The survival mechanisms underlying caspofungin-induced Candida albicans biofilm persisters

Abstract

OBJECTIVES: Candida infection has increasingly posed a threat to oral and systemic health worldwide, due to formation of robust biofilms against high concentrations of antifungals. Emerging evidence shows that a subpopulation of multidrug-tolerant Candida persisters accounts for the biofilm recalcitrance and resilience. This pioneering study identified the phenotypic characteristics of Candida biofilm persisters and investigated their underlying survival mechanisms with shotgun proteomics. METHODS: Candida albicans biofilms were formed on 24-well polystyrene plates for 48h and then treated with caspofungin for 24h. The minimum inhibitory concentration (MIC) against biofilms was determined by XTT reduction assay and the viable cells were quantified by CFU counting. For acquisition of persisters, biofilms were challenged with 128μg/ml of caspofungin. Next, proteins were harvested from biofilm persisters, biofilms treated with caspofungin at MIC (BCM) and the controls. LC-MS/MS was performed on an LTQ-Orbitrap system to identify and quantify the proteins. The proteome profiles were subjected to statistical analysis and Gene Ontology enrichment analysis. RESULTS: The caspofungin treatment at 128μg/ml produced two distinct subpopulations of Candida biofilms, with the majority killed and a small amount of biofilm persisters unaffected. Over 900 proteins were identified and those from persisters and BCM were significantly up- or down-regulated with reference to the controls. Numerous biological processes were overrepresented, such as glycolysis, tricarboxylic acid cycle and oxidation-reduction process. Notably, significantly differentially expressed proteins were detected between persisters and BCM. Proteins involved in cell wall integrity (e.g., PSA1, PCK1, PNG2, CSH1 and PIL1) were significantly enriched in persisters. Proteins associated with cell cycle (GSP1), Ty3 transposition (HRT2) and PPIase activity (FKBP5) were significantly down-regulated. CONCLUSIONS: This pioneering study shows that the caspofungin-induced Candida albicans biofilm persisters exhibit specific proteomic signature. The regulatory response to cell wall damage may be essential for survival of the persisters.