Impact of Erg11 Amino Acid Substitutions Identified in Candida auris Clade III Isolates on Triazole Drug Susceptibility

Abstract

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 the long-tailed triazoles posaconazole and itraconazole for the V125A/F126L, F126L, Y132F, and K143R alleles.

Keywords: African clade; Candida auris; ERG11; azole resistance; clade III; fluconazole resistance; heterologous expression; mutagenesis; short- and long-tailed triazoles.

FIG 1

Molecular dissection of C. auris Erg11 V125A and F126L amino acid substitutions. (A) Region of C. auris clade III ERG11 DNA that displays nucleotide mutations (T374C/T376C) in red and resulting protein alterations (V125A/F126L) in yellow highlight. (B) Forward mutagenic primer used to revert leucine (L) back to phenylalanine (F). After mutagenesis, this construct contained only V125A (pCauErg11-V125A). (C) Forward mutagenic primer used to revert alanine (A) back to wild-type valine (V). After mutagenesis, this construct contained only F126L (pCauErg11-F126L). (D) Forward mutagenic primer used to revert leucine (L) back to phenylalanine (F) using the pCauErg11-F126L plasmid as a template. After mutagenesis, this construct contained both wild-type nucleotides and amino acids (pCauErg11-‘wt’). (E) Plasmid sequencing chromatograms of relevant codons corresponding to the 125^th and 126^th amino acids following mutagenesis and propagation in Escherichia coli.