Adaptive response to fluconazole in candida parapsilosis: Ergosterol pathway mutations, gene expressions, and metabolomic shifts

Candida parapsilosis complex is the second most common cause of bloodstream infection among Candida species both in our country and in many centers in Southern Europe and become a major health-care problem due to the increasing antifungal resistance. Azoles, an important group of antifungal drugs are bind to lanosterol demethylase and inhibit the enzyme. Resistance is usually associated with point mutations in the ERG11 gene and ERG3 genes which play role in ergosterol biosynthesis. This study aims to analyze the effects of fluconazole on C. parapsilosis cells in metabolomic, transcriptomic and genomic level especially focusing on the critical steps in ergosterol synthesis pathway. In this study, 15 fluconazole non-susceptible C. parapsilosis isolates are included. Analysis of the resistance profile is performed with broth microdilution test according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Fluconazole non-susceptible isolates are treated with the 0.5 Minimum Inhibitory Concentration (MIC) and 1 MIC of fluconazole and the mRNA expression level changes are measured with quantitative real-time polymerase chain reaction (qRT-PCR) method. Effects of fluconazole on the metabolomic profiles of C. parapsilosis cells are examined with Nucleic Magnetic Resonance Spectroscopy (NMR). Sanger sequence analysis showed that 10/15 (66%) of the isolates were harboring Y132F mutations in ERG11 gene and all of them were carrying missense mutation (C825T) in ERG3 gene. Although, an increase in ERG11 mRNA expression levels is observed regardless of the mutation type, some of the selected isolates behaved controversial under fluconazole exposure. Together with all isolates, especially K478 had a decreasing ERG11 mRNA expression level trend under higher fluconazole concentration. Even if the highest ERG3 and UPC2 mRNA expression levels were observed in K478, the same decreasing trend were also detected under higher fluconazole concentrations in these genes. According to metabolomic analysis results, under the fluconazole exposure, the metabolic pathways most significantly altered are: 1) alanine, aspartate, and glutamate metabolism, 2) arginine and proline metabolism, 3) homocysteine, methionine metabolism, and 4) arabinitol metabolism. We assumed that the metabolomic changes observed in our study might be related with two important biochemical processes which are nitrogen assimilation and oxidative stress responses. In conclusion, in drug-resistant C. parapsilosis isolates, the response to fluconazole exposure in terms of mRNA expression of ergosterol biosynthesis enzyme genes is strain-specific, clearly indicating that there are numerous different regulations besides point mutations in the enzyme genes of ergosterol synthesis and control. These regulations lead to a wide range of metabolomic changes within the cell, while a common metabolic response is formed in almost all isolates to neutralize the toxic effect of fluconazole and ensure survival. A detailed in-depth investigation of this survival response will uncover new drug targets in the future. Keywords: Candida parapsilosis, azole resistance, ERG11 gene, ERG3 gene, UPC2 gene, quantitative real-time PCR (qRT-PCR), Sanger sequencing, NMR spectroscopy