Clubroot, caused by the soil-borne protist Plasmodiophora brassicae, is a major disease of Brassica crops, resulting in severe root malformations and yield losses. While most research has centred on immune signalling and hormone dynamics, plant-pathogen interactions also dramatically reshape primary metabolism, often modifying source activity and converting infected tissues into strong metabolic sinks. The SnRK1 (SNF1-related kinase 1) protein kinase acts as a cellular fuel gauge in plants, integrating metabolic status and environmental and developmental cues to maintain carbon and energy homoeostasis. Here, we explored SnRK1-mediated quantitative resistance against clubroot disease in the related crucifer model Arabidopsis thaliana. Both soil- and hydroponics-based disease assays revealed how especially increased nuclear SnRK1α1 activity antagonizes clubroot development, suggesting a pivotal role for transcriptional regulation. qRT-PCR analysis and quantification of soluble sugar contents and invertase activity in roots indicate that SnRK1-mediated resistance coincides with reduced sucrose transporter expression as well as cell wall invertase expression and activity, likely limiting clubroot development by reducing sink strength. Consistently, cellular assays indicate that the recently identified SnRK1α1-targeting P. brassicae effector PBZF1 interferes with SnRK1α1 nuclear translocation. Our study thus corroborates that SnRK1 is a primary effector target and shows that SnRK1-mediated reprogramming of gene expression and sink activity is an effective mechanism against clubroot disease development.

Fungal infections represent a growing global health concern, aggravated by the paucity of effective antifungal therapies and the rapid rise of drug resistance. The development of new treatments is challenging due to the shared eukaryotic biology of fungi and human hosts, which restricts selective molecular targets and contributes to issues such as host toxicity and fungistatic activity. These challenges underscore the pressing need for new agents with distinct modes of action. In this study, we identified four promising compounds with novel antifungal activity from a high-throughput screen of 20,000 compounds against the opportunistic fungal pathogen, Candida albicans, namely Z1199266541, Z1024453766, Z56842335, and Z126932704. Subsequently, the compounds were characterized in vitro to assess their spectrum of activity, efficacy against a fluconazole-resistant isolate (CaCi-17), and effectiveness in combination treatments, followed by in vivo evaluation in a Galleria mellonella infection model. Among the candidates, Z56842335 emerged as a lead compound, effectively clearing infection caused by the Candida albicans in this invertebrate model. Mechanistic analyses revealed that the antifungal activity of Z56842335 is mitigated by supplementation with iron, copper, and zinc, indicating a metal-dependent mode of action. Notably, despite its apparent metal-binding capacity, Z56842335 displays a narrow spectrum of activity, targeting only C. albicans and, to a lesser extent C. dubliniensis. This selectivity suggests that, in addition to metal availability, species-specific factors such as differences in metal acquisition, intracellular metal buffering, or compound uptake or accumulation may contribute to susceptibility, highlighting a promising yet mechanistically intriguing avenue for further investigation.

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.

The opportunistic pathogenic fungus Nakaseomyces glabratus inhabits diverse host niches with fluctuating nutrient availability. Therefore, efficient control of glycolytic entry is essential, yet the regulatory principles governing hexose phosphorylation in this species remain incompletely understood. Here, we investigated the functional organization of sugar kinases in N. glabratus. Among five predicted sugar kinases, only three (Hxk2, Hxk2b and Glk1) catalyzed phosphorylation of glucose, fructose or mannose, whereas Hxk1 and Glk1b lacked detectable activity. Kinetic analyses revealed a functional specialization, with the hexokinases acting as high-capacity enzymes and Glk1 functioning as a high-affinity, low-capacity kinase optimized for low-sugar concentrations. Despite similar intrinsic kinetics among the hexokinases, Hxk2b emerged as the physiologically dominant enzyme, reflecting differential regulation rather than catalytic properties alone. Both hexokinases, but not Glk1, were strongly inhibited by trehalose-6-phosphate, linking glycolytic entry to trehalose metabolism. Consistent with this, perturbation of trehalose synthesis modulated hexose uptake, revealing that phosphorylation capacity is a major driver of sugar import, while metabolic feedback further constrains uptake. Nuclear localization of sugar kinases and condition-dependent expression patterns indicate additional regulatory layers. Together, our results demonstrate that N. glabratus controls glycolytic entry through a multilayered architecture integrating enzyme specialization, transcriptional tuning, trehalose-6-phosphate–mediated feedback and uptake coupling. This systems-level organization results in robust growth across fluctuating and often sugar-limited host environments.

The oral cavity is colonized by many species of bacteria and fungi without causing serious infections of hosts. However, upon loss of homeostasis, some of these colonizers have the potential to invade the host and cause life-threatening infections. The fungus Candida albicans and the gram-positive bacterium Staphylococcus aureus represent well-studied examples. In immunocompromised mice, oral co-colonization with these two species can result in dissemination of S. aureus through the body, causing S. aureus bacteremia and subsequent sepsis. In the proof-of-principle experiments described here, we show effective prevention of dissemination of methicillin-resistant S. aureus (MRSA) using an innovative endolysin-derived chimeric protein, XZ.700. Using the established murine model for oral co-colonization, we evaluated the effect of treatment with XZ.700 in drinking water (ad libitum) and applied as a gel formulation twice a day. XZ.700 did not show any adverse reaction in the mice treated and did not show a reduction of C. albicans colonization. XZ.700 in drinking water completely prevented detectable dissemination of MRSA in co-infected mice and significantly reduced weight loss. The gel formulation showed a trend in decreased MRSA colonization of the tongue and dissemination to the kidneys, although this was not significantly different from the placebo control. Taken together, the results presented here indicate that selective removal of MRSA from the oral cavity of mice with XZ.700 during co-infection with C. albicans is effective in preventing dissemination. Further studies need to show clinical applicability of XZ.700 and related compounds in prevention of sepsis in at-risk patients.

The emergence of multidrug-resistant fungal pathogens from urinary tract infections (UTIs) poses a growing challenge in clinical settings. Here, we report a case of a complicated UTI caused by Nakaseomyces glabratus (Candida glabrata) that progressed to urosepsis, leading to the emergence of an isolate carrying simultaneous loss-of-function mutations in ERG3 and ERG11, and abrogated ergosterol biosynthesis. Together with a missense mutation in FUR1-likely responsible for 5-fluorocytosine resistance-this constellation confers resistance to all viable UTI antifungals: azoles, amphotericin B, and flucytosine. Engineered ERG3Δ + ERG11Δ strains recapitulated this multidrug resistance and revealed profound fitness costs that come with it, challenging the assumption that high-cost mutations are unlikely to persist during infection. Among fitness trade-offs, we detected collateral sensitivity to nitroxoline, a commonly used urinary tract antibiotic with potent antifungal activity and a unique mechanism of action. This study provides the first clinical evidence of an elusive mechanism of hyper-multidrug resistance in N. glabratus and highlights nitroxoline as a promising repurposing agent for treating multidrug-resistant fungal infections of the urinary tract. IMPORTANCE:Evolutionary theory states that fitness determines survival. In a drug-treatment environment, resistance increases fitness, but it often comes at a cost, such as slower growth or reduced stress tolerance. If these costs are too severe, they can undermine virulence, making resistance unlikely to persist. Our study challenges this assumption. We describe the first clinical case of Nakaseomyces glabratus evolving multidrug resistance through loss-of-function mutations in ERG3 and ERG11, despite severe fitness trade-offs. This case suggests that certain infection niches, such as the urinary tract, can provide conditions where even highly impaired yet resistant strains persist under strong antifungal pressure. Importantly, we show that this extreme resistance induces collateral sensitivity to nitroxoline, a urinary tract infection antibiotic with potent antifungal activity and a unique mechanism of action. These findings open promising therapeutic avenues to counter multidrug-resistant fungal infections of the urinary tract.

Gim3 is an evolutionarily conserved component of the prefoldin chaperone complex, involved in protein folding. We previously found that GIM3 genetically interacts with many de novo mutations in Saccharomyces cerevisiae. Removing GIM3 from mutagenized S. cerevisiae cells significantly affected the fitness effect of mutations. This indicates that Gim3 might change the evolutionary impact of de novo mutations by either buffering (hiding) or potentiating (increasing) their phenotypic effects, depending on the environmental or genetic context. Here, we investigated Gim3's role in shaping the evolutionary fate of de novo mutations under fluconazole stress, an antifungal drug used to combat fungal infections. Applying both strong and moderate fluconazole stress in the presence or absence of GIM3 revealed that Gim3 potentiates fluconazole susceptibility (resistance and tolerance) by enabling mutations to have immediate phenotypic effects. Deleting GIM3 reduced growth in fluconazole in most mutants, indicating that GIM3 could be a promising target for new antifungal therapies against drug-resistant infections. Importantly, Gim3 also modulates fluconazole susceptibility of the fungal pathogen Nakaseomyces glabratus, further highlighting Gim3's role in fluconazole resistance and tolerance.

INTRODUCTION:Vulvovaginal candidiasis (VVC) is a highly prevalent fungal vaginal infection with a substantial impact on women's quality of life. Increasing evidence indicates that VVC pathogenesis is driven not only by high fungal burden but also by dysregulated host-immune responses, highlighting the need for therapeutic strategies that extend beyond conventional antifungal treatment. AREAS COVERED:In this review, recent advances elucidating the antifungal and immunomodulatory properties of Saccharomyces cerevisiae in the context of (recurrent) VVC are critically examined. Evidence from in vitro, ex vivo, in vivo, and clinical studies is integrated to assess S. cerevisiae-mediated attenuation of Candida virulence, modulation of host inflammatory responses, and interactions with the vaginal microbiota. In addition, translational challenges, safety considerations, regulatory constraints, and the therapeutic potential of yeast-based combination strategies are discussed. EXPERT OPINION:S. cerevisiae treatment will play a role in both the prevention of recurrence/occurrence of Candida infections and the integration into combination regimens, where they can complement antifungal and host-directed therapies rather than function as standalone treatments. Future advances will depend on patient stratification, strain-specific optimization, and the integration of microbial therapies into combination treatment frameworks aimed at restoring and maintaining mucosal homeostasis.

Vulvovaginal candidiasis (VVC) affects millions of women globally and is characterized by multifactorial immunopathology, with Candida albicans virulence driving disease progression through epithelial tissue damage and neutrophil hyperactivation and dysfunction contributing to disease severity. Here, we explored the multifaceted nature of S. cerevisiae as a live-biotherapeutic to attenuate C. albicans virulence and modulate host immune responses during VVC. We identified an S. cerevisiae isolate (Sc3458) that targets multiple aspects of C. albicans virulence, including fungal proliferation, adhesion, and hyphal morphogenesis, collectively impairing biofilm formation and disrupting pathogenic potential. These effects were linked to transcriptional reprogramming in C. albicans, marked by metabolic stress and downregulation of virulence- and biofilm-related genes. Additionally, S. cerevisiae (Sc3458) reduced inflammatory responses and neutrophil hyperactivation, while preserving neutrophil antimicrobial functionality. Altogether, this translated to an improved control of infection and dampened VVC-associated hyperinflammation in a murine VVC model. These findings position S. cerevisiae strain Sc3458 as a promising candidate live biotherapeutic for VVC treatment. Further clinical validation in human cohorts is essential to confirm its therapeutic efficacy and to assess how Sc3458 performs relative to established market-standard formulations.

Candidiasis pose a serious health threat, stimulating efforts to develop new antifungal agents and alternative therapies. Given the high mortality of fungal infections and the historical use of natural remedies, there is a growing interest in integrating natural substances into modern treatments. It is particularly important to explore interactions between home remedies and clinically approved antifungals to avoid harmful combinations or enhance beneficial effects. In this study, the chemical composition of the ethanolic extract of propolis (EEP) using UHPLC-DAD-QqTOF-MS was analyzed. The interactions of this extract with several antifungal agents against four yeast pathogens causing candidiasis: Candida albicans, Nakaseomyces glabratus, Pichia kudriavzevii, and Candida auris were investigated using Checkerboard Titration Assay, Growth Kinetics, and Disc-diffusion assay. Also, a novel simulated infection model was proposed. The results showed synergistic interactions between EEP and amphotericin B, and additive effects with nystatin. Synergy and additivity with fluconazole and voriconazole were observed, but limited to C. albicans and N. glabratus. In contrast, antagonistic interactions were noted with caspofungin, clotrimazole, and ketoconazole, which may have clinical relevance. Additionally, positive interactions with 2-phenoxyethanol and silver nanoparticles (AgNPs) suggest potential practical applications. Propolis’s synergistic properties could expand antifungal strategies and support the development of multi-target, resistance-preventing therapies.

Fungal infections are combatted using three main classes of antifungals, of which the azoles, considered to be fungistatic, are the most widely used. Slow growth of Candida albicans at supra-minimal inhibitory concentrations (MIC) of fluconazole (FLC), termed tolerance, is routinely observed. A combination therapy resulting in the eradication of this fungistatic character would be a valid therapeutic strategy, and indeed, the synergistic combination of the antibiotic doxycycline and FLC has such an effect. We hypothesized that iron-requiring mitochondrial functions may be the targets of the synergistic combination. The proteome enriched for mitochondria obtained from FLC + Fe-treated cells hinted that iron alleviated the FLC stress and that intracellular iron homeostasis, more specifically the vacuolar iron exporter Smf3, might be a key factor during FLC treatment, as its expression was induced. Moreover, a ROS assay revealed that a smf3Δ/Δ strain treated with FLC accumulated ROS to a similar extent as that displayed by the WT undergoing a FLC+DOX combination treatment. Thus, deletion of SMF3 mimics the addition of doxycycline in wild-type cells. The ROS accumulation can be attenuated through overexpression of the mitochondrial superoxide dismutase SOD2, and this restored the synergy between DOX and FLC in the smf3Δ/Δ background. ROS accumulation, in part through altered iron availability from the vacuolar storage pool, is thus the molecular mechanism underlying the synergy between doxycycline and FLC. Furthermore, no effect on either cidality or tolerance was observed in the smf3Δ/Δ strain, highlighting that synergy is not necessarily an indication of cidal therapies.IMPORTANCEAzoles are widely used against Candida albicans, yet many cells survive above the minimal inhibitory concentrations (MIC) by growing slowly, which can prolong infection and foster resistance. We show that intracellular iron homeostasis alters the fluconazole characteristics by affecting ROS accumulation in mitochondria, and that this is the molecular mechanism underlying the combination therapy of fluconazole and doxycycline. These results place iron release from the vacuole at the center of azole responses, suggesting novel ways to boost azole efficacy.

The human commensal, Candida albicans, adapts to glucose-limited niches by utilizing alternative carbon sources such as carboxylates. Saccharomyces cerevisiae can assimilate monocarboxylates via Ato1 (Ady2), a member of the Acetate Uptake Transporter (AceTr) family. In C. albicans, this Ato family has expanded significantly to 10 members (Ato1-Ato10), most with unknown functions. Therefore, we investigated the roles of C. albicans Ato proteins in carboxylate utilization. Functional diversification of C. albicans Atos (CaAtos), suggested by in silico analyses of their AceTr motifs, pore radii, and substrate-binding sites, was confirmed by experimental dissection of their carboxylate transport capacities, revealing CaAto1 as the major acetate transporter, driven by the proton motive force. CaAto1-3 and CaAto6 showed carboxylate-dependent expression and plasma membrane localization. Furthermore, CaATO1 deletion resulted in endoplasmic reticulum (ER) retention of CaAto2 and loss of CaAto3 expression, indicating a central regulatory role for CaAto1. Our analyses reveal further evolutionary diversification of the Ato family in vertebrates.

The human vaginal microbiome, particularly with lactobacilli as the main inhabitants, plays a key role in maintaining women’s health. While lactic acid-mediated pathogen exclusion is well known, broader metabolic functions of vaginal lactobacilli remain underexplored. In this study, we analyzed the vaginal microbiome and metabolome of 258 healthy women from the Isala program. Using targeted metabolomics analysis, we detected a high prevalence with strong interpersonal differences of most B-vitamins, their precursors, and vitamin A in the vaginal microenvironment. Riboflavin (B2) and biotin (B7) showed strong associations with Lactobacillus crispatus and Limosilactobacillus sp . Comparative genomics, phenotypic assays, and in vivo metatranscriptomic data (VIRGO2) collectively confirmed riboflavin biosynthesis by these taxa. Using a riboflavin overproducing Lim. reuteri as a functional model, we showed that microbially derived riboflavin and its pathway intermediates are transported across the vaginal epithelium and modulate host redox balance, cytokine production, and activation of mucosal-associated invariant T (MAIT) cells via induction of MR1 (Major histocompatibility complex, class I-related protein receptor), revealing a potential immunometabolic interface between the vaginal microbiota and its host. ### Competing Interest Statement S.L. declares to be a voluntary academic board member of the International Scientific Association on Probiotics and Prebiotics (ISAPP, www.isappscience.org), cofounder of YUN and aMylla, and scientific advisor for Freya Biosciences. She declares research funding from YUN, Bioorg, Puratos, DSM I-Health, DSM-Firmenich, Fonterra and Lesaffre/Gnosis. S.L., I.S. and S.A. are co-inventors on a patent application related to strain Lim. reuteri AMBV339 used in this work. European Research Council, https://ror.org/0472cxd90, 26850, 101213306, 101078353 Research Foundation - Flanders, https://ror.org/03qtxy027, 1S28622N, 1S89826N, 1277222N, 1271225N, G049022N, G031222N Research Foundation - Flanders, https://ror.org/03qtxy027, S006424N, GOH421GN, AUHA-08-004

ABSTRACT The incidence of non- albicans Candida infections has witnessed a substantial rise in recent decades. Candida glabrata (Nakaseomyces glabratus ), an opportunistic human fungal pathogen, is accountable for both superficial mucosal and life-threatening bloodstream infections, particularly in immunocompromised individuals. Distinguished by its remarkable resilience to environmental stressors, C. glabrata exhibits intrinsic tolerance to azoles and a high propensity to swiftly develop azole resistance during treatment. The molecular mechanism for the high tolerance is not fully understood. In this work, we investigated the possible role of trehalose in this tolerance. We generated mutants in the C. glabrata TPS1 , TPS2 , and NTH1 genes, encoding trehalose 6-phosphate synthase (Tps1), trehalose 6-phosphate phosphatase (Tps2), and neutral trehalase (Nth1), respectively. As expected, the tps1∆ strain cannot grow on glucose. The tps2 ∆ strain demonstrated diminished trehalose accumulation and very high levels of trehalose 6-phosphate (T6P), the biosynthetic intermediate, in comparison to the wild-type (WT) strain. Whereas these higher T6P levels did not affect growth, the lower trehalose levels clearly resulted in lower environmental stress tolerance and a lower susceptibility to fluconazole. More interestingly, the tps2∆ strain completely lost tolerance to fluconazole, characterized by the absence of slow growth at supra-MIC concentrations of this drug. All these phenotypes are reversed in the nth1 ∆ strain, which accumulates high levels of trehalose. Our findings underscore the role of trehalose in enabling tolerance toward fluconazole in C. glabrata . We further show that the change in tolerance is a result of the effect that trehalose has on the sterol pattern in the cell.

Candidozyma auris is an emerging multidrug-resistant fungal pathogen that poses a major public-health challenge owing to high mortality and the limited efficacy of current therapies. Echinocandins, which inhibit β-glucan synthesis, are first-line therapy for invasive C. auris infection; however, resistance to this class is rising, underscoring the urgent need for new antifungal targets. Here we show that enzymes in the trehalose-biosynthetic pathway regulate stress responses, antifungal resistance/tolerance and virulence in C. auris. The tps2Δ strain displays heightened susceptibility to echinocandins, whereas tps1Δ and tps1Δ tps2Δ strains show resistance and tolerance comparable to wild type (WT). Mechanistically, the tps2Δ strain accumulates trehalose 6-phosphate (T6P), which inhibits hexokinase activity and reduces the flux of glucose 6-phosphate (G6P) into the chitin biosynthesis pathway, leading to substantially decreased cell wall chitin. During echinocandin exposure, the tps2Δ strain fails to compensate for reduced β-glucan with increased chitin, thereby rendering it highly susceptible to these drugs. In a systemic mouse infection model, deletion of the TPS2 gene results in lower tissue fungal burdens after treatment with caspofungin. Together, these findings identify Tps2 as a potential therapeutic target that can potentiate echinocandin efficacy in C. auris via a distinct mechanism of action. Echinocandins are the first-line therapy for invasive Candidozyma auris infections, but the resistance to this drug is increasing. Here, the authors identify a potential target with a distinct mechanism of action for improving echinocandin treatment in C. auris.

The Hxk1 protein of Candida albicans phosphorylates N-acetylglucosamine (GlcNAc) which is necessary for various cellular functions, including energy production and chitin synthesis. Further, this protein also regulates morphogenesis independently of its role in GlcNAc catabolism. When HXK1 is deleted, cells are hyperfilamentous on serum-containing medium. Furthermore, Hxk1 translocates to the nucleus in the presence of glucose. To gain a broad understanding of the effect of Hxk1 on gene expression in C. albicans, we performed genome-wide transcriptional profiling of the hxk1 mutant strain by RNA-Seq. The analysis of these RNA-Seq data showed that Hxk1 affects gene expression in both a carbon source-dependent and -independent manner. However, the effect on gene expression occurs via an indirect mechanism, as genome-wide CUT&RUN binding experiments demonstrated that Hxk1 does not bind to the upstream intergenic regions of the differentially expressed genes. Deletion of HXK1 not only resulted in differences in gene expression of genes present in the GlcNAc and galactose regulons, but also in glucose transporter genes, including HGT13. Hxk1 also negatively influences the expression of virulence-associated genes, including HWP1, BRG1, and UME6. Consequently, an hxk1 mutant strain showed higher toxicity toward gut epithelial cells compared to the WT strain. Furthermore, the hxk1 mutant strain had higher expression levels of SOD4 and SOD5 and showed higher resistance toward H2O2. These findings highlight the multiple functions of Hxk1 in different cellular processes.IMPORTANCECandida albicans is a fungus that lives in the human body but does not cause any harm in healthy individuals. However, when the immune system is weakened, C. albicans can spread via the bloodstream all over the body and can lead to severe illness and even death. To infect the human body, multiple proteins hold distinct functions. Hxk1 is one of these proteins. This protein is involved in N-acetylglucosamine (GlcNAc) phosphorylation, as well as hyphae formation and glucose transport. To obtain a complete view of the processes regulated by Hxk1, we performed an RNA-Seq experiment. These data revealed that Hxk1 influences the regulation of genes involved in metabolic and virulence-related processes, such as GlcNAc metabolism, sterol metabolism, and oxidative stress resistance. These findings are important to better understand how C. albicans adapted itself to infect the host.

Mycelium-based composites (MBC) are valued for their ability to convert low-value organic materials into sustainable building resources, making them a promising solution for decarbonizing the construction sector [1-5]. MBC performance is influencing by various factors including mycelium species, substrate composition, growth conditions, and post-processing techniques [1,6]. Conventional fabrication methods involve combining grain spawn with loose organic material to achieve specific functional properties, such as strength, acoustic absorption, or thermal insulation [7,8]. Recent advancements have increasingly focused on digital biofabrication methods to enhance growth, improve material properties, and shape mycelium materials with a primary focus on additive manufacturing (AM) [9-14]. This approach integrates lignocellulosic substrates with fungal inoculation to create complex composite structures. Most experiments use an extrudable paste to construct scaffolds, with studies investigating the effects of inoculating the paste either before or after fabrication [15-20]. Liquid spawn, however, holds significant potential, particularly in AM, due to its ease of deposition and greater precision compared to grain spawn. Combining liquid spawn and AM has the potential to control localized growth within MBC, enabling more precise material customization. I propose a new digital biofabrication framework that consists of three key components: (1) computational design, which analyzes material functionalities and generates robotic actions; (2) robotic fabrication, which executes inoculation and time-dependent actions; and (3) biological material selection, encompassing mycelium species and substrate composition.

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 UTIs. 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. ### Competing Interest Statement KL received consultancy fees from Mundipharma, speaker fees from Pfizer, Gilead, Mundipharma and FUJIFILM Wako chemicals Europe GmbH, a service fee from TECOmedical, a fee for Advisory Board participation from Pfizer and travel support from Pfizer, Gilead and AstraZeneca. All other authors declare no competing interests.