Intrinsic short-tailed azole resistance in mucormycetes is due to an evolutionary conserved aminoacid substitution of the lanosterol 14α-demethylase

Abstract

Mucormycoses are emerging and potentially lethal infections. An increase of breakthrough infections has been found in cohorts receiving short-tailed azoles prophylaxis (e.g. voriconazole (VCZ)). Although VCZ is ineffective in vitro and in vivo, long-tailed triazoles such as posaconazole remain active against mucormycetes. Our goal was to validate the molecular mechanism of resistance to short-tailed triazoles in Mucorales. The paralogous cytochrome P450 genes (CYP51 F1 and CYP51 F5) of Rhizopus arrhizus, Rhizopus microsporus, and Mucor circinelloides were amplified and sequenced. Alignment of the protein sequences of the R. arrhizus, R. microsporus, and M. circinelloides CYP51 F1 and F5 with additional Mucorales species (n = 3) and other fungi (n = 16) confirmed the sequences to be lanosterol 14α-demethylases (LDMs). Sequence alignment identified a pan-Mucorales conservation of a phenylalanine129 substitution in all CYP51 F5s analyzed. A high resolution X-ray crystal structure of Saccharomyces cerevisiae LDM in complex with VCZ was used for generating a homology model of R. arrhizus CYP51 F5. Structural and functional knowledge of S. cerevisiae CYP51 shows that the F129 residue in Mucorales CYP51 F5 is responsible for intrinsic resistance of Mucorales against short-tailed triazoles, with a V to A substitution in Helix I also potentially playing a role.

Figure 1

Sequence alignment of Saccharomyces cerevisiae lanosterol 14-α demethylase with six Mucorales CYP51s. R. arrhizus F1 (n = 17) and F5 CYP51s (n = 3), R. microsporus F1 and F5 CYP51s (n = 13 for both LDMs) and M. circinelloides F1 and F5 CYP51s (n = 18 for both LDMs) were aligned against S. cerevisiae CYP51 using T-coffee (Expresso). Residues within 4 Å of voriconazole in the structure S. cerevisiae CYP51 (PDBID: 5HS1) are highlighted in green. Those highlighted in red correspond to resistance mutations in CYP51 F5. The V to A substitution is shown in blue. Helices from the voriconazole CYP51 complex are shown above the alignment in gray and the heme coordinating cysteine is shown in yellow. The final two residues of the S. cerevisiae sequence are not shown. MH, Membrane-associated helix; TMD, transmembrane domain.

Figure 2

Amino acid sequence alignment of lanosterol 14-α demethylase in human pathogenic fungi. Proteins belonging to sixteen fungi with consensus sequences of six Mucorales CYP51s. (A) Alignment using Saccharomyces cerevisiae Erg11 as reference (partial amino acid sequence colored) compared with wild-type ascomycete strains (n = 13) (in light green) with phylum consensus (dark green); wild-type basidiomycetes (n = 3) (light orange) with phylum consensus sequence (dark orange); sequences of R. arrhizus (n = 3), R. microsporus (n = 13), and M. circinelloides (n = 18) CYP51 F5 sequences, together with Mucor ambiguus, Parasitella parasitica, and Absidia glauca LDM sequences (light blue), with subphylum consensus sequence (dark blue). The frequently mutated tyrosine residue homologous to S. cerevisiae CYP51 Y140 is marked in bold, and the change to phenylalanine is seen in all our CYP51 F5 Mucorales species (original position F129) shown in red. The V to A substitution in position 311 (S. cerevisiae numbering) is only observed among Mucorales LDM F5 sequences, and are depicted in red. The Q/N side chains aligning with S. cerevisiae LDM S382 are marked in bold. Superscript numbers show the positions of the 5′ and 3′ amino acids for each sequence. The last column gives references reporting a mutation homologous to Y140F/H as responsible for short-tailed azole resistance in the species indicated; ^aaccording to S. cerevisiae numbering. (B) Shows the same alignment with consensus sequences of six Mucorales CYP51 F1s. The residue homologous to ScErg11p Y140 is marked in bold and is seen also in the protein sequence of the six Mucorales CYP51 F1s. In position 311 (S. cerevisiae numbering), a V is also present in all Mucorales CYP51 F1s. The Q/N side chains are also present in Mucorales CYP51 F1, marked in bold, and in both LDMs do not affect VCZ binding.

Figure 3

Homology model of R. arrhizus LDM F5. (A) Superimposition showing the active site of R. arrhizus LDM F5 (green) showing the position of F129 in relation to S. cerevisiae Y140 (purple, PDB ID: 5HS1). Voriconazole (green carbons, blue nitrogens, red oxygens and cyan fluorines) is shown coordinating to the heme (orange) in the homology model. The key water-mediated hydrogen bonding network centered on the water molecule (red sphere) with hydrogen bonds is shown by dashed lines. The orange dashed line shows the hydrogen bond that is lost in the presence of phenylalanine (F129) substitution compared with tyrosine (Y140). The water molecule is present only in the crystal structure. (B) Substitution of serine (S. cerevisiae S382) for asparagine (R. arrhizus LDM F5 N362) can be seen adjacent to VCZ and shows no polar interactions. This is highlighted by the superimposition of the homology model (green) onto the crystal structure (purple). The water molecule is present only in the crystal structure.