Species in the genus Armillaria (fungi, basidiomycota) are well-known as saprophytes and pathogens on plants. Many of them cause white-rot root disease in diverse woody plants worldwide. Mitochondrial genomes (mitogenomes) are widely used in evolutionary and population studies, but despite the importance and wide distribution of Armillaria, the complete mitogenomes have not previously been reported for this genus. Meanwhile, the well-supported phylogeny of Armillaria species provides an excellent framework in which to study variation in mitogenomes and how they have evolved over time. Here we completely sequenced, assembled, and annotated the circular mitogenomes of four species: A. borealis, A. gallica, A. sinapina, and A. solidipes (116,443, 98,896, 103,563, and 122,167 bp, respectively). The variation in mitogenome size can be explained by variable numbers of mobile genetic elements, introns, and plasmid-related sequences. Most Armillaria introns contained open reading frames (ORFs) that are related to homing endonucleases of the LAGLIDADG and GIY-YIG families. Insertions of mobile elements were also evident as fragments of plasmid-related sequences in Armillaria mitogenomes. We also found several truncated gene duplications in all four mitogenomes. Our study showed that fungal mitogenomes have a high degree of variation in size, gene content, and genomic organization even among closely related species of Armillara. We suggest that mobile genetic elements invading introns and intergenic sequences in the Armillaria mitogenomes have played a significant role in shaping their genome structure. The mitogenome changes we describe here are consistent with widely accepted phylogenetic relationships among the four species.

Echinocandins, which target the fungal β-1,3-glucan synthase (Fks), are essential for treating invasive fungal infections, yet resistance is increasingly reported. While resistance typically arises through mutations in Fks hotspots, emerging evidence suggests a contributing role of changes in membrane sterol composition due to ERG3 mutations. Here, we present a clinical case of Nakaseomyces glabratus ( Candida glabrata ) in which combined mutations in ERG3 and FKS2 , but not FKS2 alone, appear to confer echinocandin resistance. Integrated analyses reveal a recurrent association between Erg3 loss-of-function and echinocandin resistance mediated by Fks variation across Candida species, but exclude Erg3 loss-of-function as an independent resistance mechanism. Advances in Fks structural biology and insights into echinocandin-Fks interactions support a model of epistatic cross-talk between membrane sterols and Fks function. Understanding this interaction is crucial, as it may underlie not only acquired echinocandin resistance but also the broader development of multidrug resistance across major antifungal drug classes. IMPORTANCE: A clinical case in which the combination of variation in a β‑1,3‑glucan synthase-encoding gene ( FKS2 ) and the sterol desaturase-encoding gene ERG3 seems to underlie echinocandin resistance, prompted us to hypothesize that membrane sterol changes may modulate, rather than independently cause, Fks‑linked resistance. We were able to explore this hypothesis due to recent developments in the field, such as the release of the FungAMR database, which enables global co‑occurrence analyses; AI‑driven variant effect predictors such as Evolutionary Scale Modeling (ESM) that can explore the impact of thousands of ERG3 alleles; the cryo‑EM resolution of the Fks1 protein; and the first mechanistic model of echinocandin‑Fks1 binding. Together, these advances provide the structural and computational framework needed to delineate our hypothesis that specific sterol variants might influence β‑1,3‑glucan synthase function and drug binding. Further surveillance of this potentially epistatic interaction can be of significant clinical importance amid rising multidrug‑resistant infections, as overlooking such interactions could lead to under‑calling resistance and misguided therapy.

Background: Nakaseomyces glabratus ( Candida gabrata ) poses a significant clinical challenge due to common drug resistance. We report a case of a complicated urinary tract infection (UTI) progressing to prostatitis and urosepsis, with the emergence of a hyper-multidrug-resistant isolate with low stress tolerance, slow growth and a short life span. This study elucidates the genetic mechanisms and phenotypic characteristics underlying antifungal hyper-resistance with strong fitness trade-offs, and explores potential alternative therapies for resistant UTI’s. Methods: Whole-genome sequencing was performed to identify resistance-associated mutations and gene knock-out strains were generated to assess the relative impact of putative loss-of-function (LoF) mutations on antifungal resistance, fitness and membrane sterol composition. Drug susceptibility testing of the antibiotic nitroxoline and related compounds was conducted to evaluate it as a therapeutic alternative and study the mechanism of action. Findings: Loss-of-function mutations in ERG3 and ERG11 were identified and linked to the accumulation of 4,14-dimethylzymosterol and lanosterol instead of ergosterol. Engineered ERG3Δ+ERG11Δ strains recapitulated the clinical isolate’s hyper-multidrug resistance and associated fitness deficits. While ERG3Δ strains showed no resistance but enhanced thermotolerance, ERG11Δ and ERG3Δ+ERG11Δ strains exhibited multidrug resistance with severe fitness trade-offs. Interestingly, ERG3Δ+ERG11Δ strains showed mild resistance to flucytosine, but an additional FUR1 mutation in the clinical isolate most probably underlies hyper-resistance to flucytosine. The UTI antibiotic nitroxoline demonstrated high antifungal activity against all strains, and the LoF of ERG3 and/or ERG11 induced collateral sensitivity to this drug. Testing of related compounds suggest a mode of action beyond iron chelation. Interpretation: This case demonstrates that hyper-resistant strains of N. glabratus can emerge despite significant fitness costs and persist under prolonged antifungal therapy in specific clinical settings. These findings underscore the importance of vigilant antifungal resistance monitoring and highlight nitroxoline as a promising alternative treatment for complicated fungal UTIs. These results challenge the notion that strains with fitness deficits are clinically irrelevant and emphasize the need for novel therapeutic strategies including repurposed agents.