Due to their eukaryotic heritage, the differences between a fungal pathogen’s molecular makeup and its human host are small. Therefore, the discovery and subsequent development of novel antifungal drugs are extremely challenging. Nevertheless, since the 1940s, researchers have successfully uncovered potent candidates from natural or synthetic sources. Analogs and novel formulations of these drugs enhanced the pharmacological parameters and improved overall drug efficiency. These compounds ultimately became the founding members of novel drug classes and were successfully applied in clinical settings, offering valuable and efficient treatment of mycosis for decades. Currently, only five different antifungal drug classes exist, all characterized by a unique mode of action; these are polyenes, pyrimidine analogs, azoles, allylamines, and echinocandins. The latter, being the latest addition to the antifungal armamentarium, was introduced over two decades ago. As a result of this limited arsenal, antifungal resistance development has exponentially increased and, with it, a growing healthcare crisis. In this review, we discuss the original sources of antifungal compounds, either natural or synthetic. Additionally, we summarize the existing drug classes, potential novel candidates in the clinical pipeline, and emerging non-traditional treatment options.

SUMMARY: Polyunsaturated fatty acid (PUFA) synthase-like enzymes are best known for their role in membrane lipid biosynthesis in marine bacteria, but have also been repurposed for the assembly of specialized lipid metabolites with unique biological functions. Here, we illuminate their broader biosynthetic potential by charting the unexplored landscape of hybrid peptide-polyketide-specialized lipid biosynthesis in bacteria. Using a targeted genome mining strategy, we identified more than 60 biosynthetic gene clusters that combine PUFA synthase-like, polyketide synthase (PKS), and nonribosomal peptide synthetase (NRPS) enzymes across diverse bacterial lineages. Comparative analysis revealed extensive diversification of these triple hybrid pathways through gene fusion, domain reshuffling and recruitment of accessory enzymes. We further expand the known repertoire of peptide-polyketide-specialized lipid hybrids by identifying the chitinimines, a new family of amphiphilic metabolites produced by Chitinimonas koreensis featuring a C22 polyunsaturated lipid chain conjugated to a cyclic peptide-polyketide and a pyruvate-derived cyclic acetal moiety. The chitinimines exhibit surfactant properties, as well as moderate antibacterial activity against Gram-positive bacteria and contribute to a growth-promoting interaction between C. koreensis and Salmonella spp. Together, these findings demonstrate that PUFA synthase-like systems are far more versatile than previously appreciated, playing a key role in combinatorial biosynthetic innovation and serving as a rich, untapped source of chemically and functionally diverse specialized lipids.

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.

Polyunsaturated fatty acid (PUFA) synthase enzymes are best known for their role in membrane lipid biosynthesis in marine psychrophilic bacteria but have also evolved to assemble specialized lipid-containing metabolites with unique biological functions. Here, we illuminate their broader biosynthetic potential by charting the unexplored landscape of hybrid peptide-polyketide-specialized lipid biosynthesis in bacteria. Using a targeted genome mining strategy, we identified more than 60 biosynthetic gene clusters that encode PUFA synthase-like, polyketide synthase (PKS), and nonribosomal peptide synthetase (NRPS) enzymes across diverse bacterial lineages. Comparative analysis revealed extensive diversification of these triple hybrid pathways through gene fusion, domain reshuffling, and enzyme recruitment. We further expand the known repertoire of peptide-polyketide-specialized lipid hybrids by identifying the chitinimines, a new family of amphiphilic metabolites produced by Chitinimonas koreensis featuring a C22 polyunsaturated lipid conjugated to a cyclic peptide-polyketide and a pyruvate-derived cyclic acetal moiety. The chitinimines exhibit surfactant properties, as well as moderate activity against Gram-positive bacteria, and contribute to a growth-promoting effect on Salmonella serovars. Together, these findings demonstrate that PUFA synthase-like systems are far more versatile than previously appreciated, playing a key role in combinatorial biosynthetic innovation and serving as a rich, untapped source of chemically and functionally diverse specialized lipids.