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Quinone outside inhibitor (QoI) fungicide resistance in Alternaria alternata populations was reported in Brazil for the first time in 2019, in São Paulo orchards, and the mutation G143A in cytochrome b (cytb) was found in resistant isolates. Our study investigated the infectious process, production of reactive oxygen species (ROS), and fungal cell death in resistant (QoI-R) and sensitive (QoI-S) A. alternata pathotype tangerine (Aapt) isolates. Morphological characterization of Aapt isolates was performed using confocal laser scanning microscopy (CLSM). Alternaria brown spot (ABS) symptoms were produced by Aapt isolates on tangelo cv. BRS Piemonte. Germination of QoI-R conidia and production of germ tubes on tangelo leaflets treated with 100 μg mL -1 of pyraclostrobin 18 h after inoculation (hai) was observed using scanning electron microscopy (SEM). At the same time, QoI-S conidial germination was inhibited on tangelo leaflets treated with pyraclostrobin. ROS production and cell death in Aapt isolates at high fungicide concentrations were observed using CLSM. QoI-S conidia exhibited high ROS production, indicating high oxidative stress. When dyed with propidium iodate (PI), QoI-S conidia emitted red fluorescence, showing cell death and confirming their sensitive phenotype. In contrast, QoI-R conidia neither produced ROS nor exhibited red fluorescence, indicating no cell death and confirming their resistant phenotype. Therefore, our findings evidence that microscopic techniques may help characterize events during fungi-plant interactions, ROS production, cell death, and Aapt phenotypes resistant and sensitive to QoIs using fluorometric protocols., (© 2024 Wiley Periodicals LLC.)

This study explores the effectiveness of various antifungal drugs in treating sporotrichosis caused by Sporothrix schenckii, especially in non-wild-type (non-WT) strains. The drugs tested include enilconazole (ENIL), isavuconazole (ISA), posaconazole (POS), terbinafine (TER), and itraconazole (ITC). The study involved in vitro and in vivo tests on 10 WT isolates and eight ITC non-WT isolates. Two isolates were assessed using time-kill assays, checkerboard assays, and Galleria mellonella infection models. In vitro studies have shown that all of these drugs were more effective than or equal to ITC against WT and non-WT isolates. No ITC resistance was observed with other azoles. All drugs inhibited fungal growth of WT and non-WT strains within 24 h at all incubations. ENIL and TER showed fungicidal effect against types at over 2x minimum inhibitory concentrations with no regrowth. POS was fungicidal against WT at high concentrations but not against non-WT. ISA was only fungicidal for non-WT. ITC did not exhibit any fungicidal activity. In checkerboard experiments, the combination of POS or ISA with TER showed enhanced activity against WT and non-WT strains, surpassing the combination of ITC with TER. In vivo model experiments demonstrated significantly reduced mortality rates with ENIL, POS, and TER against WT and with ENIL, ISA, POS, and TER against non-WT. The study concludes that monotherapy with ENIL, ISA, POS, and TER, and combinations of POS/TER or ISA/TER, show promise as effective antifungal treatments against S. schenckii, including ITC-non-WT isolates., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval: This study has obtained permission from the MUVS Application for Permission of Animal Care and Use (MUVS-ACU F01). The protocol has been approved with the number MUVF-2022-01-02., (© 2025. The Author(s).)

Invasive fungal infections (IFIs) pose significant challenges in clinical settings, particularly due to their high morbidity and mortality rates. The rising incidence of these infections, coupled with increasing antifungal resistance, underscores the urgent need for novel therapeutic strategies. Current antifungal drugs target the fungal cell membrane, cell wall, or intracellular components, but resistance mechanisms such as altered drug-target interactions, enhanced efflux, and adaptive cellular responses have diminished their efficacy. Recent research has highlighted the potential of dual inhibitors that simultaneously target multiple pathways or enzymes involved in fungal growth and survival. Combining pharmacophores, such as lanosterol 14α-demethylase (CYP51), heat shock protein 90 (HSP90), histone deacetylase (HDAC), and squalene epoxidase (SE) inhibitors, has led to the development of compounds with enhanced antifungal activity and reduced resistance. This dual-target approach, along with novel chemical scaffolds, not only represents a promising strategy for combating antifungal resistance but is also being utilized in the development of anticancer agents. This review explores the development of new antifungal agents that employ mono-, dual-, or multi-target strategies to combat IFIs. We discuss emerging antifungal targets, resistance mechanisms, and innovative therapeutic approaches that offer hope in managing these challenging infections., (© 2025 The Author(s). Chemical Biology & Drug Design published by John Wiley & Sons Ltd.)

In recent years, the widespread emergence of drug resistance in yeasts and filamentous fungi to existing antifungal armamentariums has become a severe threat to global health. There is also concern regarding increased rates of azole resistance in Aspergillus fumigatus and Terbinafine resistance in Trichophyton species. To overcome this concern of resistance to regular therapies, new antifungal drugs with novel and effective mechanisms are crucially needed. Herbal remedies may be promising strategies for the treatment of resistant infections. We aimed to investigate the in vitro and silico activity of piperlongumine against clinical azole susceptible/resistant A. fumigatus and terbinafine-susceptible/resistant Trichophyton species. In the current study, piperlongumine demonstrated potent antifungal activity, with minimum inhibitory concentrations (MICs) ranging from 0.016-4 μg/mL against Trichophyton isolates and 0.25-2 μg/mL for A. fumigatus isolates. Additionally, molecular docking studies indicated that piperlongumine has a strong binding affinity to the active sites of squalene epoxidase and sterol 14-alpha demethylase. However, further studies are warranted to correlate these findings with clinical outcomes and provide the basis for further investigations to pave the way for developing novel antifungal agents., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Iman Haghani reports financial support was provided by Mazandaran University of Medical Sciences. This research was financially supported by grant number 17588 from Mazandaran University of Medical Sciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The development of innovative and effective strategies to combat fungal pathogens is critical to sustainable crop protection. Fungicides have been used for over two centuries, with traditional copper- and sulfur-based formulations still in use due to their broad-spectrum, multisite mode of action, which minimizes the risk of pathogen resistance. In contrast, modern systemic fungicides, though potent, often target a single site of action, leading to the accelerated emergence of resistant fungal strains. This study explores synergistic interactions between chitosan (CS) and selected fungicides, focusing on their antifungal activity against Fusarium graminearum . Among the fungicides tested, azoxystrobin (Amistar) exhibited the highest 44.88 synergy score when combined with CS (30 kDa, degree of deacetylation ≥ 90), resulting in significantly improved antifungal efficacy. Furthermore, the combination of CS and Amistar with double-stranded RNA (dsRNA) targeting selected ABC transporter genes further amplified antifungal activity by silencing genes critical for fungal tolerance to treatment. This dual synergy highlights the potential of RNA interference (RNAi) as both a functional tool to investigate fungal physiology and an effective antifungal strategy. These findings reveal a promising and environmentally friendly approach to mitigate resistance while improving fungal control. Furthermore, the remarkable synergy between azoxystrobin and CS presents a novel mechanism with significant potential for sustainable agricultural applications, which warrants further investigation to elucidate its molecular basis.

In recent years, the incidence of fungal infections has been rising annually, especially among immunocompromised populations, posing a significant challenge to public health. Although antifungal medications provide some relief, the escalating problem of resistance sharply curtails their effectiveness, presenting an urgent clinical dilemma that demands immediate attention. Research has shown that fungal resistance is closely related to quorum sensing (QS), and QS inhibitors (QSIs) are considered an effective solution to this issue. Garlic, as a natural QSI, has demonstrated significant effects in inhibiting fungal growth, preventing biofilm formation, enhancing immunity, and combating resistance. This study explores the potential of garlic in mitigating fungal drug resistance and identifies its key role in inhibiting the QS mechanism, these findings offer a new perspective for the treatment of fungal infections, especially in addressing the increasingly severe problem of resistance. However, the clinical application of garlic still faces several challenges, such as ensuring the standardization of active ingredient extraction, as well as issues of safety and stability. Future research should focus on the QS mechanism and promote interdisciplinary collaboration to develop more natural, effective, and safe QSI drugs like garlic, while actively conducting clinical trials to validate their efficacy and safety. Additionally, incorporating advanced technologies such as nanotechnology to enhance drug stability and targeting, provide a more comprehensive strategy for the treatment of fungal infections. Overall, Our study provides scientific evidence supporting the potential of garlic as a novel antifungal treatment and lays the groundwork for the development of future natural QSIs for therapeutic use. It offers new insights, particularly for the treatment of immunocompromised populations and drug-resistant fungal strains., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Li et al.)

Luliconazole (LCZ) is a topical imidazole antifungal agent with broad-spectrum activity. However, LCZ encounters challenges such as low aqueous solubility, skin retention, and penetration, which reduce its dermal bioavailability and hinder its efficacy in drug delivery. The aim of the present study was to formulate, characterize, and evaluate the in vitro antifungal efficacy of luliconazole-loaded nanostructured lipid carriers (LCZ-NLCs) against a panel of resistant fungal strains. The LCZ-NLCs were synthesized using a modified emulsification-solvent evaporation technique. Characterization involved assessing parameters such as poly-dispersity index (PDI), zeta potential, encapsulation efficiency (EE %), Field Emission Scanning Electron Microscopy (FESEM), Differential Scanning Calorimetry (DSC) analysis, and Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR). Furthermore, in vitro drug release experiments, analysis of release kinetics, cytotoxicity assessments, and in vitro antifungal susceptibility tests were performed as part of the study. The findings indicated that LCZ-NLCs displayed nanoscale dimensions, uniform dispersion, and a favorable zeta potential. The encapsulation efficiency of LCZ in NLCs was approximately 90%. FESEM analysis revealed spherical nanoparticles with consistent shape. ATR-FTIR analysis indicated no chemical interaction between LCZ and excipients. In vitro drug release experiments demonstrated that LCZ-NLCs notably improved the drug's dissolution rate. The stability testing confirmed consistent colloidal nanometer ranges in the LCZ-NLCs samples. Additionally, cytotoxicity tests revealed no toxicity within the tested concentration. Moreover, in vitro antifungal susceptibility tests demonstrated potent antifungal activity of LCZ-NLCs against the tested resistant fungal isolates. The study findings suggest that the LCZ-NLCs formulation developed in this research could be a promising topical treatment for superficial fungal infections, especially in cases of resistant infections. However, the study needs further ex vivo and in vivo tests to ensure safety and efficacy., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The growing threat of antimicrobial resistance (AMR) necessitates innovative strategies beyond conventional antibiotics. In response, we developed a rapid one-step method to sythesize antimicrobial peptide (AMP) ε-poly- L -lysine stabilized selenium nanoparticles (ε-PL-Se NPs). These polycrystalline NPs with highly positive net surface charges, exhibited superior antimicrobial activity against a broad panel of pathogens, including the Gram-positive and -negative bacteria Staphylococcus aureus , Enterococcus faecalis , Escherichia coli , and Pseudomonas aeruginosa and their drug-resistant counterparts, as well as the yeast Candida albicans . Notably, 10PL-Se NPs exhibited 6-log reduction of methicillin-resistant S. aureus (MRSA) at a concentration of 5 μg/mL within 90 min, with minimum bactericidal concentrations (MBCs) below 50 μg/mL for all tested bacterial strains. The minimum fungicidal concentration (MFC) of 10PL-Se NPs against C. albicans was 26 ± 10 μg/mL. Crucially, bacteria exposed to ε-PL-Se NPs exhibited significantly delayed resistance development compared to the conventional antibiotic kanamycin. S. aureus developed resistance to kanamycin after ∼72 generations, whereas resistance to 10PL-Se NPs emerged after ∼216 generations. Remarkably, E. coli showed resistance to kanamycin after ∼39 generations but failed to develop resistance to 10PL-Se NPs even after 300 generations. This work highlights the synergistic interactions between ε-PL and Se NPs, offering a robust and scalable strategy to combat AMR.

Berberine (BBR), an isoquinoline alkaloid found in medicinal plants such as Coptidis rhizoma, Berberis sp., and Hydrastis canadensis, is a distinctive compound known for its dual ability to exhibit broad-spectrum antifungal activity while offering beneficial effects to the host. These attributes make it a highly valuable candidate for antifungal therapy and as an antibiotic adjuvant. This review provides a comprehensive evaluation of BBR's antifungal properties, focusing on its in vitro and in vivo activity, underlying mechanisms, and its influence on fungal pathogenicity, including virulence, biofilm formation, and resistance. Additionally, the antifungal potential of BBR extracts, derivatives, and nanoformulations is examined in detail. BBR demonstrates fungicidal effects through multiple mechanisms. It targets critical fungal components such as mitochondria, cell membranes, and cell walls, while also inhibiting enzymatic activity and transcription processes. Furthermore, it suppresses the expression of virulence factors, effectively diminishing fungal pathogenicity. Beyond its direct antifungal activity, BBR exerts beneficial effects on the host by modulating gut microbiota, thereby bolstering host defenses against fungal infections and reducing potential adverse effects. BBR's interaction with conventional antifungal drugs presents a unique complexity, particularly in the context of resistance mechanisms. When used in combination therapies, conventional antifungal drugs enhance the intracellular accumulation of BBR, thereby amplifying its antifungal potency as the primary active agent. These synergistic effects position BBR as a promising candidate for combination strategies, especially in addressing drug-resistant fungal infections and persistent biofilms. As antifungal resistance and biofilm-associated infections continue to rise, the multifaceted properties of BBR and its advanced formulations highlight their significant therapeutic potential. However, the scarcity of robust in vivo and clinical studies limits a full understanding of its efficacy and safety profile. To bridge this gap, future investigations should prioritize well-designed in vivo and clinical trials to thoroughly evaluate the therapeutic effectiveness and safety of BBR in diverse clinical settings. This approach could pave the way for its broader application in combating fungal infections., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Today, Candida albicans is still the most common cause of both local and life-threatening systemic candidiasis. The spread of resistant fungal strains has resulted in an urgent need to search for new promising antimycotics. Here, we investigated the antifungal action of the tobacco defensin NaD1 against susceptible and resistant to azoles and echinocandins strains of C. albicans . We demonstrated that NaD1 was equally effective and fungicidal against all tested strains. The MIC and MFC values were 6.25 and 12.5 µM, respectively. We showed for the first time that NaD1 could act synergistically not only with caspofungin but also with human host defense antimicrobial peptides cathelicidin LL-37 and β-defensin-2 (HBD2) against susceptible and resistant fungal strains. Using flow cytometry, we demonstrated that NaD1 in combinations with LL-37 or HBD2 can reinforce each other by enhancing membrane disruption. Using the Caco-2 cell monolayer model, we demonstrated that NaD1 impaired the adhesion of C. albicans cells to the human epithelium. Moreover, NaD1 inhibited the formation of fungal biofilms in Sabouraud broth and less markedly in nutrient-rich RPMI-1640 medium, and enhanced the antibiofilm activity of caspofungin. Thus, we hypothesized that NaD1 might affect the development of candidiasis in vivo, including that caused by resistant fungal strains.

3,3'-Diindolylmethane (DIM), a compound derived from natural fruits and vegetables, is widely recognized for its anti-cancer activity. However, its action mechanisms remain ambiguous. In this study, to study the molecular mechanism of 3,3'-Diindolylmethane, we identified a novel mutation in the gene of mitochondrial translation elongation factor EF-Ts (tsf1 + ), a key factor in mitochondrial protein translation, that conferred DIM resistance to Schizosaccharomyces pombe. The tsf1Δ also conferred DIM resistance. Decreased mitochondrial translation was found to be responsible for conferring DIM resistance to Schizosaccharomyces pombe, as the cells gained DIM resistance after treatment with chloramphenicol, a specific mitochondrial translation inhibitor. Notably, tsf1Δ conferred DIM resistance in the absence of either autophagy-related protein, Atg7, or nuclear envelope protein, Lem2, two proteins that have been reported to be required for cell survival in the presence of DIM. Overall, this study revealed novel biological functions of DIM and highlighted its potential as an anti-cancer agent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tan spot caused by Pyrenophora tritici-repentis is a severe threat to wheat production in all major wheat-growing regions. Sustainable tan spot control can be achieved by an integrated approach, including responsible management of fungicide sprays. The data about the sensitivity of P. tritici-repentis to various fungicides in the Baltic Sea region are rare. In this study, we described the variation of P. tritici-repentis sensitivity to four fungicide active ingredients to detect the formation of resistance to the most commonly used quinone-outside inhibitor (QoI) and 14α-demethylase inhibitor (DMI) fungicides in the pathogen's population in Latvia. The effect of prothioconazole, mefentrifluconazole, pyraclostrobin, and azoxystrobin on 93 P. tritici-repentis strains from various hosts was tested in vitro by assessing mycelium linear growth inhibition at three different active ingredient concentrations (0 0.01, 0.1 and 0.5 mg L -1 ). Pathogen sensitivity significantly ( p < 0.001) varied between the fungicide active ingredients and strains. The prothioconazole (concentration 0.5 mg L -1 ) had the most significant effect, with a median mycelial growth inhibition of 70.34%, followed by pyraclostrobin (47.02%), azoxystrobin (24.24%), and mefentrifluconazole (11.11%). Mutation G143A was detected in cytb gene sequences and confirmed the resistance formation in Latvia's P. tritici-repentis population, while F129L and G137R mutations were absent. This study provided insight into P. tritici-repentis population's sensitivity to active ingredients of DMI and OoI fungicide groups, helping to fill the knowledge gap about the pathogen fungicide sensitivity in this region.

Aspergillus fumigatus is a widely distributed pathogen responsible for severe infections, particularly in immunocompromised individuals. Triazoles are the primary treatments options for Aspergillus infections; however, the emergence of acquired resistance to this antifungal class is becoming a growing concern. In this study, we investigated the potential of the antiviral drug, lopinavir (LPV) to restore the susceptibility of A. fumigatus strains to a set of azoles, while also reducing the required azole dosage for treatment of susceptible isolates. The combination of LPV with either itraconazole (ITC) or posaconazole (POS) demonstrated potent synergistic interactions against 16 out of 23 (~70%) and 21 out of 23 (~91%) A. fumigatus isolates, respectively. Moreover, the combination showed synergistic activity against other clinically important Aspergillus species, including A. niger, A. flavus, and A. brasiliensis. The fractional inhibitory concentration index (FICI) for the combinations ranged from 0.18 to 0.313 for ITC and 0.091 to 0.313 for POS, indicating strong synergistic effects. Further investigation revealed that efflux pump inhibition contributed to the synergy observed between azole and LPV. Morphological examination of the fungal cells subjected to this combinational therapy at sub-inhibitory doses showed the presence of carbohydrate granules/patches. The identification of LPV as a promising adjunct therapy holds promise for addressing the emerging challenge of azole resistance in Aspergillus species and improving treatment outcomes for patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Burns et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Candida spp., causes invasive fungal infections, especially in immune-compromised patients and the propensity of antifungal resistance against azole-based drugs need to be addressed. This study is thus aimed to characterize the anticandidal effect of the cinnamic acid extracted from the barks of Cinnamomum cassia. Five species of Fluconazole-resistant Candida sp. were retrieved from the department repertoire. The extraction of CA was performed by three different methods followed by silica gel column chromatography. Eluant was subjected to FTIR and XRD analysis for confirmation. The anticandidal activity of the CA was checked by the agar disc diffusion method and the MIC and MFC were determined. The anti-biofilm effect of CA was assessed using the CLSM technique followed by the biocompatibility check using MTT assay in normal HGF cell lines. CA was best extracted with the hot maceration method using ethanol with a maximum yield of 6.73 mg. Purification by column chromatography was achieved using benzene, acetic acid, and water (6:7:3) mobile phase. CA was confirmed by FTIR with absorption peaks and by XDR based on strong intensity. CA was found to possess promising anticandidal activity at 8 µg/mL with MIC and MFC values determined as 0.8 µg/mL and 0.08 µg/mL respectively. Antibiofilm activity by CLSM analysis revealed biofilm inhibition and was biocompatible at 8.5 µg/ml concentrations in HGF cell lines until 24 h. The study findings conclude that CA is the best alternative to treat candidal infection warranting further experimental preclinical studies., Competing Interests: Declarations. Conflict of interest: The authors declare that is no conflict of interest in association with the research study., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Black Sigatoka is the most widespread banana disease worldwide. It is caused by Pseudocercospora fijiensis, a fungal pathogen known for developing resistance to fungicides such as thiabendazole. Despite the increasing costs associated with the use of chemicals to control this disease, the pathogen's mechanisms for fungicide resistance are not fully understood. The metabolite profiles of P. fijiensis isolates with different levels of resistance to thiabendazole were characterized by GC-MS. A total of 33 isolates were obtained from symptomatic banana plants and the sensitivity of each isolate to thiabendazole was assessed at 0, 1, 10, 100, 1000, and 10000 μg.mL-1. Then, the metabolite profile of each isolate was assessed using GC-MS. Metabolites such as hexadecanoic acid, tetradecanoic acid, octadecadienoic acid and octadecanoic acid were significantly over-accumulated in the presence of thiabendazole at 10 μg.mL-1. Phosphoric acid, L-proline, and D-allose increased in concentration with time in the presence of 100 μg.mL-1 of thiabendazole, and mannonic acid, 1-hexadecanol, D-sorbitol and tetracosanoic acid were only detected in the presence of the fungicide. Metabolic pathways including that of fructose, mannose metabolism, the biosynthesis of unsaturated fatty acids, and ABC transporters were upregulated in resistant isolates. Our findings show an increment of tetracosanoic (myristic) acid suggesting a possible β-tubulin-compensation mechanism in resistant isolates. The presence of myristic acid promoted the generation of diacylglycerol kinase δ which facilitated the production of β-tubulin in other studies. Additionally, important changes in the metabolite profiles were observed as soon as six hours after exposure to the fungicide showing an early response of the pathogen. To the best of our knowledge, this is the first report that describes the changes in the metabolite profile of P. fijiensis resistant to thiabendazole when exposed to the fungicide., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Maridueña-Zavala et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Antifungal drug resistance is a critical concern, demanding innovative therapeutic solutions. The dual-targeting mechanism of action (MoA), as an effective strategy to reduce drug resistance, has been validated in the design of antibacterial agents. However, the structural similarities between mammalian and fungal cells complicate the development of such a strategy for antifungal agents as the selectivity can be compromised. Herein, we introduce a dual-targeting strategy addressing fungal infections by selectively introducing DNA binding molecules into fungal nuclei. We incorporate rigid hydrophobic units into a DNA-binding domain to fabricate antifungal luminogens of TPY and TPZ, which exhibit enhanced membrane penetration and DNA-binding capabilities. These compounds exhibit dual-targeting MoA by depolarizing fungal membranes and inducing DNA damage, amplifying their potency against fungal pathogens with undetectable drug resistance. TPY and TPZ demonstrated robust antifungal activity in vitro and exhibited ideal therapeutic efficacy in a murine model of C. albicans -induced vaginitis. This multifaceted approach holds promise for overcoming drug resistance and advancing antifungal therapy.

Fungal infections in humans are difficult to treat, with very limited drug options. Due to a confluence of factors, there is an urgent need for innovation in the antifungal drug space, particularly to combat increasing antifungal drug resistance. Our previous studies showed that Cdc50, a subunit of fungal lipid translocase (flippase), is essential for Cryptococcus neoformans virulence and required for antifungal drug resistance, suggesting that fungal lipid flippase could be a novel drug target. Here, we characterized an antifungal peptide, Cryptomycinamide (KKOO-NH 2 ), derived from a 9-amino acid segment of the C. neoformans Cdc50 protein. A fungal killing assay indicated that KKOO-NH 2 is fungicidal against C. neoformans . The peptide has antifungal activity against multiple major fungal pathogens with a minimum inhibitory concentration (MIC) of 8 μg/mL against C. neoformans and Candida glabrata , 16 μg/mL against Candida albicans and C. auris , and 32 μg/mL against Aspergillus fumigatus . The peptide has low cytotoxicity against host cells based on our hemolysis assays and vesicle leakage assays. Strikingly, the peptide exhibits strong drug synergy with multiple antifungal drugs, including amphotericin B, itraconazole, and caspofungin, depending on the specific species on which the combinations were assayed. The fluorescently labeled peptide was detected to localize to the plasma membrane, likely inhibiting key interactions of Cdc50 with membrane proteins such as P4 ATPases. Cryptococcus cells exposed to sub-MIC of peptide showed increased reactive oxygen species production and intracellular calcium levels, indicating a peptide-induced stress response. Decreased intracellular proliferation within macrophages was observed after 30 min of peptide exposure and 24 h coincubation with macrophages, providing a potential translational mechanism to explore further in vivo. In aggregate, the synergistic activity of our KKOO-NH 2 peptide may offer a potential novel candidate for combination therapy with existing antifungal drugs.

Fungal disease affects over a billion people worldwide. Naamidine A inhibits the growth of diverse fungal pathogens through an unknown mechanism. Here, we show that the supplementation of medium with excess zinc abolishes the antifungal activity of naamidine A. Furthermore, we highlight that naamidine A has in vitro activity against terbinafine-resistant Trichophyton spp . and in vivo efficacy in a mouse model of dermatomycosis caused by T. mentagrophytes, highlighting its therapeutic potential as a topical treatment., Competing Interests: L.E.C. and L.W. are co-founders and shareholders in Bright Angel Therapeutics, a platform company for the development of novel antifungal therapeutics. L.E.C. is a Science Advisor for Kapoose Creek, a company that harnesses the therapeutic potential of fungi.

Numerous Candida species are responsible for fungal infections; however, Candida albicans stands out among the others. Treatment with fluconazole is often ineffective due to the resistance phenotype mediated by transmembrane transporters and/or biofilm formation, mechanisms of resistance commonly found in C. albicans strains. A previous study by our group demonstrated that batzelladine D can inhibit the Pdr5p transporter in Saccharomyces cerevisiae . In the present study, our aim was to investigate the efficacy of batzelladine D in inhibiting the main efflux pumps of Candida albicans , CaCdr1p and CaCdr2p, as well as to evaluate the effect of the compound on C. albicans biofilm. Assays were conducted using a clinical isolate of Candida albicans expressing both transporters. Additionally, to allow the study of each transporter, S. cerevisiae mutant strains overexpressing CaCdr1p or CaCdr2p were used. Batzelladine D was able to reverse the fluconazole resistance phenotype by acting on both transporters. The compound synergistically improved the effect of fluconazole against the clinical isolate when tested in the Caenorhabditis elegans animal model. Moreover, the compound disrupted the preformed biofilm. Based on the obtained data, the continuation of batzelladine D studies as a potential new antifungal agent and/or chemosensitizer in Candida albicans infections can be suggested.

The rise in non- albicans Candida species, exhibiting unpredictable antifungal resistance, complicates treatment and contributes to the growing threat of invasive, life-threatening infections. This study evaluates the antifungal activity of four benzo[ a ]phenoxazine derivatives ( C34 , C35 , A42 , and A44 ) against 14 Candida strains following EUCAST standards. Fluconazole interactions are analysed through fractional inhibitory concentration index (FICI) calculation and response surface analysis based on the Bliss model. Macrophage-like J774A.1 cells are used to assess Candida killing in the presence of synergistic compounds. The MIC values against the different strains vary, with C34 showing the strongest activity, followed by C35 , while A42 has the highest MIC values, indicating lower efficacy. However, A42 demonstrates the best synergy with fluconazole against fluconazole-resistant Candida strains. Cytotoxicity assays reveal that the chloropropyl group present in C35 and A42 enhances cytocompatibility. Co-culture with macrophages shows significant yeast killing for C. albicans and C. auris when fluconazole and A42 are combined, requiring concentrations 4 and 16 times lower than their MIC values, enhancing antifungal activity. Given fluconazole's fungistatic nature and the emergence of drug-resistant strains, benzo[ a ]phenoxazine derivatives' ability to enhance fluconazole's efficacy present a promising strategy to address antifungal resistance in critical pathogens. These findings align with global research priorities, offering new potential avenues for developing more effective antifungal therapies.

We compared the minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of azoles in antifungal drug-susceptible, terbinafine-resistant, and lowly itraconazole (ITCZ)-susceptible strains of dermatophytes. To assess the MICs of ITCZ, ravuconazole (RVCZ), efinaconazole (EFCZ), and luliconazole (LUCZ) in the isolates, broth microdilution assays were performed based on the Clinical and Laboratory Standards Institute M38-A2 guidelines with modifications. After the assays for determining the MICs, the inoculum suspensions in wells were resuspended, then 10 μL of the growth solution in each well was inoculated onto potato dextrose agar with the use of a pipette. After 7 days of incubation at 28°C, the MFCs were determined as the lowest concentration of a drug that allowed the growth of colonies on the potato dextrose agar. The MICs in the dermatophytes were <0.03 to >32 mg/L for ITCZ, <0.03 to 4 mg/L for RVCZ, <0.03 to 2 mg/L for EFCZ, and <0.03 mg/L for LUCZ. The MFCs in the dermatophytes were 1 to >32 mg/L for ITCZ, 0.06 to >32 mg/L for RVCZ, <0.03 to 4 mg/L for EFCZ, and <0.03 to 2 mg/L for LUCZ. If the drug susceptibility test shows that the fungi are resistant to the drug, the treatment can be changed to a susceptible drug in advance, or if the fungi are low-susceptible, the treatment can be done with the recognition that it may require a longer treatment period than usual., (© 2024 Japanese Dermatological Association.)

Amphotericin B (AmB) is an antifungal agent administered for the management of serious systemic fungal infections. However, its clinical application is limited because of its water insolubility and side effects. Herein, to apply the minimum dose of AmB that can be used to manage fungal infections, a targeted drug delivery system was designed using lipopeptides and poly(lactide-co-glycolide) (PLGA). Lipopeptides conjugated with PEGylated distearoyl phosphoethanolamine (DSPE) and short peptides via a maleimide-thiol reaction formed nanosized micelles with PLGA and AmB. The antifungal effects of AmB-loaded micelles containing lipopeptides were remarkably enhanced both in vitro and in vivo. Moreover, the intravenous injection of these micelles demonstrated their in vivo targeting capacity of short peptides in a mouse model infected with drug-resistant Candida albicans. Our findings suggest that short antifungal peptides displayed on the surfaces of micelles represent a promising therapeutic candidate for targeting drug-resistant fungal infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Sarcococca hookeriana var. digyna Franch. has been widely utilized in folk medicine by the Miao people in the southwestern region of China for treating skin sores which may be associated with microbial infection., Aim of the Study: To investigate the antifungal bioactivity of S. hookeriana var. digyna against fluconazole-resistant Candida albicans in vitro and in vivo, as well as its underlying mechanism and the key bioactive component., Materials and Methods: The antifungal bioactivity of 80% ethanol extract of S. hookeriana var. digyna (SHE80) was investigated in vitro using the broth microdilution method, time-growth curve, and time-kill assay. Its key functional component and antifungal mechanism were explored with combined approaches including UPLC-Q-TOF-MS, network pharmacology and metabolomics. The antifungal pathway was further supported via microscopic observation of fungal cell morphology and examination of its effects on fungal biofilm and cell membranes using fluorescent staining reagents. In vivo assessment of antifungal bioactivity was conducted using a mouse model infected with C. albicans on the skin., Results: S. hookeriana var. digyna suppressed fluconazole-resistant C. albicans efficiently (MIC = 16 μg/mL, MFC = 64 μg/mL). It removed fungal biofilm, increased cell membrane permeability, induced protein leakage, reduced membrane fluidity, disrupted mitochondrial membrane potential, induced the release of reactive oxygen species, promoted cell apoptosis, and inhibited the transformation of fungi from the yeast state to the hyphal state significantly. In terms of mechanism, it affected sphingolipid metabolism and signaling pathway. Moreover, the predicted bioactive component, sarcovagine D, was supported by antifungal bioactivity evaluation in vitro (MIC = 4 μg/mL, MFC = 16 μg/mL). Furthermore, S. hookeriana var. digyna promoted wound healing, reduced the number of colony-forming units, and reduced inflammation effectively in vivo., Conclusions: The traditional use of S. hookeriana var. digyna for fungal skin infections was supported by antifungal bioactivity investigated in vitro and in vivo. Its mechanism and bioactive component were predicted and confirmed by experiments, which also provided a new antifungal agent for future research., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Candida species undeniably rank as the most prevalent opportunistic human fungal pathogens worldwide, with Candida albicans as the predominant representative. However, the emergence of non-albicans Candida species (NACs) has marked a significant shift, accompanied by rising incidence rates and concerning trends of antifungal resistance. The search for new strategies to combat antifungal-resistant Candida strains is of paramount importance. Recently, our research group reported the anti-Candida activity of a coordination compound containing copper(II) complexed with theophylline (theo) and 1,10-phenanthroline (phen), known as "CTP" - Cu(theo) 2 phen(H 2 O).5H 2 O. In the present work, we investigated the mechanisms of action of CTP against six medically relevant, antifungal-resistant NACs, including C. auris, C. glabrata, C. haemulonii, C. krusei, C. parapsilosis and C. tropicalis. CTP demonstrated significant efficacy in inhibiting mitochondrial dehydrogenases, leading to heightened intracellular reactive oxygen species production. CTP treatment resulted in substantial damage to the plasma membrane, as evidenced by the passive incorporation of propidium iodide, and induced DNA fragmentation as revealed by the TUNEL assay. Scanning electron microscopy images of post-CTP treatment NACs further illustrated profound alterations in the fungal surface morphology, including invaginations, cavitations and lysis. These surface modifications significantly impacted the ability of Candida cells to adhere to a polystyrene surface and to form robust biofilm structures. Moreover, CTP was effective in disassembling mature biofilms formed by these NACs. In conclusion, CTP represents a promising avenue for the development of novel antifungals with innovative mechanisms of action against clinically relevant NACs that are resistant to antifungals commonly used in clinical settings., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Candida albicans is an opportunistic pathogen commonly found in human mucous membranes. In light of the escalating challenge posed by antibiotic resistance of C. albicans strains worldwide, it is an urgently necessary to explore alternative therapeutic options., Objective: This study aims to assess the efficacy of two Cinnamaldehyde derivatives, 2-Cl Cinnamaldehyde (2-Cl CA) and 4-Cl Cinnamaldehyde (4-Cl CA), against C. albicans through both in vitro experiments and in vivo murine models and to evaluate their potential as new drug candidates for treating C. albicans., Methods and Results: The minimum inhibitory concentrations (MICs) of Cinnamaldehyde 2-Cl and 4-Cl benzene ring derivatives against C. albicans were 25 μg/mL. Time-killing experiments revealed that both Cinnamaldehyde derivatives exhibited fungicidal activity against C. albicans at concentrations of 5 MIC and 10 MIC. In the checkerboard experiment, 4-Cl CA did not show any antagonistic effect when combined with first-line antifungal drugs. Instead, it exhibited additive effects in combination with nystatin. Both 2-Cl and 4-Cl CA demonstrated inhibitory activity against C. albicans biofilm formation, especially at 8 MIC and 16 MIC concentrations. In C. albicans biofilm eradication experiments, although high drug concentrations of 2-Cl and 4-Cl CA were unable to eradicate the biofilm completely, they were still effective in killing C. albicans cells within the biofilm. Moreover, sub-inhibitory concentrations of 4-Cl CA (ranging from 5 to 20 μg/mL) significantly inhibited cell aggregation and hyphal formation. Furthermore, 4-Cl CA effectively inhibited intracellular C. albicans infection in macrophages. Lastly, the effectiveness of 4-Cl CA was evaluated in a mouse model of hematogenous disseminated candidiasis caused by C. albicans, which revealed that 4-Cl CA significantly reduced fungal burden and improved mouse survival compared to the untreated controls., Conclusion: The 4-Cl CA exhibited inhibitory effects against C. albicans through both in vivo and in vitro models, demonstrating its therapeutic potential as a promising new drug candidate for treating drug-resistant candidiasis albicans., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Candida parapsilosis was introduced as the second most responsible for nail involvement. The colonization of biotic and abiotic surfaces by Candida spp. can result in the formation of biofilms, which possess a high level of resistance to typical antifungal agents. Since Candida spp. can produce biofilm mass on the surface of the nails, dermatologists should consider appropriate antifungals to eliminate both the planktonic and biofilm cells. The aim of this research was to determine the antifungal efficacy of itraconazole against C. parapsilosis sensu lato biofilm formations, in addition to its static effects. Ten C. parapsilosis sensu lato isolates were enrolled in this study. The use of itraconazole results in the accumulation of reactive oxygen species (ROS) during treatment. In order to verify the correlation between ROS and itraconazole-induced cell death, the viability of cells was analyzed by administering the ROS scavenger Ascorbic acid. The apoptotic features of itraconazole were analyzed using the Annexin V-FITC method. Based on current data, it was found that the generation of intracellular stresses by itraconazole is not observed in cells upon ROS inhibition, emphasizing the importance of intracellular ROS in the apoptotic mechanism of itraconazole. Targeting the oxidative defense system is a powerful point to use ROS-inducing antifungals as a superior choice for more effective therapies in case of recalcitrant onychomycosis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Research on fungi under anaerobic conditions is limited but crucial for understanding their ecological and pathological impacts. Here, we present a protocol for enriching, isolating, and characterizing anaerobic fungi from environmental and clinical samples. We also describe steps for evaluating the anaerobic growth potential and drug susceptibility of fungal pathogens. This protocol can contribute to the need for initiating effective antifungal therapy to address and manage fungal infections or mycosis in oxygen-limited environments. For complete details on the use and execution of this protocol, please refer to Yadav et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Systemic infection with Candida albicans poses a significant risk for people with weakened immune systems and carries a mortality rate of up to 60%. However, current therapeutic options have several limitations, including increasing drug tolerance, notable off-target effects, and severe adverse reactions. Over the past four decades, the progress in developing drugs to treat Candida albicans infections has been sluggish. This comprehensive review addresses the limitations of existing drugs and summarizes the efforts made toward redesigning and innovating existing or novel drugs through nanotechnology. The discussion explores the potential applications of nanomedicine in Candida albicans infections from four perspectives: nano-preparations for anti-biofilm therapy, innovative formulations of "old drugs" targeting the cell membrane and cell wall, reverse drug resistance therapy targeting subcellular organelles, and virulence deprivation therapy leveraging the unique polymorphism of Candida albicans. These therapeutic approaches are promising to address the above challenges and enhance the efficiency of drug development for Candida albicans infections. By harnessing nano-preparation technology to transform existing and preclinical drugs, novel therapeutic targets will be uncovered, providing effective solutions and broader horizons to improve patient survival rates., (© 2024. The Author(s).)

Biofilms are composed of complex multi-species in nature, potentially threatening drinking water safety. In this work, the formation of single- and multi-species fungal biofilms formed by Aspergillus niger (A. niger) and Aspergillus flavus (A. flavus), and the inactivation of mature biofilms using chlor(am)ine were firstly investigated. Results revealed that the antagonistic interaction occurred between A. niger and A. flavus. Chloramination at 20 mg/L for 30 min achieved 74.74 % and 76.04 % inactivation of A. flavus and multi-species biofilm, which were 1.69- and 1.84-fold higher than that of chlorine at the same condition. However, no significant difference was observed in the inactivation of A. niger biofilm between chlorine and monochloramine disinfection due to the lower amount of extracellular polymeric substance produced by it (p > 0.05). The inactivation of biofilm by monochloramine fitted the Weibull model well. According to the Weibull model, the monochloramine resistance of biofilm were as follows: A. flavus > multi-species > A. niger biofilm. Besides, an increase in reactive oxygen levels, damage of cell membrane, and leakage of intracellular substances in biofilms were observed after chlor(am)ination. More intracellular polysaccharides and proteins were leaked in chloramination inactivation (p < 0.05). This study provides important implications for controlling fungal biofilm., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Life-threatening invasive fungal infections pose a serious threat to human health. A series of novel triazole derivatives bearing a pyrazole-methoxyl moiety were designed and synthesized in an effort to obtain antifungals with potent, broad-spectrum activity that are less susceptible to resistance. Most of these compounds exhibited moderate to excellent in vitro antifungal activities against Candida albicans SC5314 and 10,231, Cryptococcus neoformans 32,609, Candida glabrata 537 and Candida parapsilosis 22,019 with minimum inhibitory concentration (MIC) values of ≤0.125 μg/mL to 0.5 μg/mL. Use of recombinant Saccharomyces cerevisiae strains showed compounds 7 and 10 overcame the overexpression and resistant-related mutations in ERG11 of S. cerevisae and several pathogenic Candida spp. Despite being substrates of the C. albicans and Candida auris Cdr1 drug efflux pumps, compounds 7 and 10 showed moderate potency against five fluconazole (FCZ)-resistant fungi with MIC values from 2.0 μg/mL to 16.0 μg/mL. Growth kinetics confirmed compounds 7 and 10 had much stronger fungistatic activity than FCZ. For C. albicans, compounds 7 and 10 inhibited the yeast-to-hyphae transition, biofilm formation and destroyed mature biofilm more effectively than FCZ. Preliminary mechanism of action studies showed compounds 7 and 10 blocked the ergosterol biosynthesis pathway at Erg11, ultimately leading to cell membrane disruption. Further investigation of these novel triazole derivatives is also warranted by their predicted ADMET properties and low cytotoxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Candida albicans is a common commensal fungus and fourth most frequent causative agent of nosocomial infections including life-threatening invasive candidiasis in humans. The effectiveness of present antifungal therapies using azoles, polyenes, flucytosine and echinocandins has plateaued in managing fungal infections. The limitations of these antifungal drugs are related to polymorphic morphology, biofilm formation, emergence of drug-resistant strains and production of several virulence factors. Development of new antifungal agents, which can particularly afflict multiple cellular targets and limiting evolving resistant strains are needed. Recently, metal nanoparticles have emerged as a source of new antifungal agents for antifungal formulations. Furthermore, green nanotechnology deals with the use of biosynthetic routes that offer new avenue for synthesizing antifungal nanoparticles coupled with less toxic chemical inventory and environmental sustainability. This article reviews the recent developments on C. albicans pathogenesis, biofilm formation, drug resistance, mode of action of antifungal drugs and antifungal activities of metal nanoparticles. The antifungal efficacy and mode of action of metal nanoparticles are described in the context of prospective therapeutic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Candida spp. can be found in the human microbiome. However, immunocompromised patients are likely to develop invasive Candida infections, with mortality rates higher than 50%. The discovery of C. auris, a species that rapidly acquire antifungal resistance, increased the concern about Candida infections. The limited number of antifungal agents and the high incidence of resistance to them make imperative the development of new antifungal drugs. β-lapachone is a biological active naphthoquinone that displays antifungal activity against C. albicans and C. glabrata. The aim of this study was to evaluate if this substance affects C. auris growth and elucidate its mechanism of action. A fluconazole-resistant C. auris isolate was used in this study. The antifungal activity of β-lapachone was determined through microbroth dilution assays, and its mechanism of action was evaluated using fluorescent probes. Interaction with fluconazole and amphotericin B was assessed by disk diffusion assay and checkerboard. β-lapachone inhibited planktonic C. auris cell growth by 92.7%, biofilm formation by 84.9%, and decrease the metabolism of preformed biofilms by 87.1% at 100 µg/ml. At 100 µg/ml, reductions of 30% and 59% of Calcofluor White and Nile red fluorescences were observed, indicating that β-lapachone affects cell wall chitin and neutral lipids content, respectively. Also, the ratio 590 nm/529 nm of JC-1 decreased 52%, showing that the compound affects mitochondria. No synergism was observed between β-lapachone and fluconazole or amphotericin B. Data show that β-lapachone may be a promising candidate to be used as monotherapy to treat C. auris resistant infections., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Candida auris, an emerging multidrug-resistant fungal pathogen discovered in Japan in 2009, poses a significant global health threat, with infections reported in about 25 countries. The escalation of drug-resistant strains underscores the urgent need for new treatment options. This study aimed to investigate the antifungal potential of 2,3,4,4a-tetrahydro-1H-xanthen-1-one (XA1) against C. auris, as well as its mechanism of action and toxic profile. The antifungal activity of XA1 was first evaluated by determining the minimum inhibitory concentration (MIC), time-kill kinetics and biofilm inhibition. In addition, structural changes, membrane permeability, reactive oxygen species (ROS) production, and in vitro and in vivo toxicity of C. auris after exposure to XA1 were investigated. The results indicated that XA1 exhibited an MIC of 50 μg/mL against C. auris, with time-kill kinetics highlighting its efficacy. Field emission scanning electron microscopy (FE-SEM) showed structural damage in XA1-treated cells, supported by increased membrane permeability leading to cell death. Furthermore, XA1 induced ROS production and significantly inhibited biofilm formation. Importantly, XA1 exhibited low cytotoxicity in human epidermal keratinocytes (HaCaT), with a cell viability of over 90 % at 6.25 μg/mL. In addition, an LD 50 of 17.68 μg/mL was determined in zebrafish embryos 24 h post fertilization (hpf), with developmental delay observed at prolonged exposure at 6.25 μg/mL (48-96 hpf). These findings position XA1 as a promising candidate for further research and development of an effective antifungal agent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aim: To evaluate the antifungal activity of mangiferin against Candida spp. resistant to fluconazole. Materials & methods: The antifungal activity of mangiferin was assessed using broth microdilution and its interaction with azoles and amphotericin B was evaluated by checkerboard. The activity of mangiferin against Candida spp. biofilms was assessed using the MTT colorimetric assay and its possible mechanism of action was evaluated using flow cytometry. Results: Mangiferin showed activity against Candida albicans, Candida tropicalis and Candida parapsilosis resistant to fluconazole and showed synergism with azoles and amphotericin B. Mangiferin increased the activity of antifungals against Candida biofilms and caused depolarization of the mitochondrial membrane and externalization of phosphatidylserine, suggesting apoptosis. Conclusion: mangiferin combined with antifungals has potential against Candida spp.

Botrytis cinerea is a notorious pathogen causing pre- and post-harvest spoilage in many economically important crops. Excessive application of site-specific fungicides to control the pathogen has led to the selection of strains possessing target site alterations associated with resistance to these fungicides and/or strains overexpressing efflux transporters associated with multidrug resistance (MDR). MDR in B. cinerea has been correlated with the overexpression of atrB and mfsM2, encoding an ATP-binding cassette (ABC) and a major facilitator superfamily (MFS) transporter, respectively. However, it remains unknown whether other transporters may also contribute to the MDR phenotype. In the current study, the transcriptome of a B. cinerea multidrug-resistant (MDR) field strain was analysed upon exposure to the fungicide fludioxonil, and compared to the B05.10 reference strain. The transcriptome of this field strain displayed significant differences as compared to B05.10, including genes involved in sugar membrane transport, toxin production and virulence. Among the induced genes in the field strain, even before exposure to fludioxonil, were several putatively encoding ABC and MFS transmembrane transporters. Overexpression of a highly induced MFS transporter gene in the B05.10 strain led to an increased tolerance to the fungicides fluopyram and boscalid, indicating an involvement in efflux transport of these compounds. Overall, the data from this study give insights towards better understanding the molecular mechanisms involved in MDR and fitness cost, contributing to the development of more efficient control strategies against this pathogen., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Previous reports have demonstrated that the peptide derived from LfcinB, R-1-R, exhibits anti- Candida activity, which is enhanced when combined with an extract from the Bidens pilosa plant. However, the mechanism of action remains unexplored. In this research, a proteomic study was carried out, followed by a bioinformatic analysis and biological assays in both the SC5314 strain and a fluconazole-resistant isolate of Candida albicans after incubation with R-1-R. The proteomic data revealed that treatment with R-1-R led to the up-regulation of most differentially expressed proteins compared to the controls in both strains. These proteins are primarily involved in membrane and cell wall biosynthesis, membrane transport, oxidative stress response, the mitochondrial respiratory chain, and DNA damage response. Additionally, proteomic analysis of the C. albicans parental strain SC5314 treated with R-1-R combined with an ethanolic extract of B. pilosa was performed. The differentially expressed proteins following this combined treatment were involved in similar functional processes as those treated with the R-1-R peptide alone but were mostly down-regulated (data are available through ProteomeXchange with identifier PXD053558). Biological assays validated the proteomic results, evidencing cell surface damage, reactive oxygen species generation, and decreased mitochondrial membrane potential. These findings provide insights into the complex antifungal mechanisms of the R-1-R peptide and its combination with the B. pilosa extract, potentially informing future studies on natural product derivatives.

Invasive fungal infections (IFIs) caused by pathogenic fungi pose a significant public health concern, particularly for immunocompromised individuals. Mortality rates for IFIs remain high, and currently available treatment options are limited. Existing antifungal agents often suffer from limited clinical efficacy, poor fungicidal activity within the host, potential toxicity, and increasing ineffectiveness due to emerging resistance, especially against triazole drugs, the current mainstay of antifungal treatment. A recent study has identified MBX-7591, a small molecule with promising antifungal activity against Aspergillus fumigatus and other pathogenic fungi, including strains resistant to triazoles (C. Gutierrez-Perez, C. Puerner, J. T. Jones, S. Vellanki, E. M. Vesely, et al., mBio e01166-24, 2024, https://doi.org/10.1128/mbio.01166-24). This novel compound appears to inhibit stearoyl-CoA 9-desaturase, a key enzyme involved in fungal fatty acid biosynthesis. By disrupting the conversion of saturated fatty acids to oleic acid, MBX-7591 offers a unique mechanism of action, potentially reducing the risk of resistance development. Here, we now discuss the implications of these groundbreaking findings for overcoming antifungal drug resistance., Competing Interests: Dr. Maurizio Del Poeta, M.D., is a co-founder and chief scientific officer (CSO). The goal of MicroRid Technologies Inc. is to develop new anti-fungal agents for therapeutic use. Dr. Nivea Pereira de Sa declares no competing interests.

Fungal keratitis (FK) is a leading cause of preventable blindness and eye loss. The poor antifungal activity, increased drug resistance, limited corneal permeability, and unsatisfactory biosafety of conventional antifungal eye drops are among the majority of the challenges that need to be addressed for currently available antifungal drugs. Herein, this study proposes an effective strategy that employs chitosan-poly(ethylene glycol)-LK 13 peptide conjugate (CPL) in the treatment of FK. Nanoassembly CPL can permeate the lipophilic corneal epithelium in the transcellular route, and its hydrophilicity surface is a feature to drive its permeability through hydrophilic stroma. When encountering fungal cell membrane, CPL dissembles and exposes the antimicrobial peptide (LK 13 ) to destroy fungal cell membranes, the minimum inhibitory concentration values of CPL against Fusarium solani ( F. solani ) are always not to exceed 8 μg peptide/mL before and after drug resistance induction. In a rat model of Fusarium keratitis, CPL demonstrates superior therapeutic efficacy than commercially available natamycin ophthalmic suspension. This study provides more theoretical and experimental supports for the application of CPL in the treatment of FK.

In our previous study, coumarin-containing CYP51 inhibitor A32 demonstrated potent antiresistance activity. However, compound A32 demonstrated unsatisfied metabolic stability, necessitating modifications to overcome these limitations. In this study, α,β-unsaturated amides were used to replace the unstable coumarin ring, which increased metabolic stability by four times while maintaining antifungal activity, including activity against resistant strains. Subsequently, the sterol composition analysis and morphological observation experiments indicated that the target of these novel compounds is lanosterol 14α-demethylase (CYP51). Meanwhile, biofilm growth was inhibited and resistance genes ( ERG11 , CDR1 , CDR2 , and MDR1 ) expression was downregulated to find out how the antiresistance works. Importantly, compound C07 demonstrated the capacity to stimulate reactive oxygen species, thus displaying potent fungicidal activity. Moreover, C07 exhibited encouraging effectiveness in vivo following intraperitoneal administration. Additionally, the most potent compound C07 showed satisfactory pharmacokinetic properties and low toxicity. These α,β-unsaturated amide derivatives, particularly C07 , are potential candidates for treating azole-resistant candidiasis.

Aspergillus fumigatus is the primary etiological agent of aspergillosis. Here, we show that the host defense peptide mimetic brilacidin (BRI) can potentiate ibrexafungerp (IBX) against clinical isolates of A. fumigatus . BRI + IBX can inhibit the growth of A. fumigatus voriconazole- and caspofungin-resistant clinical isolates. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against viruses, bacteria, and fungi. In vitro , combination of BRI + IBX plays a fungicidal role, increases the fungal cell permeability, decreases the fungal survival in the presence of A549 epithelial cells, and appears as a promising antifungal therapeutic alternative against A. fumigatus ., Importance: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Aspergillus fumigatus causes a series of distinct invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. A. fumigatus causes a spectrum of distinct clinical entities named aspergillosis, which the most severe form is the invasive pulmonary aspergillosis. There are few therapeutic options for treating aspergillosis and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a synergizer o fibrexafungerp (IBX) against A. fumigatus . BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. We propose the combination of BRI and IBX as a new antifungal combinatorial treatment against aspergillosis., Competing Interests: G.H.G. is a scientific consultant for Innovation Pharmaceuticals Inc. The other authors declare no other competing interests.

Invasive fungal infections are rapidly increasing, and the opportunistic pathogenic Candida species are the fourth most common cause of nosocomial systemic infections. The current antifungal classes, of which azoles are the most widely used, all have shortcomings. Azoles are generally considered fungistatic rather than fungicidal, they do not actively kill fungal cells and therefore resistance against azoles can be rapidly acquired. Combination therapies with azoles provide an interesting therapeutic outlook and agents limiting iron are excellent candidates. We summarize how iron is acquired by the host and transported towards both storage and iron-utilizing organelles. We indicate whether these pathways alter azole susceptibility and/or tolerance, to finally link these transport mechanisms to mitochondrial iron availability. In this review, we highlight putative novel intracellular iron shuffling mechanisms and indicate that mitochondrial iron dynamics in relation to azole treatment and iron limitation is a significant knowledge gap., (© 2023 Federation of European Biochemical Societies.)

Vulvovaginal candidiasis (VVC) is an opportunistic infection caused by a fungus of the Candida genus, affecting approximately 75 % of women during their lifetime. Fungal resistance cases and adverse effects have been the main challenges of oral therapies. In this study, the topical application of thin films containing fluconazole (FLU) and thymol (THY) was proposed to overcome these problems. Vaginal films based only on chitosan (CH) or combining this biopolymer with pectin (PEC) or hydroxypropylmethylcellulose acetate succinate (HPMCAS) were developed by the solvent casting method. In addition to a higher swelling index, CH/HPMCAS films showed to be more plastic and flexible than systems prepared with CH/PEC or only chitosan. Biopolymers and FLU were found in an amorphous state, contributing to explaining the rapid gel formation after contact with vaginal fluid. High permeability rates of FLU were also found after its immobilization into thin films. The presence of THY in polymer films increased the distribution of FLU in vaginal tissues and resulted in improved anti-Candida activity. A significant activity against the resistant C. glabrata was achieved, reducing the required FLU dose by 50 %. These results suggest that the developed polymer films represent a promising alternative for the treatment of resistant vulvovaginal candidiasis, encouraging further studies in this context., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors also thank Professor Adailton Bortoluzzi for his help with XRD analyses., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pathogenic fungi are an increasing public health concern. The emergence of antifungal resistance coupled with the scarce antifungal arsenal highlights the need for novel therapeutics. Fortunately, the past few years have witnessed breakthroughs in antifungal development. Here, we discuss pivotal interdisciplinary approaches for the discovery of novel compounds with efficacy against diverse fungal pathogens. We highlight breakthroughs in improving current antifungal scaffolds, as well as the utility of compound combinations to extend the lifespan of antifungals. Finally, we describe efforts to refine candidate chemical scaffolds by leveraging structure-guided approaches, and the use of functional genomics to expand our knowledge of druggable antifungal targets. Overall, we emphasize the importance of interdisciplinary collaborations in the endeavor to develop innovative antifungal strategies., Competing Interests: Declaration of Interests L.E.C. is a co-founder and shareholder in Bright Angel Therapeutics, a platform company for the development of novel antifungal therapeutics, and is also a science advisor for Kapoose Creek, a company that harnesses the therapeutic potential of fungi. All other authors have no competing interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aims: Candida albicans is the most prevalent pathogenic fungus, exhibiting escalating multidrug resistance (MDR). Antimicrobial peptides (AMPs) represent promising candidates for addressing this issue. In this research, five antimicrobial peptides, ACP1 to ACP5 which named ACPs were studied as alternative fungicidal molecules., Main Methods: CD assay was used to analyze the 2D structures, Absorbance method was used to test the antimicrobial activity, haemolytic activity, time-kill kinetics, biofilm inhibition and reduction activity, resistance induction activity and assessment against fluconazole-resistant C. albicans. SEM, TEM, CLSM, flow cytometer and FM were carried out to provide insight into the mechanisms of anti-Candida action., Key Findings: ACPs possessed an α-helical structure and strong anti-Candida activities, with minimum inhibitory concentrations (MICs) from 3.9 to 15.6 μg/mL. In addition, ACPs did not produce hemolysis at concentrations lower than 10 or 62 × MIC, indicating their low cytotoxicity. Fungicidal kinetics showed that they completely killed C. albicans within 8 h at 2 to 4 × MIC. Notably, ACPs were highly fungicidal against fluconazole-resistant C. albicans and showed low resistance. In addition, they were effective in inhibiting mycelium and biofilm formation. Fluorescence microscopy revealed that while fluconazole had minimal to no inhibitory effect on biofilm-forming cells, ACPs induced apoptosis in all of them. The research on mechanism of action revealed that ACPs disrupted the cell membranes, with ROS increasing and cellular mitochondrial membrane potential decreasing., Significance: ACPs could be promising candidates for combating fluconazole-resistant C. albicans infections., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Heteroleptic metal complexes containing Cu II , Co II , and Zn II , incorporating curcumin and a Schiff base ligand (L), were synthesized and characterized, and their antifungal activity was evaluated. Their antifungal activities were investigated individually and in combination with fluconazole. Utilizing various analytical techniques such as UV-Vis, FT-IR, NMR, ESI-MS, TGA-DTG, elemental analyses, conductance, and magnetic susceptibility measurements, complex C1 ([Cu(Cur)LCl(H 2 O)]) was assigned a distorted octahedral geometry, while complexes C2 ([Co(Cur)LCl(H 2 O)]) and C3 ([Zn(Cur)LCl(H 2 O)]) were assigned octahedral geometries. Among these complexes, C2 exhibited the highest inhibitory activity against both FLC-susceptible and resistant strains of Candida albicans . Furthermore, C2 demonstrated candidicidal activity and synergistic interactions with fluconazole, effectively inhibiting the growth and survival of both FLC-resistant and FLC-sensitive C. albicans strains. The complex displayed a dose-dependent inhibition of drug efflux pumps in FLC-resistant C. albicans strains, indicating its potential to disrupt the cell membrane of these strains. The significant role of membrane efflux transporters in the development of antifungal drug resistance within Candida species has been extensively documented and our findings indicate that complex C2 specifically targets this crucial factor, thereby playing a pivotal role in mitigating drug resistance in C. albicans .

Antifungal peptides are an appealing alternative to standard antifungal medicines due to their unique mechanism of action and low-level resistance. However, their susceptibility to protease degradation keeps hindering their future development. Herein, a library was established to design peptides with protease resistance and high antifungal activity. The peptides were incorporated with minimal D-amino acids to further improve the protease stability. The most active peptide, IR3, demonstrated good antifungal activity and low toxicity, and its molecular integrity was maintained after protease hydrolysis for 8 h at 2 mg/mL. Furthermore, IR3 could permeate the fungal cell wall, disrupt the cell membrane, produce reactive oxygen species, and induce apoptosis in fungal cells. In vivo experiments confirmed that IR3 could effectively treat fungal keratitis. Collectively, these findings suggest that IR3 is a promising antifungal agent and may be beneficial in the design and development of protease-resistant antifungal peptides.

Introduction. The development of new antifungal drugs has become a global priority, given the increasing cases of fungal diseases together with the rising resistance to available antifungal drugs. In this scenario, drug repositioning has emerged as an alternative for such development, with advantages such as reduced research time and costs. Gap statement. Propafenone is an antiarrhythmic drug whose antifungal activity is poorly described, being a good candidate for further study. Aim. This study aims to evaluate propafenone activity against different species of Candida spp. to evaluate its combination with standard antifungals, as well as its possible action mechanism. Methodology. To this end, we carried out tests against strains of Candida albicans , Candida auris , Candida parapsilosis , Candida tropicalis , Candida glabrata and Candida krusei based on the evaluation of the MIC, minimum fungicidal concentration and tolerance level, along with checkerboard and flow cytometry tests with clinical strains and cell structure analysis by scanning electron microscopy (SEM). Results. The results showed that propafenone has a 50% MIC ranging from 32 to 256 µg ml -1 , with fungicidal activity and positive interactions with itraconazole in 83.3% of the strains evaluated. The effects of the treatments observed by SEM were extensive damage to the cell structure, while flow cytometry revealed the apoptotic potential of propafenone against Candida spp. Conclusion. Taken together, these results indicate that propafenone has the potential for repositioning as an antifungal drug.

Aims: Azoles have been widely employed for the treatment of invasive fungal diseases; however, their efficacy is diminished as pathogenic fungi tolerate them due to their fungistatic properties. Geldanamycin (GdA) can render azoles fungicidal by inhibiting the ATPase and molecular chaperone activities of heat shock protein 90 (Hsp90). Nonetheless, the clinical applicability of GdA is restricted due to its cytotoxic ansamycin scaffold structure, its induction of cytoprotective heat shock responses, and the conservative nature of Hsp90. Hence, it is imperative to elucidate the mechanism of action of GdA to confer fungicidal properties to azoles and mitigate the toxic adverse effects associated with GdA., Materials and Methods: Through various experimental methods, including the construction of gene-deleted Candida albicans mutants, in vitro drug sensitivity experiments, Western blot analysis, reactive oxygen species (ROS) assays, and succinate dehydrogenase activity assays, we identified Hsp90 client proteins associated with the tolerance of C. albicans to azoles., Key Findings: It was observed that GdA effectively hindered the entry of Hsp90 into mitochondria, resulting in the alleviation of inhibitory effect of Hsp90 on succinate dehydrogenase. Consequently, the activation of succinate dehydrogenase led to an increased production of ROS. within the mitochondria, thereby facilitating the antifungal effects of azoles against C. albicans., Significance: This research presents a novel approach for conferring fungicidal properties to azoles, which involves specifically disrupting the interaction of between Hsp90 and succinate dehydrogenase rather than employing a non-specific inhibition of ATPase activity of Hsp90., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Objectives: This study investigated the anti-cryptococcal potential of certain essential oils (EOs)/compounds alone and in combination with fluconazole., Materials and Methods: We investigated the antifungal activity of oils of Cinnamomum verum, Cymbopogon citratus, Cymbopogon martini, and Syzygium aromaticum, and their major active ingredients cinnamaldehyde, citral, eugenol, and geraniol against clinical and standard strains of Cryptococcus neoformans (CN). Disc diffusion, broth microdilution, checkerboard methods, and transmission electron microscopy were employed to determine growth inhibition, synergistic interaction, and mechanism of action of test compounds., Results: EOs/compounds showed pronounced antifungal efficacy against azole-resistant CN in the order of cinnamaldehyde > eugenol > S. aromaticum > C. verum > citral > C. citratus > geraniol ≥ C. martini, each exhibiting zone of inhibition >15 mm. These oils/compounds were highly cidal compared to fluconazole. Eugenol and cinnamaldehyde showed the strongest synergy with fluconazole against CN by lowering their MICs up to 32-fold. Transmission electron microscopy indicated damage of the fungal cell wall, cell membrane, and other endomembranous organelles., Conclusion: Test oils and their active compounds exhibited potential anti-cryptococcus activity against the azole-resistant strains of CN. Moreover, eugenol and cinnamaldehyde significantly potentiated the anti-cryptococcal activity of fluconazole. It is suggested that multiple sites of action from oils/compounds could turn static fluconazole into a cidal drug combination in combating cryptococcosis., (Copyright © 2024 Copyright: © 2024 Annals of African Medicine.)