Hmg1 mutations in Aspergillus fumigatus and their contribution to triazole susceptibility

Abstract

Triazole-resistant Aspergillus fumigatus is a global health concern. In general, each triazole resistance pattern caused by the specified amino acid substitution of Cyp51A has a typical pattern depending on the mutation site. We evaluated the contribution of both Cyp51A and Hmg1 mutations to atypical triazole resistance in A. fumigatus. We used clinical triazole-resistant A. fumigatus strains collected in Japan and investigated the sequences of cyp51A and hmg1 genes. To delineate the association between the hmg1 mutation and atypical triazole resistance, the mutant hmg1 alleles in clinical multi-azole resistant strains were replaced with the wild-type hmg1 allele by CRISPR/Cas9 system. In our study, the combination of Cyp51A mutation and Hmg1 mutation was shown to additively contribute to triazole resistance. We also demonstrated that the triazole resistance conferred by the Hmg1 mutation showed a different pattern depending on the mutation site, similar to the Cyp51A mutation. Our results indicate that focusing on the phenotypes of multiple genes is essential to clarify the overall picture of the triazole resistance mechanism of A. fumigatus. Lay Summary The number of triazole-resistant Aspergillus fumigatus is increasing. We confirmed thatmutation in a hydroxymethylglutaryl-CoA reductase (Hmg1) in the fungus contributesto the resistance separately from Cyp51A mutation, and that susceptibility patterns aredifferent based on mutation site.

hapE and hmg1 Mutations Are Drivers of cyp51A -Independent Pan-Triazole Resistance in an Aspergillus fumigatus Clinical Isolate

“…The same hmg1 F262del mutation introduced in a susceptible A. fumigatus laboratory strain induced increases of at least 4-fold in the MICs for all triazoles, accumulation of ergosterol precursors, and significant increases in total cellular ergosterol levels ( 26 ). In a similar way, amino acid substitutions in other residues of the Hmg1 SSD have also been identified, and they are often associated with triazole-resistant isolates ( 25 – 27 , 35 , 36 ). Some of these mutations, namely, S305P, I412S, S269F, and E306K, have been shown to impart decreased susceptibility to multiple triazoles when genetically inserted in a triazole-susceptible laboratory strain ( 25 , 26 , 36 ).…”

“…Hmg1 initiates ergosterol biosynthesis by catalyzing the reduction of HMG-CoA into mevalonate and has a conserved motif known as the sterol-sensing domain (SSD), which is predicted to participate in the regulation of HMG-CoA reductase activity ( 32 – 34 ). Although the clinical prevalence of hmg1 mutations has not been assessed in a systematic manner, such mutations have been identified in over 150 isolates thus far reported in the literature, many of which are resistant to triazole drugs and lack cyp51A -associated mutations ( 25 – 28 , 35 – 44 ). The most frequent Hmg1 amino acid substitutions associated with triazole drug resistance occur in residues putatively located in the Hmg1 SSD, such as S269, S305, G307, and I412, with several studies showing that some of these mutations, namely, F262del, S305P, I412S, S269F, and E306K, impart decreased susceptibility to multiple triazoles when genetically introduced into a susceptible laboratory strain ( 25 , 26 , 36 , 40 ).…”

“…Although the clinical prevalence of hmg1 mutations has not been assessed in a systematic manner, such mutations have been identified in over 150 isolates thus far reported in the literature, many of which are resistant to triazole drugs and lack cyp51A -associated mutations ( 25 – 28 , 35 – 44 ). The most frequent Hmg1 amino acid substitutions associated with triazole drug resistance occur in residues putatively located in the Hmg1 SSD, such as S269, S305, G307, and I412, with several studies showing that some of these mutations, namely, F262del, S305P, I412S, S269F, and E306K, impart decreased susceptibility to multiple triazoles when genetically introduced into a susceptible laboratory strain ( 25 , 26 , 36 , 40 ). The mechanism by which hmg1 mutations alter triazole resistance remains to be defined; however, it is speculated that amino acid substitutions in the SSD impair its ability to sense sterols and signal for Hmg1 degradation ( 25 , 26 , 28 ).…”

Intimate genetic relationships and fungicide resistance in multiple strains of Aspergillus fumigatus isolated from a plant bulb

“…The mutation in hmg1 gene encoding a 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A reductase has been recently reported to be involved in resistance to triazoles (Rybak et al ., 2019 ; Arai et al ., 2021 ). From the genome sequences, SNPs in hmg1 were extracted.…”

CRISPR/Cas9-Based Genome Editing and Its Application in Aspergillus Species

“…Generally, triazole resistance generated by Cyp51A specific amino acid substitution exhibits a typical pattern depending on the mutation site. In a recent study, Cyp51A and Hmg1 mutations that contribute to atypical triazole resistance were assessed using the established RNP-based CRISPR/Cas9 approach in A. fumigatus [ 96 , 97 ]. Then, in another study of antifungal drugs, researchers used the RNP-based CRISPR/Cas9 system to disrupt genes encoding putative protein kinases in A. fumigatus to identify the genes required for fungal survival under the stress of echinocandin, an antifungal with a limited effect on invasive aspergillosis [ 98 ].…”