Identification and Characterization of Mediators of Fluconazole Tolerance in Candida albicans.

Sadri Znaidi,Eric Delarze,Fabio Maranzano,Claire Pralong,Murielle Chauvel,Christophe d’Enfert,Mélanie Legrand,Marie-Elisabeth Bougnoux,Dominique Sanglard,Marion Patxot,Emilie Trachsel,Ludivine Brandt

doi:10.3389/fmicb.2020.591140

Abstract

Candida albicans is an important human pathogen and a major concern in intensive care units around the world. C. albicans infections are associated with a high mortality despite the use of antifungal treatments. One of the causes of therapeutic failures is the acquisition of antifungal resistance by mutations in the C. albicans genome. Fluconazole (FLC) is one of the most widely used antifungal and mechanisms of FLC resistance occurring by mutations have been extensively investigated. However, some clinical isolates are known to be able to survive at high FLC concentrations without acquiring resistance mutations, a phenotype known as tolerance. Mechanisms behind FLC tolerance are not well studied, mainly due to the lack of a proper way to identify and quantify tolerance in clinical isolates. We proposed here culture conditions to investigate FLC tolerance as well as an easy and efficient method to identity and quantify tolerance to FLC. The screening of C. albicans strain collections revealed that FLC tolerance is pH- and strain-dependent, suggesting the involvement of multiple mechanisms. Here, we addressed the identification of FLC tolerance mediators in C. albicans by an overexpression strategy focusing on 572 C. albicans genes. This strategy led to the identification of two transcription factors, CRZ1 and GZF3. CRZ1 is a C2H2-type transcription factor that is part of the calcineurin-dependent pathway in C. albicans, while GZF3 is a GATA-type transcription factor of unknown function in C. albicans. Overexpression of each gene resulted in an increase of FLC tolerance, however, only the deletion of CRZ1 in clinical FLC-tolerant strains consistently decreased their FLC tolerance. Transcription profiling of clinical isolates with variable levels of FLC tolerance confirmed a calcineurin-dependent signature in these isolates when exposed to FLC.

Highlights

Candida species are major fungal pathogens which can cause life-threatening systemic infection known as invasive candidiasis
We here focused on the identification of tolerance using standardized susceptibility assays in microtiter plates as it could be implemented and reproduced in most laboratories
The re-identification of CRZ1 here underscores that the selection of FLC mediators by the undertaken experimental approaches is feasible, even if limitations have to be considered.We focused only on CRZ1 and GZF3, since the Dox-dependent overexpression of these genes in C. albicans reached the threshold of tolerance (TI ≥ 0.2) that we previously defined for clinical isolates

Summary

Introduction

Candida species are major fungal pathogens which can cause life-threatening systemic infection known as invasive candidiasis. Despite an extensive knowledge of these mechanisms, the acquisition of resistance does not explain all therapeutic failures. It is known that some clinical isolates of C. albicans tolerate the presence of FLC more than others. These are characterized by an increased growth, known as residual growth, at drug concentrations above their minimum inhibitory concentration (MIC). This residual growth, which is a characteristic of drug tolerance, is in general not coupled to an increased MIC (unlike resistance) and is thought to favor the acquisition of resistance mechanisms due to extended survival of some C. albicans isolates upon treatment. Tolerant isolates may to be a source of reinfection upon arrest of antifungal treatment (Delarze and Sanglard, 2015)

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