Azole resistance mechanisms in Candida albicans. Exposure to fluconazole and the role of the Erg11 Y477C, Tac1 S758F and Mrr2 A311V new mutations by CRISPR-Cas9 gene editing.

Abstract

Invasive fungal infections caused by Candida species, among which Candida albicans is the predominant one, are the fourth cause of nosocomial infections. Most Candida infections are treated with azoles, thus, acquisition of resistance to these compounds extremely hinders the treatment options. In the recent years, there has been an increase in azole resistance, and azole tolerance is also gaining importance since it is considered to facilitate resistance development and recurrent or persistent infections. There are several well-established azole resistance mechanisms, but there are also reports of resistant clinical isolates without any of the aforementioned. In this work we have determined the presence of these known mechanisms in a collection of C. albicans strains and concluded that further research in this area is still needed to elucidate alternative resistance mechanisms. In addition, we have identified new mutations in resistance-associated genes that had not yet been reported in the literature and tested their involvement in azole resistance by means of CRISPR-Cas9 gene editing; nevertheless, we could not link these new mutations to fluconazole resistance in the C. albicans SC5314 background. We also performed an alternative approach to investigate the development of resistance to antifungals by means of an in vitro evolution experiment with two azole-susceptible C. albicans strains, SC5314 and BE-47, that wereexposed to increasing concentrations of fluconazole for 23 days. After only 4 days of fluconazole exposure, the C. albicans SC5314 strain acquired a mutation in Tac1 known to be associated to azole resistance that became homozygous by the 7th day of exposure. The occurrence of this mutation was correlated with an increase in MIC values for azoles and expression of target genes and was maintained throughout the whole experiment. Conversely, the BE-47 strain did not acquire azole resistance nor presented any of the described resistance mechanisms, but it increased its tolerance to fluconazole after the exposure period. These populations constitute a very interesting material warranting future studies of the mechanisms involved in the acquisition of azole tolerance and/or resistance.