Zinc deficiency reverses biofilm azole resistance in Candida albicans

Biofilm formation is one of the causes of azole resistance in Candida albicans . Although zinc is an essential trace element involved in biofilm regulation, its specific mechanistic role remains unclear. Here, we systematically evaluated the effects and mechanisms of zinc deficiency on biofilm formation and drug resistance. Intracellular zinc deficiency was induced using the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and a CSR1 knockout strain, as confirmed using zinquin fluorescence. Biofilm formation and susceptibility were assessed using standardized microdilution techniques, including sessile minimum inhibitory concentration (sMIC) determinations via the XTT reduction assay, while drug interactions were assessed using a checkerboard assay. Efflux pump activity was measured using a Rhodamine 6G assay, and transcriptomic analysis was performed to elucidate underlying mechanisms. Pathogenicity was validated using a Galleria mellonella infection model. The results show that zinc deficiency inhibited biofilm development at all stages. Low-concentration TPEN (5µM) reduced the sMIC of fluconazole by more than 16-fold and ultimately reversed its azole resistance. This effect was mechanistically associated with the downregulation of key biofilm-related transcription factors and multidrug efflux pumps, as revealed by transcriptomic analysis, which also indicated that zinc deficiency triggered ribosomal remodeling and activated glucose metabolism. Survival analysis in the G. mellonella infection model confirmed that zinc deficiency reduced the overall pathogenicity of C. albicans biofilms. These results validate zinc homeostasis as a novel therapeutic strategy against drug-resistant and recurrent fungal infections, especially those involving biofilms.