Translation Inhibition by Rocaglates Activates a Species-Specific Cell Death Program in the Emerging Fungal Pathogen Candida auris

Abstract

Fungal infections are a major contributor to infectious disease-related deaths worldwide. Recently, global emergence of the fungal pathogen Candida auris has caused considerable concern because most C. auris isolates are resistant to fluconazole, the most commonly administered antifungal, and some isolates are resistant to drugs from all three major antifungal classes. To identify novel agents with bioactivity against C. auris, we screened 2,454 compounds from a diversity-oriented synthesis collection. Of the five hits identified, most shared a common rocaglate core structure and displayed fungicidal activity against C. auris. These rocaglate hits inhibited translation in C. auris but not in its pathogenic relative Candida albicans. Species specificity was contingent on variation at a single amino acid residue in Tif1, a fungal member of the eukaryotic initiation factor 4A (eIF4A) family of translation initiation factors known to be targeted by rocaglates. Rocaglate-mediated inhibition of translation in C. auris activated a cell death program characterized by loss of mitochondrial membrane potential, increased caspase-like activity, and disrupted vacuolar homeostasis. In a rocaglate-sensitized C. albicans mutant engineered to express translation initiation factor 1 (Tif1) with the variant amino acid that we had identified in C. auris, translation was inhibited but no programmed cell death phenotypes were observed. This surprising finding suggests divergence between these related fungal pathogens in their pathways of cellular responses to translation inhibition. From a therapeutic perspective, the chemical biology that we have uncovered reveals species-specific vulnerability in C. auris and identifies a promising target for development of new, mechanistically distinct antifungals in the battle against this emerging pathogen. IMPORTANCE Emergence of the fungal pathogen Candida auris has ignited intrigue and alarm within the medical community and the public at large. This pathogen is unusually resistant to antifungals, threatening to overwhelm current management options. By screening a library of structurally diverse molecules, we found that C. auris is surprisingly sensitive to translation inhibition by a class of compounds known as rocaglates (also known as flavaglines). Despite the high level of conservation across fungi in their protein synthesis machinery, these compounds inhibited translation initiation and activated a cell death program in C. auris but not in its relative Candida albicans. Our findings highlight a surprising divergence across the cell death programs operating in Candida species and underscore the need to understand the specific biology of a pathogen in attempting to develop more-effective treatments against it.

A Zinc Cluster Transcription Factor Contributes to the Intrinsic Fluconazole Resistance of Candida auris

“…Rybak et al (16) also used a CRISPR-Cas9 system in combination with a replacement cassette containing the caSAT1 selection marker and short (50-bp) flanking homology regions to delete CDR1 and MDR1 in two clade I isolates but did not report the frequency of specific gene replacement. Integration specificity was also not detailed in two very recent studies in which the NAT1 marker and long flanking regions were used for gene deletions in C. auris (23,26). The reason for the low frequency of specific marker integration into the target locus in our experiments is not evident.…”

“…Since C. auris is a haploid species, we anticipated that the generation of specific gene deletion mutants would be straightforward, as also inferred from the successful construction of C. auris mutants by other researchers (15,16,(23)(24)(25)(26). Unexpectedly, the vast majority of our transformants had unspecifically inserted the five different gene deletion cassettes at ectopic sites in the genome instead of integrating them at the target locus, suggesting that homologous recombination is much less efficient in C. auris than in C. albicans.…”

“…Nourseothricin resistance was used for the selection of transformants in all six previous studies, and Rybak et al (16) also used the same nourseothricin resistance gene (caSAT1) as we did. The use of electroporation for transformation of C. auris also does not seem to be the problem, because electroporation was also used in the studies by Grahl et al, Kim et al,Rybak et al,and Iyer et al (15,16,25,26). It is possible that homologous recombination is less efficient in strains from clade III and clade IV, used to generate mutants in our present study, as opposed to clade I strains, which were the parents of the mutants generated in the six previous studies (15,16,(23)(24)(25)(26).…”

An oxindole efflux inhibitor potentiates azoles and impairs virulence in the fungal pathogen Candida auris

“…Through Sanger sequencing we confirmed this strain harbored both an Erg11 K143R substitution and Tac1b A640V substitution. Compounds that reduced growth after 48 h compared to the control by 7-median absolute deviations from the median alone were classified as single agent antifungals; their mechanism of action has been described elsewhere 29 . Compounds for which antifungal activity was only observed in combination with fluconazole were classified as fluconazole potentiators (Fig.…”

Candida auris Urinary Tract Infections and Possible Treatment

“…Novel agents for the treatment of C. auris are needed. Potential agents for investigation include ibrexafungerp, nitroxoline, and the rocaglates class [ 52 , 53 , 54 ].…”