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To persist in their dynamic human host environments, fungal pathogens must sense and adapt by modulating their gene expression to fulfill their cellular needs. Understanding transcriptional regulation on a global scale would uncover cellular processes linked to persistence and virulence mechanisms that could be targeted for antifungal therapeutics. Infections associated with the yeast Candida albicans , a highly prevalent fungal pathogen, and the multiresistant related species Candida auris are becoming a serious public health threat. To define the set of a gene regulated by a transcriptional regulator in C. albicans , chromatin immunoprecipitation (ChIP)-based techniques, including ChIP with microarray technology (ChIP-chip) or ChIP-DNA sequencing (ChIP-seq), have been widely used. Here, we describe a new set of PCR-based micrococcal nuclease (MNase)-tagging plasmids for C. albicans and other Candida spp. to determine the genome-wide location of any transcriptional regulator of interest using chromatin endogenous cleavage (ChEC) coupled to high-throughput sequencing (ChEC-seq). The ChEC procedure does not require protein-DNA cross-linking or sonication, thus avoiding artifacts related to epitope masking or the hyper-ChIPable euchromatic phenomenon. In a proof-of-concept application of ChEC-seq, we provided a high-resolution binding map of the SWI/SNF chromatin remodeling complex, a master regulator of fungal fitness in C. albicans , in addition to the transcription factor Nsi1 that is an ortholog of the DNA-binding protein Reb1 for which genome-wide occupancy was previously established in Saccharomyces cerevisiae The ChEC-seq procedure described here will allow a high-resolution genomic location definition which will enable a better understanding of transcriptional regulatory circuits that govern fungal fitness and drug resistance in these medically important fungi. IMPORTANCE Systemic fungal infections caused by Candida albicans and the "superbug" Candida auris are becoming a serious public health threat. The ability of these yeasts to cause disease is linked to their faculty to modulate the expression of genes that mediate their escape from the immune surveillance and their persistence in the different unfavorable niches within the host. Comprehensive knowledge on gene expression control of fungal fitness is consequently an interesting framework for the identification of essential infection processes that could be hindered by chemicals as potential therapeutics. Here, we expanded the use of ChEC-seq, a technique that was initially developed in the yeast model Saccharomyces cerevisiae to identify genes that are modulated by a transcriptional regulator, in pathogenic yeasts from the genus Candida This robust technique will allow a better characterization of key gene expression regulators and their contribution to virulence and antifungal resistance in these pathogenic yeasts., (Copyright © 2020 Tebbji et al.)

A poorly exploited paradigm in the antimicrobial therapy field is to target virulence traits for drug development. In contrast to target-focused approaches, antivirulence phenotypic screens enable identification of bioactive molecules that induce a desirable biological readout without making a priori assumption about the cellular target. Here, we screened a chemical library of 678 small molecules against the invasive hyphal growth of the human opportunistic yeast Candida albicans. We found that a halogenated salicylanilide (N1-(3,5-dichlorophenyl)-5-chloro-2-hydroxybenzamide) and one of its analogs, Niclosamide, an FDA-approved anthelmintic in humans, exhibited both antifilamentation and antibiofilm activities against C. albicans and the multi-resistant yeast C. auris. The antivirulence activity of halogenated salicylanilides were also expanded to C. albicans resistant strains with different resistance mechanisms. We also found that Niclosamide protected the intestinal epithelial cells against invasion by C. albicans. Transcriptional profiling of C. albicans challenged with Niclosamide exhibited a signature that is characteristic of the mitochondria-to-nucleus retrograde response. Our chemogenomic analysis showed that halogenated salicylanilides compromise the potential-dependant mitochondrial protein translocon machinery. Given the fact that the safety of Niclosamide is well established in humans, this molecule could represent the first clinically approved antivirulence agent against a pathogenic fungus.