Azole-resistant Aspergillus infections are a source of increasing concern with limited alternative therapeutic options. However, as most infections are still caused by azole-susceptible Aspergillus strains, there is a need to better understand fungal responses to azole antifungals. To this end, we discover that a long non-coding RNA, afu-182, is a major regulator of cyp51-independent sub-MIC azole response. We observe that loss of afu-182 leads to increased surface attached growth and poor treated disease outcomes in a murine model of invasive pulmonary aspergillosis upon azole treatment. In contrast, overexpression of afu-182 significantly reduces fungal burden in animals treated with the azole drug, posaconazole. Importantly, afu-182 levels decrease upon azole exposure and in an azole adaptation experiment, continuous exposure to low dose azole led to MIC increase in an afu-182 dependent manner. Whole transcriptome analyses revealed that azole drug treatment leads to an increase in transcripts of genes encoding 7-transmembrane domain proteins of the RTA1 family, and these proteins are negatively regulated by afu-182. Two RTA1 family genes have individual and combined effects and are sufficient to increase fungal susceptibility to azole drugs in the WT strain. Taken together, our data show a role of the long non-coding RNA afu-182 in regulating Aspergillus fumigatus response to azole drugs both in vitro and in vivo.

Antibiotic tolerance paves the way for acquired resistance in bacterial pathogens. However, the mechanisms of tolerance and its evolutionary role in acquired resistance in pathogenic fungi, and particularly in filamentous fungi, remain elusive. Here, we identified an Inhibitor of Growth domain-containing protein (IngB) as a novel epigenetic regulator of azole tolerance in Aspergillus fumigatus. The loss of ingB promotes supra-MIC growth on agar surfaces despite susceptible MICs in standardized assays. Moreover, established ΔingB biofilms are also less susceptible to azoles in vitro. In a murine model of invasive pulmonary aspergillosis, loss of ingB results in higher pulmonary fungal levels when animals are treated with voriconazole compared to the wild-type control. Subsequent exposure of the ΔingB-tolerant strain to high azole concentrations in vitro resulted in rapid acquired resistance, most notably driven by a frameshift mutation in a putative 20S proteasome maturation protein-encoding gene, umpA, while the susceptible wild-type strain failed to acquire adaptive mutations. The data suggest that loss of IngB provides an epistatic background for the emergence of azole resistance. Our work shows that drug tolerance in a critical fungal pathogen can facilitate azole resistance emergence.IMPORTANCEWhile antimicrobial drug resistance causes adverse effects on human health, drug tolerance can also lead to insufficient pathogen clearance, resulting in infection relapse. However, the mechanisms of antifungal drug tolerance and its evolutionary role in acquired drug resistance in pathogenic fungi, particularly the molds, remain elusive. We identified IngB as a novel regulator of azole tolerance in Aspergillus fumigatus. In a murine model of invasive pulmonary aspergillosis treated with voriconazole, loss of ingB facilitated higher fungal burden levels than the wild-type control, suggesting the observed in vitro tolerance translates to the murine pulmonary environment. Importantly, loss of IngB leads to rapid azole drug resistance under azole-selective pressure in vitro and led to the discovery of a new gene associated with azole resistance, umpA. Our work identifies a novel regulator of antifungal tolerance in a critical human fungal pathogen and suggests that drug tolerance can pave the way for resistance emergence.

Evaluating machine learning models for accurate solubility prediction of azole drugs in binary solvents at various temperatures

Fungal pathogens are adapting to increased temperatures altering host-pathogen interactions, disease patterns, and response to the antimicrobial drugs. Here, we show that thermal adaptation to 42°C leads to reversible changes in fungal colony size and azole drug response in the human pathogenic fungus Aspergillus fumigatus. Importantly, this adaptation is mediated by the lncRNA, afu-182, whose RNA levels negatively correlate with temperature. Growth at a lower temperature or ectopic upregulation of afu-182 RNA levels reverses the temperature adaptation. Previously, we have shown that Δafu-182 strains produce worse disease outcomes in a murine model of invasive pulmonary aspergillosis (IPA). Here, more importantly, we show that the overexpression of afu-182 in clinically azole-resistant isolates increased survival in a murine model of IPA. Taken together, fungal adaptation to increased temperature leads to a decrease in afu-182 RNA levels that is associated with worse disease outcomes upon azole treatment. This provides a framework to take temperature into account when analyzing the rise in azole MIC in environmental and clinical isolates.

Aspergillus fumigatus is a challenging fungal pathogen in the clinic, in part due to increasing azole drug resistance. In this study, we observed that the loss of the A. fumigatus gene arvA results in increased azole susceptibility and significant in vitro morphological changes highlighted by hyper-swollen conidia that yield stunted and polarity-deficient hyphae. Importantly, despite these severe in vitro morphological and growth abnormalities, ∆arvA surprisingly retains full pathogenicity and virulence in two immunologically distinct murine models of invasive pulmonary aspergillosis. These results challenge our understanding of the in-host environment and how it mediates fungal morphogenesis and pathogenesis. These results, consequently, not only enhance our understanding of the role of arvA in A. fumigatus morphogenesis and drug susceptibility but also further emphasize the importance of in vivo animal models in fully evaluating potential antifungal drug targets.

The increasing use of fungicides in the agricultural hub of the Central Valley of California to fight plant pathogens has led to concerns about fungal pathogens developing resistance against these agents. The soil environment harbors many opportunistic fungal species that can cause disease in plants, animals, and humans. Among them are Coccidioides spp. known to cause Valley fever, an orphan disease, endemic to the arid regions of the Southwestern U.S. The disease is often misdiagnosed, delaying treatment with antifungal agents in the early stages of the disease, which has led to the dissemination of the disease in many patients. In this study we found and tested a large cache of fungal isolates, identified as members of ten fungal families and obtained from the air of Bakersfield, Kern County, CA. A large percentage showed strong resistance against three different azole drugs, namely fluconazole, itraconazole, and voriconazole, that are used to treat fungal infections including aspergillosis and Valley fever in humans and animals. Especially fluconazole, one of the most commonly used azole drugs prescribed for treatment, showed no or only minimal effect against most fungal isolates, in contrast to posaconazole which strongly reduced fungal mycelium growth of most isolates in azole challenge assays on Sabouraud Dextrose medium. These results were statistically significant. Two-way ANOVA were used to compare the effects of four azole drugs on fungal mycelium growth among members of ten fungal families. The ANOVA revealed a significant difference in efficacy when comparing the impact of individual drugs on fungal mycelium growth, p < 0.05. Post-hoc comparisons showed that posaconazole significantly inhibited fungal growth more than all other azoles (p < 0.05). Members of most fungal families tested showed a high measure of resistance to azole drugs and 20-39% showed even an increased growth in the presence of fluconazole. The results of this study are concerning in times where Valley fever incidence is increasing due to increased soil disturbance and climate change in the Central Valley of California. KEYWORDS: Coccidioidomycosis; Valley fever; agriculture; fungal pathogen; azole drug; drug resistance; antifungal susceptibility; airborne fungal spores; fungal resistance mechanisms; fungicide

Computational molecular docking and simulation-based prediction of natural compounds from nyctanthes arbor-tristis as potential antifungal agents

Background: Cancer patients are highly susceptible to microbial infections due to immune suppression, necessitating therapeutic strategies that integrate anticancer efficacy with effective antimicrobial intervention. Chalcone-derived nitrogen-fused heterocycles represent a promising platform for developing multi-target agents with relevance to antimicrobial drug delivery, particularly for localized infections. Methods: A series of chalcone-based pyrazoline-thiadiazole nitrogen-fused azole hybrids was synthesized via thiosemicarbohydrazide-functionalized intermediates and fully characterized. Antiproliferative activity was evaluated against MCF-7, HepG-2, HeLa, and HCT-116 cell lines, alongside selectivity toward WI-38 normal fibroblasts. Antibacterial, antibiofilm, and in vivo efficacy were assessed against methicillin-resistant Staphylococcus aureus (MRSA USA300) and Acinetobacter baumannii AB5057. Mechanistic investigations included cell-cycle analysis, apoptosis assays, ERK2, RIPK3, p53, BAX/Bcl-2 quantification, DNA gyrase inhibition, molecular docking, molecular dynamics simulations, and density functional theory calculations. Results: Compound 13 exhibited potent cytotoxicity, particularly against MCF-7 (IC50 = 3.87 ± 0.2 µM), outperforming doxorubicin (IC50 = 4.17 ± 0.2 µM), with high selectivity indices (SI = 10.7 for MCF-7). Mechanistically, compound 13 induced G2/M arrest (40.16% vs. 14.15% control), increased apoptosis to 32.89%, up-regulated ERK2 (3.17-fold), RIPK3 (11.97-fold), and p53 (3.54-fold), and markedly increased the BAX/Bcl-2 ratio (~42-fold). Compounds 7 and 13 displayed bactericidal activity against MRSA and A. baumannii (MIC/MBC = 10 mg/mL), potent antibiofilm effects, and significant in vivo efficacy in an MRSA skin infection model. Compound 13 reduced bacterial load by ~5 log units, outperforming vancomycin. DNA gyrase inhibition (IC50 = 17.10 ± 0.17 µM) and computational studies supported target engagement. Conclusions: Pyrazoline-thiadiazole-based nitrogen-fused azole hybrids, particularly compound 13, demonstrated quantifiable anticancer and antimicrobial efficacy with strong in vivo validation, supporting their potential as multi-target candidates relevant to antimicrobial drug delivery in infection-prone cancer patients.

The microbiome-associated fungus Candida albicans is an opportunistic pathogen. Virulence traits include its ability to produce biofilm, a surface-associated growth form that persists on mucosae and implanted medical devices. C. albicans clinical isolates vary in ability to produce biofilm and the constituent filamentous cell types. Here, we focus on two transcription factors that promote filamentation and biofilm formation, Ndt80 and Ume6. We address two questions. First, how variable is the impact of Ndt80 among C. albicans strains? Second, what is the genetic interaction between NDT80 and UME6? We find that Ndt80 is required for filamentation and biofilm formation in five clinical isolates in addition to the reference strain SC5314, where Ndt80 function has been well established. RNA-sequencing (RNA-seq) data indicate that UME6 RNA levels are reduced in an ndt80Δ/Δ mutant, possibly a result of altered RME1 and WOR1 expression, both of which control UME6. Increased expression of UME6 in ndt80Δ/Δ mutants of three strain backgrounds restores filamentation and biofilm formation, though RNA-seq assays indicate that it does not suppress the overall ndt80Δ/Δ gene expression defect. Ndt80 has an additional role in promoting tolerance to the antifungal drug fluconazole, an inhibitor of ergosterol synthesis. This ndt80Δ/Δ phenotype varies considerably among clinical isolates. In three strains tested, increased expression of UME6 in ndt80Δ/Δ mutants enhances their susceptibility to fluconazole. Therefore, our results show an unexpected relationship between Ume6 expression and azole drug sensitivity. To our knowledge, Ume6 has previously been understood to function only in filamentation, biofilm formation, and related processes.IMPORTANCEOur focus is the fungal pathogen Candida albicans. Two traits, biofilm/hypha formation and azole resistance, are major drivers of its infection ability. We examine the roles of two biofilm transcriptional regulators, Ndt80 and Ume6, in several C. albicans clinical isolates. Prior studies in one strain background (SC5314) indicated that Ndt80 controls both biofilm/hypha formation and azole drug susceptibility and that Ume6 controls biofilm/hypha formation. The four new findings here are that (i) Ndt80 effects on fluconazole sensitivity vary considerably with strain background; (ii) Ndt80 is required for filamentation and biofilm formation in multiple clinical isolates; (iii) the Ndt80 target Ume6 contributes to Ndt80 control of filamentation and biofilm formation in multiple clinical isolates; and (iv) Ume6 influences fluconazole vulnerability, the first Ume6 function to our knowledge that is unrelated to filamentation.

Five new multicomponent crystalline forms of trithiocyanuric acid with azole active pharmaceutical ingredients (APIs) are described. The azole APIs include imidazole-derived econazole and luliconazole, as well as 1,2,4-triazole-derived isavuconazole, voriconazole and posaconazole, and the crystalline forms reported are econazolium trithiocyanurate, C18H16Cl3N2O+·C3H2N3S3-, luliconazole-trithiocyanuric acid (1/1), C14H9Cl2N3S2·C3H3N3S3, isavuconazole-trithiocyanuric acid (1/1), C22H17F2N5OS·C3H3N3S3, voriconazole-trithiocyanuric acid-water (1/2/3), C16H14F3N5O·2C3H3N3S3·3H2O, and posaconazole-trithiocyanuric acid (1/1), C37H42F2N8O4·C3H3N3S3. The crystal architectures are analyzed in the context of the supramolecular assemblies they generate. Robust acid-base pairs, governed by N-H...N hydrogen bonds, are combined into well-defined four-molecule assemblies through N-H...S hydrogen bonds, which form the characteristic ring R22(8) synthon between two TTCA molecules. Additional N-H...O or N-H...S interactions, involving the remaining azole functionalities, propagate these four-molecule units into extended chain motifs, columns or layered architectures. To elucidate the nature of these key interactions, a QTAIM analysis was performed and pairwise interaction energies were evaluated to clarify the stability and energetics of the resulting assemblies. The influence of chiral azole molecules, their specific hydrogen-bond functionalities and the solvation conditions on the formation of the supramolecular motifs is also discussed.

While antimicrobial drug resistance causes a significant adverse effect on human health, drug tolerance can also lead to insufficient pathogen clearance, resulting in infection relapse. However, the mechanisms of antifungal drug tolerance and its evolutionary role in acquired drug resistance in pathogenic fungi, particularly the molds, remains elusive. We identified IngB as a novel regulator of azole tolerance in Aspergillus fumigatus . Importantly, loss of IngB leads to rapid azole drug resistance under azole-selective pressure. Our work identifies a novel regulator of antifungal tolerance and suggests antifungal drug tolerance can pave the way for resistance emergence in a critical fungal pathogen.

We aimed to analyze the genomic variations associated with high azole resistance in the C. albicans isolates of intensive care unit (ICU) patients with Candida bloodstream infections (BSIs) and those from oropharyngeal colonization in HIV patients. The genomic DNA of azole-resistant C. albicans isolates was analyzed using the Oxford Nanopore platform. Subsequent analyses included ERG11 alignment to determine the extent and distribution of missense substitutions, Ka/Ks calculations to test for positive selection on ERG11, and detailed CDR1 and CDR2 mutational analysis across the coding sequence. Efflux function was assessed by measuring the fold reduction in the minimum inhibitory concentration (MIC) of azole drugs in the presence of milbemycin. A total of 27 azole-resistant C. albicans isolates from ICU patients with Candida BSIs in Linkou Chang Gung Memorial Hospital in Taiwan and HIV patients' oropharyngeal colonization were identified and analyzed. The in-depth, core analyses were performed on seven representative C. albicans isolates. The C. albicans isolates of HIV-infected patients had notably higher azole resistance (fluconazole MICs > 256mg/L) when compared with those of the ICU patients (fluconazole MICs 16-64mg/L), suggesting the involvement of additional mechanisms. The central role of ERG11 mutations was supported by the presence of ERG11 missense mutations in all azole-resistant C. albicans isolates. The ERG11 coding regions analyses showed no evidence of positive selection (Ka/Ks < 1.0) and no specific mutation unique to the C. albicans isolates of the HIV patients, but unique missense mutations only in CDR1/CDR2 were noted. Milbemycin substantially decreased the azole MICs in all C. albicans isolates and confirmed the efflux pump involvement. The effects of milbemycin were much lower in C. albicans of the HIV patients than those from the ICU patients. Furthermore, unique ERG11 promoter variants, including changes at the Hap43p/Hap5p sites, were noted in the C. albicans isolates of the HIV patients. While ERG11 structural mutations are foundational, the elevated azole resistance in the C. albicans isolates of HIV-infected patients is associated with specific CDR1/CDR2 mutations and distinct ERG11 promoter variants, highlighting genomic features that warrant further functional validation.

Organometallic and coordination gold(I)-azole drugs compounds: Synthesis, characterization, antitumor evaluation, and interactions with biomolecules.

Antimicrobial resistance is a momentous global threat, demanding innovative approaches to combat drug-resistant pathogens. As a prevalent fungal pathogen, Candida albicans exhibits increasing resistance to conventional antifungals, especially the azoles. This study explores a novel approach combining sophorolipids (SLs), a glycolipid biosurfactant, with clinical azoles, including fluconazole (FLZ), itraconazole (ITZ), and ketoconazole (KCZ), against C. albicans biofilms. SLs from the yeast Starmerella riodocensis exhibited promising metabolic reduction and antibiofilm activity against Candida biofilms, with a biofilm inhibitory concentration (BIC50) of 512mg/L. Among the tested azoles, ITZ exhibited the highest antibiofilm efficacy, prompting further investigation of SLs combinations. The ITZ-SLs combination markedly enhanced antibiofilm activity against preformed biofilms, with ITZ and SLs concentrations reduced by 16-fold and 4-fold, respectively, compared with their individual treatments (achieving a BIC₅₀ of 1mg/L ITZ and 128mg/L SLs). Quantitative real-time polymerase chain reaction analysis revealed significant downregulation of essential biofilm-associated genes such as BCR1, EFG1, and CDC28, demonstrating SLs's ability to inhibit various stages of biofilm development and stability. Thus, the synergy observed with azole drugs, particularly ITZ and SLs, was highly effective in biofilm removal, highlighting the compatibility of anti-biofilm biosurfactant SLs with some antifungal agents.

The fungal disease mucormycosis, while generally regarded as rare and not transmitted between individuals, has become increasingly prevalent in disaster areas, among the immunocompromised, and in diabetics especially in response to COVID-19. Treatment options are limited. These include debridement of necrotizing tissue followed by complicated multicomponent therapies with amphotericin B and selected azole drugs, usually having poor outcomes. Mucormycetes are intrinsically resistant to the widely used short-tailed azole drugs fluconazole and voriconazole, but susceptible to the long-tailed, though expensive, azole posaconazole. Knowledge of the crystal structure of Saccharomyces cerevisiae sterol 14α-demethylase (Erg11, Cyp51) led to the hypothesis that this pattern of intrinsic azole resistance and susceptibility is due to the Rhizopus arrhizus CYP51-F5 isoform residues F129 and A291, while the CYP51-F1 isoform residues Y127 and V291 confer susceptibility to both short- and long-tailed azole drugs. The heterologous overexpression of individual recombinant R. arrhizus CYP51 isoforms in a S. cerevisiae host, with or without the cognate NADPH-cytochrome P450 reductase (RaCPR), and selective genetic modification of CYP51-F5 have tested this hypothesis. Complementary gene deletion experiments in Rhizopus microsporus confirm that the amino acid residues that align with R. arrhizus CYP51-F5 F129 and A291 determine the resistance or susceptibility pattern of R. arrhizus to short-, medium-, and long-tailed azoles.

Amphotericin: B (AmpB)-resistant Candida (C.) species, such as C. parapsilosis, are among the most common causes of invasive fungal infections, posing significant challenges in hospital settings. Although AmpB is considered the first-line treatment owing to its broad-spectrum fungicidal activity, its use is hampered by severe side effects and the emergence of acquired resistance, particularly in C. parapsilosis, which exhibits reduced susceptibility to polyene, azole, and echinocandin-based antifungal drugs. Here, we present findings on photodynamic therapy (PDT) that targets the opportunistic fungal pathogens C. parapsilosis and C. albicans via the use of photosensitizers from the iodocyanine and newly developed iodinated Methylene blue families. These compounds contain heavy iodine atoms that increase the production of reactive oxygen species (ROS), the agents responsible for oxidative cellular damage, via the heavy-atom effect, which promotes intersystem crossing (ISC) and triplet-state formation. A strong antifungal effect was observed against AmpB-resistant C. parapsilosis, indicating a correlation between the quantum yield of ROS generation and the photosensitizing efficacy under near-infrared (NIR) light irradiation. The combination of efficient cellular uptake and enhanced ROS generation positions iodinated photosensitizers as promising candidates for the treatment of drug-resistant Candida strains.

Aspergillus fumigatus is a challenging fungal pathogen in the clinic in part due to increasing azole drug resistance. In this study, we observe that loss of the A. fumigatus gene arvA results in increased azole susceptibility and significant in vitro morphological changes highlighted by hyper-swollen conidia that yield stunted and polarity deficient hyphae. Importantly, despite these severe in vitro morphological and growth abnormalities, ΔarvA surprisingly retains full pathogenicity and virulence in two immunologically distinct murine models of invasive pulmonary aspergillosis. These results challenge our understanding of the in-host environment and how it mediates fungal morphogenesis and pathogenesis. These results, consequently, not only enhances our understanding of the role of arvA in A. fumigatus morphogenesis and drug susceptibility, but further emphasizes the importance of in vivo animal models in fully evaluating potential antifungal drug targets.

The increasing incidence of invasive fungal infections-particularly disseminated candidiasis caused by Candida albicans-highlights the need for innovative therapeutic strategies. This study evaluates the antifungal activity of a curcumin nanosuspension (CNS), both alone and in combination with azole drugs, focusing on its potential to counteract drug resistance. CNS substantially improved the aqueous solubility and enabled sustained release of curcumin in vitro, overcoming its inherent pharmacokinetic limitations. Checkerboard assays identified synergistic effects between CNS and fluconazole against planktonic cells (fractional inhibitory concentration index [FICI]: 0.38-0.5) and early biofilms of C. albicans (FICI < 0.5). Morphological analyses revealed that the combination suppressed hyphal growth and disrupted biofilm integrity-critical virulence attributes of C. albicans. Further mechanistic observations suggested that the enhanced antifungal activity may involve facilitated drug uptake and suppression of efflux function, alongside reduced expression of adhesion-related genes (ALS1, ALS3, HWP1, and EFG1). These results underscore a promising nanotechnology-based approach to enhance the efficacy of conventional azoles through combination therapy.IMPORTANCEThis study provides a novel nanotechnology-based strategy to overcome azole resistance in C. albicans, a major clinical pathogen responsible for life-threatening systemic infections. By formulating curcumin into a stable nanosuspension (CNS), we significantly enhanced its solubility and bioavailability, overcoming a fundamental limitation that has hindered its clinical application. More importantly, we demonstrate that CNS acts synergistically with fluconazole, effectively restoring its efficacy against resistant planktonic cells and biofilms through dual mechanisms: increasing intracellular drug accumulation by inhibiting efflux pumps and suppressing key virulence traits including hyphal formation and adhesion. This combinatory approach not only reduces the required drug dosage and potential toxicity but also provides a promising therapeutic avenue against biofilm-associated refractory candidiasis. Our findings highlight the potential of harnessing natural product-nanocarrier systems to extend the lifespan of existing antifungals and combat drug-resistant fungal infections.

Potential synergistic antifungal activity of microbially synthesized silver nanoparticles and vitamin D3 against Candida albicans: In vitro and Galleria mellonella model studies.

Home sampling for therapeutic drug monitoring (TDM) shows promise as a valuable tool, particularly for stable patients with limited access to health care. Quantitative dried blood spot (qDBS) technology has emerged as a promising solution. This study aimed to develop a method for simultaneous quantification of fluconazole, voriconazole, isavuconazole, posaconazole, itraconazole, and hydroxyitraconazole and to investigate key technical issues such as transport stability and the conversion formulas between different matrices. The extraction protocol was optimized using spiked qDBS samples with a series of methanol/water and acetonitrile/water mixtures. Stability tests were performed at 2-8°C and 45°C to mimic environmental conditions of regular mail delivery. Linear regression for converting voriconazole concentrations from qDBS to plasma samples was established using 101 routine TDM samples. The optimal extraction solvent was acetonitrile/water (70/30). The method validation demonstrated excellent linearity (R 2 > 0.99), accuracy (recovery of 92.1%-104.2%), and precision (intraday and interday variability <3.3% and 5.7%, respectively). Stability tests confirmed that all azole drugs remained stable, even at 45°C for 7 days. Conversion formula indicated that qDBS sample concentration was equivalent to that in plasma, as supported by Bland-Altman analysis, which revealed a mean bias of -8.49% and 95% limits of agreement ranging from -20.11% to +3.13%. This study established a sensitive, accurate, precise, and robust quantification method in qDBS samples containing only 10 μL of whole blood. Stability tests showed that samples could be safely mailed, offering a cheaper alternative to cold chain logistics. A conversion formula using routine TDM samples was developed, providing a straightforward and cost-effective approach for establishing a conversion formula that is potentially applicable to other TDM analytes. This study represents an in vitro analytical validation, and future clinical research is needed to translate the analytical method from bench to bedside.