Sterol regulatory element-binding proteins mediate intrinsic fungicide tolerance and antagonism in the fungal biocontrol agent Clonostachys rosea IK726.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors governing various biological processes in fungi, including virulence and fungicide tolerance, by regulating ergosterol biosynthesis and homeostasis. While studied in model fungal species, their role in fungal species used for biocontrol remains elusive. This study delves into the biological and regulatory function of SREBPs in the fungal biocontrol agent (BCA) Clonostachys rosea IK726, with a specific focus on fungicide tolerance and antagonism. Clonostachys rosea genome contains two SREBP coding genes (sre1 and sre2) with distinct characteristics. Deletion of sre1 resulted in mutant strains with pleiotropic phenotypes, including reduced C. rosea growth on medium supplemented with prothioconazole and boscalid fungicides, hypoxia mimicking agent CoCl2 and cell wall stressor SDS, and altered antagonistic abilities against Botrytis cinerea and Rhizoctonia solani. However, Δsre2 strains showed no significant effect. Consistent with the gene deletion results, overexpression of sre1 in Saccharomyces cerevisiae enhanced tolerance to prothioconazole. The functional differentiation between SRE1 and SRE2 was elucidated by the yeast-two-hybridization assay, which showed an interaction between SREBP cleavage-activating protein (SCAP) and SRE1 but not between SRE2 and SCAP. Transcriptome analysis of the Δsre1 strain unveiled SRE1-mediated expression regulation of genes involved in lipid metabolism, respiration, and xenobiotic tolerance. Notably, genes coding for antimicrobial compounds chitinases and polyketide synthases were downregulated, aligning with the altered antagonism phenotype. This study uncovers the role of SREBPs in fungal BCAs, providing insights for C. rosea IK726 application into integrated pest management strategies.

Are there conserved biosynthetic genes in lichens? Genome-wide assessment of terpene biosynthetic genes suggests ubiquitous distribution of the squalene synthase cluster.

Lichen-forming fungi (LFF) are prolific producers of functionally and structurally diverse secondary metabolites, most of which are taxonomically exclusive and play lineage-specific roles. To date, widely distributed, evolutionarily conserved biosynthetic pathways in LFF are not known. However, this idea stems from polyketide derivatives, since most biochemical research on lichens has concentrated on polyketide synthases (PKSs). Here, we present the first systematic identification and comparison of terpene biosynthetic genes of LFF using all the available Lecanoromycete reference genomes and 22 de novo sequenced ones (111 in total, representing 60 genera and 23 families). We implemented genome mining and gene networking approaches to identify and group the biosynthetic gene clusters (BGCs) into networks of similar BGCs. Our large-scale analysis led to the identification of 724 terpene BGCs with varying degrees of pairwise similarity. Most BGCs in the dataset were unique with no similarity to a previously known fungal or bacterial BGC or among each other. Remarkably, we found two BGCs that were widely distributed in LFF. Interestingly, both conserved BGCs contain the same core gene, i.e., putatively a squalene/phytoene synthase (SQS), involved in sterol biosynthesis. This indicates that early gene duplications, followed by gene losses/gains and gene rearrangement are the major evolutionary factors shaping the composition of these widely distributed SQS BGCs across LFF. We provide an in-depth overview of these BGCs, including the transmembrane, conserved, variable and LFF-specific regions. Our study revealed that lichenized fungi do have a highly conserved BGC, providing the first evidence that a biosynthetic gene may constitute essential genes in lichens.

Design and synthesis of novel mitochondria-targeted ergosterol peroxide derivatives as potential anti-cancer agents.

Ergosterol peroxide (EP) is a natural steroid compound that has been reported to have significant antitumor activity. However, its poor water solubility and cellular uptake mean that it has weak efficacy against tumor cells. Herein, we designed and synthesized a series of EP derivatives with mitochondrial targeting properties. Of these, compound 15a showed an IC50 value of 0.32 µM against MCF-7 cells, which was 67-fold higher than that of the parental EP (IC50 = 21.46 µM), and was better than cisplatin (IC50 = 4.23 µM), had a selectivity index of 25.28 (IC50MCF-10A/IC50MCF-7). Additionally, compound 15a promoted an increase in intracellular reactive oxygen species levels and a decrease in mitochondrial membrane potential, and blocked the cell cycle in the G0/G1 phase. In a mouse model of breast cancer, 15a showed 89.85 % tumor inhibition at a dose of 20 mg/kg, which is similar to the therapeutic effect of the cisplatin. On the basis of these results, 15a could be considered for further preclinical evaluation for cancer therapy.

Uncovering the Hidden Potential of Phytoene Production by the Fungus Blakeslea trispora.

Phytoene is an uncommon linear carotene within the carotenoid group as it is colorless due to its short chromophore. Recent research constitutes a relatively new area which has emerged from phytoene's importance as a major dietary carotenoid promoting health and appearance. Its resources point to the potential of biotechnological production systems. Our work has been designed to study the efficacy of two colored carotenoid biosynthesis inhibitors, diphenylamine and 2-methyl imidazole, and one sterol biosynthesis inhibitor, terbinafine, to modify the metabolic flux in mated cultures of Blakeslea trispora to achieve maximum phytoene production. Bioprocess kinetics optimized by response surface methodology and monitored by high-performance liquid chromatography revealed maximum phytoene content (5.02 mg/g dry biomass) and yield (203.91 mg/L culture medium) comparable or even higher than those reported for other potent phytoene microbial producers. The in vivo antioxidant activity of phytoene-rich carotenoid extract from fungal cells was also considered and discussed.

Inhibitory Effect of Sorbus aucuparia Extracts on the Fusarium proliferatum and F. culmorum Growth and Mycotoxin Biosynthesis.

Fungal infections are among the most common diseases of crop plants. Various species of the Fusarium spp. are naturally prevalent and globally cause the qualitative and quantitative losses of farming commodities, mainly cereals, fruits, and vegetables. In addition, Fusarium spp. can synthesize toxic secondary metabolites-mycotoxins under high temperature and humidity conditions. Among the strategies against Fusarium spp. incidence and mycotoxins biosynthesis, the application of biological control, specifically natural plant extracts, has proved to be one of the solutions as an alternative to chemical treatments. Notably, rowanberries taken from Sorbus aucuparia are a rich source of phytochemicals, such as vitamins, carotenoids, flavonoids, and phenolic acids, as well as minerals, including iron, potassium, and magnesium, making them promising candidates for biological control strategies. The study aimed to investigate the effect of rowanberry extracts obtained by supercritical fluid extraction (SFE) under different conditions on the growth of Fusarium (F. culmorum and F. proliferatum) and mycotoxin biosynthesis. The results showed that various extracts had different effects on Fusarium growth as well as ergosterol content and mycotoxin biosynthesis. These findings suggest that rowanberry extracts obtained by the SFE method could be a natural alternative to synthetic fungicides for eradicating Fusarium pathogens in crops, particularly cereal grains. However, more research is necessary to evaluate their efficacy against other Fusarium species and in vivo applications.

Metconazole inhibits fungal growth and toxin production in major Fusarium species that cause rice panicle blight.

Rice panicle blight (RPB) caused by various Fusarium spp. is an emerging disease in the major rice-growing regions of China. Epidemics of this disease cause significant yield loss and reduce grain quality by contaminating panicles with different Fusarium toxins. However, there is currently no registered fungicide for the control of RPB in China. The 14α-demethylation inhibitor (DMI) fungicide metconazole has been shown to be effective against several Fusarium spp. that cause wheat head blight, wheat crown rot and maize ear rot. In this study, we investigated the specific activity of metconazole against six Fusarium spp. that cause RPB. Metconazole significantly inhibited mycelial growth, conidium formation, germination, germ tube elongation and major toxin production in Fusarium strains collected from major rice-growing regions in China, as well as disrupting cell membrane function by inhibiting ergosterol biosynthesis. Greenhouse experiments indicated a significant reduction in blight occurrence and toxin accumulation in rice panicles treated with metconazole. Overall, our study demonstrated the potential of metconazole for managing RPB and toxin contamination, as well as providing insight into its bioactivities and modes of action of metconazole against distinct Fusarium spp.

Discovery and Optimization of Ergosterol Peroxide Derivatives as Novel Glutaminase 1 Inhibitors for the Treatment of Triple-Negative Breast Cancer.

In this study, novel ergosterol peroxide (EP) derivatives were synthesized and evaluated to assess their antiproliferative activity against four human cancer cell lines (A549, HepG2, MCF-7, and MDA-MB-231). Compound 3g exhibited the most potent antiproliferative activity, with an IC50 value of 3.20 µM against MDA-MB-231. This value was 5.4-fold higher than that of the parental EP. Bioassay optimization further identified 3g as a novel glutaminase 1 (GLS1) inhibitor (IC50 = 3.77 µM). In MDA-MB-231 cells, 3g reduced the cellular glutamate levels by blocking the glutamine hydrolysis pathway, which triggered reactive oxygen species production and induced caspase-dependent apoptosis. Molecular docking indicated that 3g interacts with the reaction site of the variable binding pocket by forming multiple interactions with GLS1. In a mouse model of breast cancer, 3g showed remarkable therapeutic effects at a dose of 50 mg/kg, with no apparent toxicity. Based on these results, 3g could be further evaluated as a novel GLS1 inhibitor for triple-negative breast cancer (TNBC) therapy.

The Kelch Repeat Protein VdKeR1 Is Essential for Development, Ergosterol Metabolism, and Virulence in Verticillium dahliae.

Verticillium dahliae is a soil-borne fungal pathogen that can cause severe vascular wilt in many plant species. Kelch repeat proteins are essential for fungal growth, resistance, and virulence. However, the function of the Kelch repeat protein family in V. dahliae is unclear. In this study, a Kelch repeat domain-containing protein DK185_4252 (VdLs.17 VDAG_08647) included in the conserved VdPKS9 gene cluster was identified and named VdKeR1. Phylogenetic analysis demonstrated a high degree of evolutionary conservation of VdKeR1 and its homologs among fungi. The experimental results showed that the absence of VdKeR1 impaired vegetative growth, microsclerotia development, and pathogenicity of V. dahliae. Osmotic and cell wall stress analyses suggested that VdKeR1-deleted mutants were more tolerant to NaCl, sorbitol, CR, and CFW, while more sensitive to H2O2 and SDS. In addition, analyses of the relative expression level of sqe and the content of squalene and ergosterol showed that VdKeR1 mediates the synthesis of squalene and ergosterol by positively regulating the activity of squalene epoxidase. In conclusion, these results indicated that VdKeR1 was involved in the growth, stress resistance, pathogenicity, and ergosterol metabolism of V. dahliae. Investigating VdKeR1 provided theoretical and experimental foundations for subsequent control of Verticillium wilt.

Deletion of PMP3 increases ketoconazole resistance by affecting plasma membrane potential in Candida albicans.

Ketoconazole is a classical antifungal drug commonly used in the clinic. With the increased use of ketoconazole in recent years, an increasing number of drug-resistant strains have emerged during clinical treatment. It is well known that fungi acquire drug resistance in multiple ways, while the molecular mechanisms underlying ketoconazole resistance remain for comprehensive exploration. In this study, we found that the expression of the small plasma membrane protein-encoding gene PMP3 was significantly down-regulated in several clinically isolated ketoconazole-resistant strains, indicating the relationship between PMP3 expression and ketoconazole resistance. By knocking out the PMP3, we found that the absence of the Pmp3 resulted in a significant increase in resistance of Candida albicans to ketoconazole, which was also confirmed in a systemic infection model in mice. We further demonstrated that various physiological properties, such as cell membrane fluidity, plasma membrane potential, permeability and ergosterol distribution were altered in the pmp3Δ/Δ mutant, which is associated with the enhanced cellular resistance to ketoconazole. In addition, overexpression rather than deletion of PMP3 alters the hyphal development and biofilm formation capacity in C. albicans. This study reveals the contribution of Pmp3 to alteration of drug resistance in fungal pathogens, which may guide the development of novel antifungal strategies.

[Anti-breast cancer effect of a mitochondrion-targeted derivative of ergosterol peroxide in vitro and in vivo].

This study aims to explore the effect and mechanism of a mitochondrion-targeted derivative of ergosterol peroxide(Mito-EP) on breast cancer. The methyl thiazolyl tetrazolium(MTT) assay was employed to examine the proliferation of MDA-MB-231 cells treated with different concentrations(0, 0.075, 0.15, 0.3, 0.6, 1.2, and 2.4 µmol·L~(-1)) of Mito-EP. Cells were grouped for treatment with water(blank control), low, medium, and high concentrations(0.15, 0.3, and 0.6 µmol·L~(-1)) of Mito-EP, and ergosterol peroxide(EP)(0.6 µmol·L~(-1)). After the cells were treated for 48 h, flow cytometry was employed to examine the apoptosis rate, reactive oxygen species(ROS) level, mitochondrial membrane potential, and cell cycle distribution, and the apoptosis, ROS, and mitochondrial membrane potential were observed by laser confocal microscopy. A mouse model bearing subcutaneous xenograft tumor was established by injecting 4T1 cell suspension and used to study the inhibitory effect of Mito-EP on breast cancer. Western blot was employed to determine the protein levels of B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), cytochrome C(Cyt C), cleaved caspase-7, and cleaved caspase-9 in cells and the tumor tissue. The results showed that Mito-EP reduced the proliferation rate of MDA-MB-231 cells in a concentration-dependent manner. Compared with the blank control group, EP(0.6 µmol·L~(-1)) caused slight changes in the apoptosis rate, ROS level, and mitochondrial membrane potential. However, Mito-EP increased the apoptosis rate, elevated the ROS level, decreased mitochondrial membrane potential, up-regulated the protein levels of Bax, Cyt C, cleaved caspase-7, and cleaved caspase-9, and down-regulated the protein level of Bcl-2(all P<0.05). Moreover, Mito-EP reduced the tumor volume and weight. In summary, Mito-EP may promote apoptosis in breast cancer cells by activating the mitochondrial apoptosis pathway.

Exploring the antifungal potential of novel carbazate derivatives as promising drug candidates against emerging superbug, Candida auris.

Candida auris (C. auris) has caused notable outbreaks across the globe in last decade and emerged as a life-threatening human pathogenic fungus. Despite significant advances in antifungal research, the drug resistance mechanisms in C. auris still remain elusive. Under such pressing circumstances, research on identification of new antifungal compounds is of immense interest. Thus, our studies aimed at identifying novel drug candidates and elucidate their biological targets in C. auris. After screening of several series of synthetic and hemisynthetic compounds from JUNIA chemical library, compounds C4 (butyl 2-(4-chlorophenyl)hydrazine-1-carboxylate) and C13 (phenyl 2-(4-chlorophenyl) hydrazine-1-carboxylate), belonging to the carbazate series, were identified to display considerable antifungal activities against C. auris as well as its fluconazole resistant isolates. Elucidation of biological targets revealed that C4 and C13 lead to changes in polysaccharide composition of the cell wall and disrupt vacuole homeostasis. Mechanistic insights further unravelled inhibited efflux pump activities of ATP binding cassette transporters and depleted ergosterol content. Additionally, C4 and C13 cause mitochondrial dysfunction and confer oxidative stress. Furthermore, both C4 and C13 impair biofilm formation in C. auris. The in vivo efficacy of C4 and C13 were demonstrated in Caenorhabditis elegans model after C. auris infection showing reduced mortality of the nematodes. Together, promising antifungal properties were observed for C4 and C13 against C. auris that warrant further investigations. To summarise, collected data pave the way for the design and development of future first-in-class antifungal drugs.

Histone deacetylase HDAC3 regulates ergosterol production for oxidative stress tolerance in the entomopathogenic and endophytic fungus Metarhizium robertsii.

Oxidative stress is encountered by fungi in almost all niches, resulting in fungal degeneration or even death. Fungal tolerance to oxidative stress has been extensively studied, but the current understanding of the mechanisms regulating oxidative stress tolerance in fungi remains limited. The entomopathogenic and endophytic fungus Metarhizium robertsii encounters oxidative stress when it infects insects and develops a symbiotic relationship with plants, and we found that host reactive oxygen species (ROSs) greatly limited fungal growth in both insects and plants. We identified a histone H3 deacetylase (HDAC3) that catalyzed the deacetylation of lysine 56 of histone H3. Deleting Hdac3 significantly reduced the tolerance of M. robertsii to oxidative stress from insects and plants, thereby decreasing fungal ability to colonize the insect hemocoel and plant roots. HDAC3 achieved this by regulating the expression of three genes in the ergosterol biosynthesis pathway, which includes the lanosterol synthase gene Las1. The deletion of Hdac3 or Las1 reduced the ergosterol content and impaired cell membrane integrity. This resulted in an increase in ROS accumulation in fungal cells that were thus more sensitive to oxidative stress. We further showed that HDAC3 regulated the expression of the three ergosterol biosynthesis genes in an indirect manner. Our work significantly advances insights into the epigenetic regulation of oxidative stress tolerance and the interactions between M. robertsii and its plant and insect hosts.IMPORTANCEOxidative stress is a common challenge encountered by fungi that have evolved sophisticated mechanisms underlying tolerance to this stress. Although fungal tolerance to oxidative stress has been extensively investigated, the current understanding of the mechanisms for fungi to regulate oxidative stress tolerance remains limited. In the model entomopathogenic and plant symbiotic fungus Metarhizium robertsii, we found that the histone H3 deacetylase HDAC3 regulates the production of ergosterol, an essential cell membrane component. This maintains the cell membrane integrity to resist the oxidative stress derived from the insect and plant hosts for successful infection of insects and development of symbiotic associates with plants. Our work provides significant insights into the regulation of oxidative stress tolerance in M. robertsii and its interactions with insects and plants.

Phospholipid biosynthesis regulation for improving pigment production by Monascus in response to ammonium chloride stress.

In the actual industrial production process, the efficient biosynthesis and secretion of Monascus pigments (MPs) tend to take place under abiotic stresses, which often result in an imbalance of cell homeostasis. The present study aimed to thoroughly describe the changes in lipid profiles in Monascus purpureus by absolute quantitative lipidomics and tandem mass tag-based quantitative proteomics. The results showed that ammonium chloride stress (15 g/L) increased MP production while inhibiting ergosterol biosynthesis, leading to an imbalance in membrane lipid homeostasis in Monascus. In response to the imbalance of lipid homeostasis, the regulation mechanism of phospholipids in Monascus was implemented, including the inhibition of lysophospholipids production, maintenance of the ratio of PC/PE, and improvement of the biosynthesis of phosphatidylglycerol, phosphatidylserine, and cardiolipin with high saturated and long carbon chain fatty acids through the CDP-DG pathway rather than the Kennedy pathway. The inhibition of lysophospholipid biosynthesis was attributed to the upregulated expression of protein and its gene related to lysophospholipase NTE1, while maintenance of the PC/PE ratio was achieved by the upregulated expression of protein and its gene related to CTP: phosphoethanolamine cytidylyltransferase and phosphatidylethanolamine N-methyltransferase in the Kennedy pathway. These findings provide insights into the regulation mechanism of MP biosynthesis from new perspectives.IMPORTANCEMonascus is important in food microbiology as it produces natural colorants known as Monascus pigments (MPs). The industrial production of MPs has been achieved by liquid fermentation, in which the nitrogen source (especially ammonium chloride) is a key nutritional parameter. Previous studies have investigated the regulatory mechanisms of substance and energy metabolism, as well as the cross-protective mechanisms in Monascus in response to ammonium chloride stress. Our research in this work demonstrated that ammonium chloride stress also caused an imbalance of membrane lipid homeostasis in Monascus due to the inhibition of ergosterol biosynthesis. We found that the regulation mechanism of phospholipids in Monascus was implemented, including inhibition of lysophospholipids production, maintenance of the ratio of PC/PE, and improvement of biosynthesis of phosphatidylglycerol, phosphatidylserine, and cardiolipin with high saturated and long carbon chain fatty acids through the CDP-DG pathway. These findings further refine the regulatory mechanisms of MP production and secretion.

Novel targets and improved immunotherapeutic techniques with an emphasis on antimycosal drug resistance for the treatment and management of mycosis.

Infections due to pathogenic fungi are endemic in particular area with increased morbidity and mortality. More than a thousand people are infected per year and the way of treatment is of high demand having a significant impact on the population health. Medical practitioners confront various troublesome analytic and therapeutical challenges in the administration of immunosuppressed sufferer at high danger of expanding fungal infections. An upgraded antimycosal treatment is fundamental for a fruitful result while treating intrusive mycoses. A collection of antimycosal drugs keeps on developing with their specific antifungal targets including cell membrane, mitochondria, cell wall, and deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) or protein biosynthesis. Some fundamental classes of ordinarily directed medications are the polyenes, amphotericin B, syringomycin, allylamines, honokiol, azoles, flucytosine, echinocandins etc. However, few immunotherapy processes and vaccinations are being developed to mark this need, although one presently can't seem to arrive at the conclusion. In this review article, there has been a trial to give details upgradation about the current immune therapeutic techniques and vaccination strategies against prevention or treatment of mycosis as well as the difficulties related with their turn of events. There has been also a visualization in the mentioned review paper about the various assorted drugs and their specific target analysis along with therapeutic interventions.

Mounting an immune response reduces male attractiveness in a lizard.

Parasites impact host fitness and constitute an important selective pressure on the host's life history. According to parasite-mediated sexual selection, ornaments are presumed to honestly indicate immune capacity or resistance against parasites, and the chooser sex (typically females) obtains an advantage by selecting more ornamented, thus more immunocompetent mates. Therefore, signalers mounting an immune response must allocate resources from the sexual signal to the immune system, hence reducing the expression of the ornament and becoming less attractive to the choosing sex. Here, we test this idea in the lizard Psammodromus algirus. We inoculated a subsample of males with lipopolysaccharide (LPS) of the cell wall of Escherichia coli, while others served as sham controls. The inoculation of LPS decreased the proportion of ergosterol (pro-vitamin D2) in femoral secretions, and chemosensory tests showed that the scent of LPS-inoculated males was less attractive to females than the scent of control males. Given that ergosterol is a precursor of vitamin D, which has physiological functions as an immune modulator, immunocompromised males likely needed to divert vitamin D to the immune system, reducing the allocation of ergosterol to secretions. In this way, females could detect "sick" males, preferring the apparently healthy males. Overall, our study shows that mounting an immune response is costly in terms of reduced attractiveness. Moreover, we disentangle the underlying mechanism, which involves an honest signal based on vitamin D allocation.

Genetic algorithm multiple linear regression and machine learning-driven QSTR modeling for the acute toxicity of sterol biosynthesis inhibitor fungicides.

Sterol Biosynthesis Inhibitors (SBIs) are a major class of fungicides used globally. Their widespread application in agriculture raises concerns about potential harm and toxicity to non-target organisms, including humans. To address these concerns, a quantitative structure-toxicity relationship (QSTR) modeling approach has been developed to assess the acute toxicity of 45 different SBIs. The genetic algorithm (GA) was used to identify key molecular descriptors influencing toxicity. These descriptors were then used to build robust QSTR models using multiple linear regression (MLR), support vector regression (SVR), and artificial neural network (ANN) algorithms. The Cross-validation, Y-randomization test, applicability domain methods, and external validation were carried out to evaluate the accuracy and validity of the generated models. The MLR model exhibited satisfactory predictive performance, with an R2 of 0.72. The SVR and ANN models obtained R2 values of 0.7 and 0.8, respectively. ANN model demonstrated superior performance compared to other models, achieving R2 cv and R2 test values of 0.74 and 0.7, respectively. The models passed both internal and external validation, indicating their robustness. These models offer a valuable tool for risk assessment, enabling the evaluation of potential hazards associated with future applications of SBIs.

Adaptative responses of Neurospora crassa by histidine kinases upon the attack of the arthropod Sinella curviseta.

Histidine kinases (HKs) are important sensor proteins in fungi and play an essential role in environmental adaptation. However, the mechanisms by which fungi sense and respond to fungivores attack via HKs are not fully understood. In this study, we utilized Neurospora crassa to investigate the involvement of HKs in responding to fungivores attack. We found that the 11 HKs in N. crassa not only affected the growth and development, but also led to fluctuations in antioxidant production. Ten mutants in the genes encoding HKs (except ∆phy1) showed increased production of reactive oxygen species (ROS), especially upon Sinella curviseta attack. The ROS burst triggered changes in conidia and perithecial beaks formation, as well as accumulation of ß-glucan, ergothioneine, ergosterol, and carotenoids. ß-glucan was increased in ∆hk9, ∆os1, ∆hcp1, ∆nik2, ∆sln1, ∆phy1 and ∆phy2 mutants compared to the wild-type strain. In parallel, ergothioneine accumulation was improved in ∆phy1 and ∆hk16 mutants and further increased upon attack, except in ∆os1 and ∆hk16 mutants. Additionally, fungivores attack stimulated ergosterol and dehydroergosterol production in ∆hk9 and ∆os1 mutants. Furthermore, deletion of these genes altered carotenoid accumulation, with wild-type strain, ∆hk9, ∆os1, ∆hcp1, ∆sln1, ∆phy2, and ∆dcc1mutants showing an increase in carotenoids upon attack. Taken together, HKs are involved in regulating the production of conidia and antioxidants. Thus, HKs may act as sensors of fungivores attack and effectively improve the adaptive capacity of fungi to environmental stimuli.

With age comes resilience: how mitochondrial modulation drives age-associated fluconazole tolerance in Cryptococcus neoformans.

Cryptococcus neoformans (Cn) is an opportunistic fungal microorganism that causes life-threatening meningoencephalitis. During the infection, the microbial population is heterogeneously composed of cells with varying generational ages, with older cells accumulating during chronic infections. This is attributed to their enhanced resistance to phagocytic killing and tolerance of antifungals like fluconazole (FLC). In this study, we investigated the role of ergosterol synthesis, ATP-binding cassette (ABC) transporters, and mitochondrial metabolism in the regulation of age-dependent FLC tolerance. We find that old Cn cells increase the production of ergosterol and exhibit upregulation of ABC transporters. Old cells also show transcriptional and phenotypic characteristics consistent with increased metabolic activity, leading to increased ATP production. This is accompanied by increased production of reactive oxygen species, which results in mitochondrial fragmentation. This study demonstrates that the metabolic changes occurring in the mitochondria of old cells drive the increase in ergosterol synthesis and the upregulation of ABC transporters, leading to FLC tolerance. IMPORTANCE: Infections caused by Cryptococcus neoformans cause more than 180,000 deaths annually. Estimated 1-year mortality for patients receiving care ranges from 20% in developed countries to 70% in developing countries, suggesting that current treatments are inadequate. Some fungal cells can persist and replicate despite the usage of current antifungal regimens, leading to death or treatment failure. Aging in fungi is associated with enhanced tolerance against antifungals and resistance to killing by host cells. This study shows that age-dependent increase in mitochondrial reactive oxygen species drive changes in the regulation of membrane transporters and ergosterol synthesis, ultimately leading to the heightened tolerance against fluconazole in old C. neoformans cells. Understanding the underlying molecular mechanisms of this age-associated antifungal tolerance will enable more targeted antifungal therapies for cryptococcal infections.

Design, Synthesis, and Bioactivity Determination of Novel Malononitrile Derivatives Containing 1,2,3-Triazole.

Using antifungal agrochemicals as the most economical solution might reduce plant diseases caused by pathogenic fungi, which have a significant negative impact on the quality and yield of food worldwide. In this work, 33 compounds (G) containing 1,2,3-triazole and malononitrile structures were synthesized. When the compounds were tested in vitro against six fungal species, they exhibited significant fungicidal activity toward Botrytis cinerea and Rhizoctonia solani. Compounds G17 and G30 displayed promising in vivo efficacy, with an EC50 of 0.19 and 0.27 mg/L respectively against R. solani. Fungal ergosterol production was suppressed by compounds G17 and G30, according to a preliminary analysis of their mechanism of action on R. solani using transcriptomics and scanning electron microscopy. It has been shown through experimentation that compounds G17 and G30 prevent R. solani from synthesizing ergosterol. Ultimately, it was anticipated that compounds G17 and G30 would be discovered to be low-toxic.

Vidarabine as a novel antifungal agent against Candida albicans: insights on mechanism of action.

Around 1.5 million mortality cases due to fungal infection are reported annually, posing a massive threat to global health. However, the effectiveness of current antifungal therapies in the treatment of invasive fungal infections is limited. Repurposing existing antifungal drugs is an advisable alternative approach for enhancing their effectiveness. This study evaluated the antifungal efficacy of the antiviral drug vidarabine against Candida albicans ATCC 90028. Antifungal susceptibility testing was performed by microbroth dilution assay and further processed to find the minimum fungicidal concentration. Investigation on probable mode of vidarabine action against C. albicans was assessed by using the ergosterol reduction assay, reactive oxygen species (ROS) accumulation, nuclear condensation, and apoptosis assay. Results revealed that C. albicans was susceptible to vidarabine action and exhibited minimum inhibitory concentration at 150 µg/ml. At a concentration of 300 µg/ml, vidarabine had fungicidal activity against C. albicans. 300 µg/ml vidarabine-treated C. albicans cells demonstrated 91% reduced ergosterol content. Annexin/FITC/PI assay showed that vidarabine (150 µg/ml) had increased late apoptotic cells up to 31%. As per the fractional inhibitory concentration index, vidarabine had synergistic activity with fluconazole and caspofungin against this fungus. The mechanism underlying fungicidal action of vidarabine was evaluated at the intracellular level, and probably because of increased nuclear condensation, enhanced ROS generation, and cell cycle arrest. In conclusion, this data is the first to report that vidarabine has potential to be used as a repurposed antifungal agent alone or in combination with standard antifungal drugs, and could be a quick and safe addition to existing therapies for treating fungal infections.