DNA checkpoint effector in Saccharomyces cerevisiae) in tolerance of various stresses and in biofilm formation. The CAGL0M02233g was cloned in p416TEF shuttle vector for constitutive expression under TEF1 promoter in BG14 strain (ura3 auxotrophic C. glabrata), and upregulated expression of the cloned ORF was confirmed by immunoblotting. The constitutive expression of CAGL0M02233g rendered cells resistant to the DNA damage stressor (MMS), replication stressor (HU) and hypoxia mimetic (CoCl₂) in plate spot and growth curve assays. Hypoxia (a low oxygen condition) is an imperative host factor that influences Candida pathogenesis. Biofilm formation by the BG14 cells transformed with p416TEF-CAGL0M02233g (REX cell) was reduced to approximately 50% under hypoxia. It is notable that biofilm formation by the REX cells was significantly lower than that of BG14 cells transformed with p416TEF vector (VC cell) under hypoxia. The biofilm of the REX cells has shown higher susceptibility to fluconazole than that of VC cells under hypoxia and REX cells at normoxia. This is the first report on the function of CAGL0M02233g in tolerance of various stressors and in modulation of the biofilm under hypoxia.