Candida Biofilm Persistence Triggered by Metabolic Inhibition and Anti-oxidative Response

Objectives:  Candida is the predominant fungal pathogen that causes superficial mucosal inflammations and even life-threatening systemic mycoses. The formation of drug-resistant biofilms has been recognized as the major virulence factor of Candida pathogenicity. A subpopulation of Candida biofilm named “persisters” can evade killing of lethal-dose antifungal drugs and therefore become a critical risk for biofilm resilience and treatment failure. This study investigated the underlying mechanisms of formation and survival of Candida biofilm persisters by analysing the proteome profile with shotgun proteomics. Methods: Candida albicans biofilms were grown on 24-well polystyrene plates and exposed to 256 μg/ml of amphotericin B for 24 h for generating persisters. Cell pellets of treated and untreated biofilms were harvested and lysed for protein extraction. Proteins were then digested with trypsin in solution and loaded for LC-MS/MS analysis. Raw data of MS/MS spectra were processed and searched against the reference proteome. The differentially expressed proteins were analysed by t-test and annotated by Gene Ontology enrichment analysis. Results: There was a considerable variation in protein expression of the treated biofilms with reference to the controls. Many crucial cellular pathways were implicated in Candida biofilm persistence, e.g. glycolysis, tricarboxylic acid cycle and oxidation-reduction process. Among the significant hits, protein enzymes involved in glycolysis were all downregulated, so did several proteins from tricarboxylic acid cycle including isocitrate dehydrogenase and malate dehydrogenase that would decrease production of reactive oxygen species eventually. Meanwhile, upregulation of antioxidants was observed, which could be beneficial for cellular adaptation to oxidative stress. Conclusion: This pioneering study on proteomics of Candida biofilm persisters suggests that inhibition of metabolism and protection against oxidative stress may contribute to the development and maintenance of Candida biofilm persistence. (Supported by Health & Medical Research Fund, Hong Kong SAR Government (12110752); and the Modern Dental Laboratory/HKU Endowment Fund to LJJ).