Comparative Ploidy Proteomics of Candida albicansBiofilms Unraveled the Role of the AHP1Gene in the Biofilm Persistence Against Amphotericin B*

Candida albicansis a major fungal pathogen causing lethal infections in immunocompromised patients. C. albicansforms antifungal tolerant biofilms contributing significantly to therapeutic failure. The recently established haploid C. albicansbiofilm model provides a new toolbox to uncover the mechanism governing the higher antifungal tolerance of biofilms. Here, we comprehensively examined the proteomics and antifungal susceptibility of standard diploid (SC5314 and BWP17) and stable haploid (GZY792 and GZY803) strains of C. albicansbiofilms. Subsequent downstream analyses identified alkyl hydroperoxide reductase 1 (AHP1) as a critical determinant of C. albicansbiofilm's tolerance of amphotericin B. At 32 μg/ml of amphotericin B, GZY803 haploid biofilms showed 0.1% of persister population as compared with 1% of the diploid biofilms. AHP1expression was found to be lower in GZY803 biofilms, and AHP1overexpression in GZY803 restored the percentage of persister population. Consistently, deleting AHP1in the diploid strain BWP17 caused a similar increase in amphotericin B susceptibility. AHP1expression was also positively correlated with the antioxidant potential. Furthermore, C. albicans ira2Δ/Δ biofilms were susceptible to amphotericin B and had a diminished antioxidant capacity. Interestingly, AHP1overexpression in the ira2Δ/Δ strain restored the antioxidant potential and enhanced the persister population against amphotericin B, and shutting down the AHP1expression in ira2Δ/Δ biofilms reversed the effect. In conclusion, we provide evidence that the AHP1gene critically determines the amphotericin B tolerance of C. albicansbiofilms possibly by maintaining the persisters' antioxidant capacity. This finding will open up new avenues for developing therapies targeting the persister population of C. albicansbiofilms. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD004274.