key: cord-0267865-x9qwi61t
authors: Williamson, Benjamin; Wilk, Adam; Guerrero, Kevin D.; Mikulski, Timothy D.; Elias, Tony N.; Sawh, Indira; Cancino-Prado, Geselle; Gardam, Dianne; Heath, Christopher H.; Govender, Nelesh P.; Perlin, David S.; Kordalewska, Milena; Healey, Kelley R.
title: Impact of Erg11 amino acid substitutions identified in Candida auris clade III isolates on triazole drug susceptibility
date: 2021-08-17
journal: bioRxiv
DOI: 10.1101/2021.08.16.456589
sha: 6b2d23978d26536c13bcf19746e48cc7749cb644
doc_id: 267865
cord_uid: x9qwi61t

ERG11 sequencing of 28 Candida auris clade III isolates revealed the presence of concomitant V125A and F126L substitutions. Heterologous expression of Erg11-V125A/F126L in Saccharomyces cerevisiae led to reduced fluconazole and voriconazole susceptibilities. Generation of single substitution gene variants through site-directed mutagenesis uncovered that F126L primarily contributes to the elevated triazole MICs. A similar, yet diminished pattern of reduced susceptibility was observed with long-tailed triazoles posaconazole and itraconazole for V125A/F126L, F126L, Y132F, and K143R alleles.

(amphotericin B), and echinocandins, have been reported, with triazole resistance being the 53 most prevalent (8) (9) (10) . 54

Triazole antifungals (e.g. fluconazole, voriconazole, itraconazole, posaconazole) target 55 the biosynthesis of fungal ergosterol, specifically through inhibition of lanosterol 14-alpha-56

Using the same approach as in (15), we cloned the Erg11 allele (V125A/F126L) from 78 isolate AR-0384 onto a low-copy plasmid (pRS416), which was then expressed in a haploid 79 strain of S. cerevisiae (BY4741). This heterologous system allowed us to focus solely on the 80 effects of ERG11 mutations on triazole susceptibilities. Multiple clones were passaged on 81 selective medium (synthetic defined medium lacking uracil; SD-Ura), screened by PCR, and 82 resulting plasmid sequences verified (for primers, see (15)). S. cerevisiae that expressed C. 83 auris Erg11-V125A/F126L demonstrated elevated MICs to fluconazole (64 µg/ml) and 84 voriconazole (1 µg/ml). In comparison, expression of an empty vector or Erg11-wild type alleles 85 from other clades yielded MICs 4 to 8-fold more susceptible (≤ 16 µg/ml to fluconazole; ≤ 0.25 86 µg/ml to voriconazole) ( Table 2) . 87

To further dissect the specific role of V125A and F126L substitutions in triazole 88 resistance, we designed mutagenic primers to individually revert each amino acid substitution 89 (Figure 1) . A Phusion site-directed mutagenesis kit (Thermo Scientific, MA, USA) was used to 90 introduce the desired wild-type mutations. The resulting C. auris Erg11-V125A and Erg11-91 F126L plasmid constructs were expressed in S. cerevisiae. In addition, we performed two 92 consecutive rounds of site-directed mutagenesis to produce a strain that carried neither 93 substitution (Erg11-V125/F126) ( Figure 1D- cerevisiae and C. albicans influence susceptibility to all triazoles, while other mutations lead to fluconazole and voriconazole (short-tailed triazoles), we tested all of our strains to determine 104 susceptibility to posaconazole and itraconazole (long-tailed triazoles) ( Table 2) . Changes in 105 posaconazole and itraconazole MICs were minimal, although consistent, with 2 to 4-fold 106 differences between the "resistant" alleles (V125A/F126L, Y132F, or K143R) and wild type 107 alleles ( Table 2) . These results are in alignment with the minimal differences observed in clinical 108 isolates ( Table 1 ) and to those of previous studies that analyzed Y132F and K143R (or 109 equivalent changes) in C. albicans and S. cerevisiae (21, 23) . 110

Crystallization of C. albicans Erg11 identified residue 126 (and the equivalent residue in 111 S. cerevisiae) as being located within the enzyme's active site and a likely player in substrate 112 binding (24, 25). Furthermore, the authors from this study predicted that alteration of this 113 residue would likely reduce affinity for all triazole drugs but would do so most extensively for 114 short-tailed azoles (24). Since all C. auris clade III isolates described in the literature contain 115 both V125A and F126L substitutions, it is likely that these two mutations occurred at nearly the 116 same time in the evolution of this clade. The V125A substitution may simply be a passenger 117 mutation. Alternatively, V125A may increase the stability of the Erg11 enzyme or be 118 advantageous for the yeast in another way and/or in combination with other alterations (e.g., 119 ERG11 copy number variants (8)). Studies have since identified TAC1b transcription factor 120 mutations, linked to increased expression of drug efflux pumps (e.g., CDR1 and/or other 121 unidentified transporters), as an alternate mechanism of triazole resistance in C. auris (26-28). 122

Additionally, a recent report demonstrated an additive effect that concomitant ERG11 (F444L) 123

and TAC1b mutations can have on triazole susceptibility (29). 124

In conclusion, the ERG11 allele found in C. auris clade III isolates directly contributes to 125 reduced triazole susceptibility, in particular to fluconazole and voriconazole. Moreover, our 126 mutagenic experiments revealed that the F126L substitution was primarily responsible for the 127 elevated triazole MICs. Results of this study further improve our understanding of triazole 

Candida auris: Epidemiology, 140 biology, antifungal resistance, and virulence

Multiple introductions 143 and subsequent transmission of multidrug-resistant Candida auris in the USA: a 144 molecular epidemiological survey

Multidrug-Resistant Candida auris 146

Critically Ill Coronavirus Disease Patients

A multicentre study of antifungal susceptibility patterns among 350

Candida auris isolates (2009-17) in India: role of the ERG11 and FKS1 genes in azole 191 and echinocandin resistance

Contribution of 193 clinically derived mutations in ERG11 to azole resistance in Candida albicans

Limited ERG11 Mutations Identified in Isolates of Candida auris Directly 197

Contribute to Reduced Azole Susceptibility

In-vitro antifungal resistance of Candida auris isolates from bloodstream infections

Govender NPfG-203 (pCauErg11-F126L). D, Forward mutagenic primer used to revert Leucine (L) back to

After mutagenesis, this 313 construct contained both wild type nucleotides and amino acids (pCauErg11-'wt'). E, Plasmid 314 sequencing chromatograms of relevant codons corresponding to the 125 th and 126 th amino 315 acids following mutagenesis