Ergosterol Synthesis Research Articles

Increasing carotenoid production in Xanthophyllomyces dendrorhous/Phaffia rhodozyma: SREBP pathway activation and promoter engineering

The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin, a high-value carotenoid with biotechnological relevance in the nutraceutical and aquaculture industries. However, enhancing carotenoid production through strain engineering remains an ongoing challenge. Recent studies have demonstrated that carotenogenesis in X. dendrorhous is regulated by the SREBP pathway, which includes the transcription factor Sre1, particularly in the mevalonate pathway that also produces precursors used for ergosterol synthesis. In this study, we explored a novel approach to enhance carotenoid synthesis by replacing the native crtE promoter, which drives geranylgeranyl pyrophosphate synthesis (the step where carotenogenesis diverges from ergosterol biosynthesis), with the promoter of the HMGS gene, which encodes 3-hydroxy-3-methylglutaryl-CoA synthase from the mevalonate pathway. The impact of this substitution was evaluated in two mutant strains that already overproduce carotenoids due to the presence of an active Sre1 transcription factor: CBS.cyp61-, which does not produce ergosterol and strain CBS.SRE1N.FLAG, which constitutively expresses the active form of Sre1. Wild-type strain CBS6938 was used as a control. Our results showed that this modification increased the crtE transcript levels more than threefold and fourfold in CBS.cyp61−.pHMGS/crtE and CBS.SRE1N.FLAG.pHMGS/crtE, respectively, resulting in 1.43-fold and 1.22-fold increases in carotenoid production. In contrast, this modification did not produce significant changes in the wild-type strain, which lacks the active Sre1 transcription factor under the same culture conditions. This study highlights the potential of promoter substitution strategies involving genes regulated by Sre1 to enhance carotenoid production, specifically in strains where the SREBP pathway is activated, offering a promising avenue for strain improvement in industrial applications.

Impact of ERG6 Gene Deletion on Membrane Composition and Properties in the Pathogenic Yeast Candida glabrata.

The ERG6 gene is crucial for the biosynthesis of ergosterol, a key component of yeast cell membranes. Our study examines the impact of ERG6 gene deletion on the membrane composition and physicochemical properties of the pathogenic yeast Candida glabrata. Specifically, we investigated changes in selected sterol content, phospholipid composition, transmembrane potential, and PDR16 gene activity. Sterol levels were measured using high-performance liquid chromatography, the phospholipid profile was analysed via thin-layer chromatography, transmembrane potential was assessed with fluorescence spectroscopy, and gene expression levels were determined by quantitative PCR. Our findings revealed a depletion of ergosterol, increased zymosterol and eburicol content, an increased phosphatidylcholine and a reduced phosphatidylethanolamine content in the Δerg6 strain compared to the wt. Additionally, the Δerg6 strain exhibited membrane hyperpolarization without changes in PDR16 expression. Furthermore, the Δerg6 strain showed increased sensitivity to the antifungals myriocin and aureobasidine A. These results suggest that ERG6 gene deletion leads to significant alterations in membrane composition and may activates an alternative ergosterol synthesis pathway in the C. glabrata Δerg6 deletion mutant.

PROTOCOLOS TERAPÊUTICOS PARA O TRATAMENTO DA ESPOROTRICOSE FELINA REFRATÁRIA E INOVAÇÕES DECORRENTES DAS PESQUISAS

Sporotrichosis in felines is much more pathogenic than in other animals or in humans, even if immunocompetent, which makes treatment a challenge in clinical medicine, as many of them are refractory to the preferred therapy such as Itraconazole or Ketoconazole. This situation also arises from the prolonged oral administration of the medication, the animal's reluctance to take the drug and the guardian's abandonment of the prescription when he notices an improvement in the lesions caused by the disease, which generates relapses and resistance to the antifungals available on the market. Itraconazole, as well as Ketoconazole, act by causing permeability of the plasma membrane and cell necrosis, however, they have adverse effects such as nausea and vomiting in the first and liver disease as relevance in the second. Terbinafine works by interrupting the synthesis of ergosterol and as a consequence the cell membrane ruptures and death, however, it also has side effects such as gastrointestinal and skin problems. Potassium iodide can be used in combination with azoles or Terbinafine, although the literature does not agree with the issue, as not all felines respond to treatment, adverse effects are due to iodism such as depression, anorexia, vomiting and diarrhea. There are also alternative treatments that can enhance oral medication, such as laser therapy, ozone therapy, cryotherapy and cryosurgery. Due to the issue of non-responsive felines, there is research being developed aiming to provide new therapeutic horizons for these animals, such as the use of intranasal infusion of 1% Clotrimazole spray associated with Itraconazole added with Potassium iodide or not, with good results, no relapses and no side effects. NikZ, the result of a substance produced by the bacterium Streptomyces tendae, called Nikkomycin Z, also appears as a new therapeutic potential for the treatment of Sporotrix spp., with a blocking effect on chitin synthase present in the cell wall of fungi, were maide research with dogs, rats and is in Phase 1 of experiments on humans. NikZ represents a new form of prescription, as it can be placed in the animals' drinking water, avoiding the desertion of those responsible for the felines, it has no observable adverse effects in the analyzes carried out and represents an alternative for cats refractory to existing medical protocols in veterinary.

Antifungal Efficacy of Ultrashort β-Peptides against Candida Species: Mechanistic Understanding and Therapeutic Implications.

Candidiasis, a condition spurred by the unchecked proliferation of Candida species, poses a formidable global health threat, particularly in immunocompromised individuals. The emergence of drug-resistant strains complicates management strategies, necessitating novel therapeutic avenues. Antimicrobial peptides (AMPs) have garnered attention for their potent antifungal properties and broad-spectrum activity against Candida species. This study assessed the antifungal effectiveness of ultrashort β-peptides against Candida strains, with a specific focus on peptide P3 (LAU-β3,3-Pip-β2,2-Ac6c-PEA). Our findings showed P3's remarkable fungistatic and fungicidal activities against Candida albicans, exhibiting an MIC of 4 μg/mL, comparable to those of standard antifungal drugs. The MIC value remained unchanged in the presence of ADC and BSA, indicating that serum albumin does not diminish the activity of P3. P3 demonstrates synergistic effects when combined with Fluconazole (FLU), Itraconazole (ITR), and Nystatin (NYS) to the extent that it becomes effective at 0.125, 0.125, and 0.03125 μg/mL, respectively. Concentration versus time-kill kinetics showed its time-dependent activity up to the first 12 h against C. albicans, and later concentration also played a role; indeed, at 24 h the whole culture was sterilized at 8× MIC. Post-antifungal effect assays confirmed prolonged suppression of pathogen growth after the removal of P3 from the media for significant durations. More importantly, P3 inhibits hyphae formation and biofilm development of Candida, outperforming Fluconazole with respect to these properties. Mechanistic insights display P3's potential to disrupt fungal cell membrane integrity and dose-dependent inhibition of ergosterol biosynthesis, essential for fungal cell wall integrity. Using the Bradford assay, it was observed that extracellular protein concentrations increased with higher doses of the compound, thereby validating the effect of P3 on membrane integrity. A comparative gene analysis using RT-PCR showed that P3 downregulates ERG3, ERG11, and HWP1, which are crucial for the survival and pathogenicity of C. albicans. The impact of P3 on ERG11 and ERG3 is more effective than that of Fluconazole. Molecular docking studies revealed strong binding of P3 to various isoforms of lanosterol 14-α-demethylase, a key enzyme in ergosterol synthesis. Furthermore, molecular dynamic simulations validated the stability of the most promising docking complex. Overall, our findings underscore P3's potential as a leading candidate for the development of innovative antifungal therapies, warranting further investigation and optimization.

Enhancement of ROS Production by Catechin Is a Primary Effect of Increased Azole Efficacy in Nakaseomyces glabratus (Candida glabrata) Cells Lacking the ERG6 Gene.

Fungal infections have become an important public health problem. Currently, there are only three available classes of antifungals for the treatment of invasive infections. Two of them, azoles and polyenes, target the synthesis of ergosterol or bind to sterols. A promising strategy to improve current therapies is the use of natural compounds in combinational therapies with the existing antifungals. In this work, we analyzed the changes in the susceptibility of the mutant strain of Nakaseomyces glabratus (Candida glabrata) lacking the ERG6 gene (encoding the sterol C-24 methyltransferase in ergosterol biosynthesis) in the presence of catechin and antifungal azoles. The reduced content of ergosterol in the Cgerg6Δ mutant resulted in the increased tolerance of the mutant cells to both azoles and polyenes. The combination of catechin with fluconazole or miconazole led to the growth inhibition of the azole-resistant Cgerg6Δ mutant strain. In the presence of catechin and miconazole, the Cgerg6Δ mutant fails to properly activate the expression of genes encoding the transcription factors CgYap1p and CgMsn4p, as well as the gene expression of CgCTA1, which are involved in oxidative stress response and lead to the intracellular accumulation of ROS. Finally, we show that catechin administration reduces mortality in a Galleria mellonella model infected with C. glabrata. Our work thus supports the use of catechin in combination therapies for fungal infections and shows that the CgERG6 gene could be a potential new drug target.

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

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.

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.

Unravelling novel molecular targets of Thymol and Carvacrol in Candida glabrata

The emergence of non-albicans Candida as a potential causative agent of candidiasis following Candida albicans is occurring globally. It is known that phenolic cresols, thymol, and carvacrol have anti-Candida characteristics. However, there are few papers that disclose the mechanism of action, which is characterized by a damaged cell wall and ergosterol synthesis, as well as calcium-induced death in C. albicans. This work utilized in-silico docking analysis to examine the molecular targets of thymol and carvacrol in Candida glabrata. The results were then validated in vitro by antifungal susceptibility testing and growth curve analysis. The study incorporated C. glabrata deletion mutants for the target proteins to gain understanding of the function of these proteins in the antifungal effects of the two monoterpenoids. After conducting molecular docking and in silico toxicity evaluations, thymol and carvacrol were chosen for in vitro investigations. Experimental tests conducted in a laboratory setting have shown that both phytocompounds exhibit strong binding affinity towards cell wall synthesis proteins (Kre1p, Kre2p, Ecm33p), calcium channel proteins (Mid1p, Ecm7p), and proteins involved in the ergosterol synthesis pathway (Erg5p). These proteins are likely to be the specific targets for the anti-Candida properties of the two isomeric monoterpenoids in C. glabrata. Minimum inhibitory concentration (MIC50) values for thymol and carvacrol range from 50 µg/ml to 75 µg/ml. The wild-type strains exhibit a minimum fungicidal concentration of 100 µg/ml for thymol and 125 µg/ml for carvacrol. The results underscore the significance of these proteins in the fungal reaction to thymol and carvacrol and also indicate a promising opportunity for the development of novel antifungal treatment methods utilizing these proteins.

Rational selection of morphological phenotypic traits to extract essential similarities in chemical perturbation in the ergosterol pathway

Terbinafine, fluconazole, and amorolfine inhibit fungal ergosterol synthesis by acting on their target enzymes at different steps in the synthetic pathway, causing the accumulation of various intermediates. We found that the effects of these three in- hibitors on yeast morphology were different. The number of morphological parameters commonly altered by these drugs was only approximately 6% of the total. Using a rational strategy to find commonly changed parameters,we focused on hidden essential similarities in the phenotypes possibly due to decreased ergosterol levels. This resulted in higher apparent morphological similarity. Improvements in morphological similarity were observed even when canonical correlation analysis was used to select biologically meaningful morphological parameters related to gene function. In addition to changes in cell morphology, we also observed differences in the synergistic effects among the three inhibitors and in their fungicidal effects against pathogenic fungi possibly due to the accumulation of different intermediates. This study provided a comprehensive understanding of the properties of inhibitors acting in the same biosynthetic pathway.

Deciphering the antiochratoxigenic activity of plant extracts and Debaryomyces hansenii against Penicillium nordicum in a “chorizo”-based medium by proteomic analysis

Penicillium nordicum is the main ochratoxin A (OTA)-producing species on the surface of dry-fermented sausages, such as the “chorizo”. New antifungal strategies are being developed using biocontrol agents (BCAs), such as plant extracts and native microorganisms. This work aimed to evaluate the antiochratoxigenic capacity and the causative modes of action of BCAs (rosemary essential oil (REO), acorn shell extract and the yeast Debaryomyces hansenii (Dh)) in a “chorizo”-based medium (Ch-DS). BCAs were inoculated on Ch-DS together with P. nordicum and incubated at 12 °C for 15 days to collect mycelia for OTA analyses and comparative proteomics. Both REO and Dh alone decreased OTA accumulation up to 99% and affected the abundance of P. nordicum proteins linked to cell wall organisation, synthesis of OTA-related metabolites and ergosterol synthesis. It is worth highlighting the increased abundance of an amidase by REO, matching with the decrease in OTA. The use of REO and Dh as BCAs could be an effective strategy to reduce the OTA hazard in the meat industry. Based on their not fully coincident modes of action, their combined application could be of interest in “chorizo” to maximise their potential against ochratoxigenic strains.

Insights into the antifungal mechanism of Bacillus subtilis cyclic lipopeptide iturin A mediated by potassium ion channel

Fungal infections pose severe and potentially lethal threats to plant, animal, and human health. Ergosterol has served as the primary target for developing antifungal medications. However, many antifungal drugs remain highly toxic to humans due to similarity in cell membrane composition between fungal and animal cells. Iturin A, lipopeptide produced by Bacillus subtilis, efficiently inhibit various fungi, but demonstrated safety in oral administration, indicating the existence of targets different from ergosterol. To pinpoint the exact antifungal target of iturin A, we used homologous recombination to knock out and overexpress erg3, a key gene in ergosterol synthesis. Saccharomyces cerevisiae and Aspergillus carbonarius were transformed using the LiAc/SS-DNNPEG and Agrobacterium-mediated transformation (AMT), respectively. Surprisingly, increasing ergosterol content did not augment antifungal activity. Furthermore, iturin A's antifungal activity against S. cerevisiae was reduced while it pre-incubation with voltage-gated potassium (Kv) channel inhibitor, indicating that Kv activation was responsible for cell death. Iturin A was found to activate the Kv protein, stimulating K+ efflux from cell. In vitro tests confirmed interaction between iturin A and Kv protein. This study highlights Kv as one of the precise targets of iturin A in its antifungal activity, offering a novel target for the development of antifungal medications.

Unraveling the Dual Role of Voriconazole as an Antifungal Agent and Precursor to Squamous Cell Carcinoma

Voriconazole, a potent triazole antifungal medication, is extensively used to treat serious fungal infections in immunocompromised patients. Despite its efficacy, recent findings suggest a potential link between long-term voriconazole therapy and the development of squamous cell carcinoma (SCC). This review examines the dual role of voriconazole, emphasizing both its therapeutic benefits and carcinogenic risks. The pharmacodynamics of voriconazole involve the inhibition of ergosterol synthesis, crucial to fungal cell integrity. However, its metabolites, such as voriconazole-N-oxide, have been implicated in phototoxic reactions that lead to DNA damage and tumor formation. This is particularly significant in patients with prolonged drug exposure, such as organ transplant recipients, where increased SCC incidence has been observed. Clinical evidence and molecular studies suggest that voriconazole may disrupt key cellular pathways like the Hedgehog pathway, affecting epidermal differentiation and increasing cancer risk. Given these concerns, the necessity for careful therapeutic monitoring and patient education about potential risks is discussed. Alternative antifungal therapies and protective measures against phototoxic effects are also recommended as strategies to mitigate SCC risk. Future research should focus on understanding the mechanisms of voriconazole-induced carcinogenesis and refining patient management protocols. This review highlights the need for a balanced approach to voriconazole therapy, weighing its antifungal benefits against the risks of adverse dermatological outcomes.

Unraveling the antifungal and anti-aflatoxin B1 mechanisms of piperitone on Aspergillus flavus

Aspergillus flavus infects important crops and produces carcinogenic aflatoxins, posing a serious threat to food safety and human health. Biochemical analysis and RNA-seq were performed to investigate the effects and mechanisms of piperitone on A. flavus growth and aflatoxin B1 biosynthesis. Piperitone significantly inhibited the growth of A. flavus, AFB1 production, and its pathogenicity on peanuts and corn flour. Differentially expressed genes (DEGs) associated with the synthesis of chitin, glucan, and ergosterol were markedly down-regulated, and the ergosterol content was reduced, resulting in a disruption in the integrity of the cell wall and cell membrane. Moreover, antioxidant genes were down-regulated, the correspondingly activities of antioxidant enzymes such as catalase, peroxidase, and superoxide dismutase were reduced, and levels of superoxide anion and hydrogen peroxide were increased, leading to a burst of reactive oxygen species (ROS). Accompanied by ROS accumulation, DNA fragmentation and cell autophagy were observed, and 16 aflatoxin cluster genes were down-regulated. Overall, piperitone disrupts the integrity of the cell wall and cell membrane, triggers the accumulation of ROS, causes DNA fragmentation and cell autophagy, ultimately leading to defective growth and impaired AFB1 biosynthesis.

Potential Anemia as a Risk Factor for Angular Cheilitis and Pseudomembranous Candidiasis: Case Report

Angular cheilitis and pseudomembranous candidiasis are oral infections caused by Candida albicans. Angular cheilitis patients also experience oral candidiasis because the Candida reservoir is intraoral. Iron deficiency anemia was closely related to this infection. A 50-year-old female patient complaint of pain in the commissure and tongue 3 weeks ago. Extraoral examination showed anemic conjunctiva and fissure erythema with crusting in bilateral commissures. Intraoral examination showed atrophy of papillae with a yellowish-white plaque that can be scraped off and leaves erythema on the tongue. A complete blood examination was performed and showed a decrease in hemoglobin, hematocrit, erythrocytes, MCV, MCH, and MCHC. The patient was diagnosed angular cheilitis and pseudomembranous candidiasis with iron deficiency anemia. The therapy was miconazole 2% cream, nystatin oral suspension 100.000 IU/ml, and iron supplements. The lesions healed after 2 weeks of therapy and the patient was consulted to an internist. The patient has Angular cheilitis and pseudomembranous candidiasis triggered by iron deficiency anemia. Iron deficiency anemia causes a decrease in salivary transferrin protein which is fungistatic so that Candida grows excessively. Angular Cheilitis therapy is miconazole cream which inhibit the synthesis of ergosterol and nystatin for candidiasis therapy which works by binding to Candida plasma membrane sterols, causing leakage and then death. Iron deficiency anemia therapy is iron supplements; however, drug therapy alone is less effective, and consultation with an internist is required for further therapy. Anemia has big potential to be a risk factor for angular cheilitis and pseudomembranous candidiasis so comprehensive treatment is needed.

Naringenin-Zinc Oxide Nanocomposites Amalgamated Polymeric Gel Augmented Drug Delivery and Attenuated Experimental Cutaneous Candidiasis in Balb/c Mice: In Vitro and In Vivo Studies.

Naringenin (NRG) inhibits the fungal 17β-hydroxysteroid dehydrogenase accountable for ergosterol synthesis in Candida albicans (C. albicans), a causative agent for cutaneous candidiasis. In present research, NRG was complexed with ZnO nanomaterial (NRG-Zn2+) to synthesize NRG-Zn2+ nanocomposites. The particle size and ζ-potential of NRG-Zn2+ nanocomposites were respectively estimated to be 180.33 ± 1.22-nm and - 3.92 ± 0.35-mV. In silico data predicted the greater affinity of NRG-Zn2+ nanocomposite for 14α-demethylase and ceramide in comparison to NRG alone. Later, NRG-Zn2+ nanocomposites solution was transformed in to naringenin-zinc oxide nanocomposites loaded chitosan gel (NRG-Zn-CS-Gel) with viscosity and firmness of 854806.7 ± 52386.43 cP and 698.27 ± 10.35g, respectively. The ex-vivo skin permeation demonstrated 70.49 ± 5.22% skin retention, significantly greater (P < 0.05) than 44.48 ± 3.06% of naringenin loaded chitosan gel (NRG-CS-Gel) and 31.24 ± 3.28% of naringenin solution (NRG Solution). NRG-Zn-CS-Gel demonstrated 6.71 ± 0.84% permeation of NRG with a flux value of 0.046 ± 0.01-µg/cm2/h. The MIC50 of NRG-Zn-CS-Gel against C. albicans was estimated to be 0.156-µg/mL with FICI (fractional inhibitory concentration index) of 0.018 that consequently exhibited synergistic efficacy. Further, NRG-Zn-CS-Gel demonstrated superior antifungal efficacy in C. albicans induced cutaneous candidiasis infection in Balb/c mice. The fungal burden in NRG-Zn-CS-Gel treated group was 109 ± 25 CFU/mL, significantly lower (P < 0.05) than positive control (2260 ± 446 CFU/mL), naringenin loaded chitosan gel (NRG-CS-Gel; 928 ± 127 CFU/mL) and chitosan gel (CS-Gel; 2116 ± 186 CFU/mL) treated mice. Further, histopathology examination and cytokine profiling of TNF-α, IL-1β and IL-10 revealed the healing of skin and inflammation associated with cutaneous candidiasis infection. In conclusion, NRG-Zn-CS-Gel may be a potential candidate for translating in to a clinical viable topical nanotherapeutic.

Phenotypic and Genotypic Characterization of Candida parapsilosis complex isolates from a Lebanese Hospital.

The opportunistic fungal pathogen Candida parapsilosis is a major causative agent of candidiasis leading to death in immunocompromised individuals. Azoles are the first line of defense in treatment by inhibiting ERG11, involved in the synthesis of ergosterol, the main sterol fungal sterol. Resistance to azoles is on the increase worldwide including in Lebanon. The purpose of this study is to characterize nine hospital isolates labeled as C. parapsilosis: four resistant and five sensitive to fluconazole. Phenotypic characterization was achieved through a battery of tests that target pathogenicity attributes such as virulence, biofilm formation, stress resistance, and ergosterol content. Genotypic analysis was done through whole genome sequencing to mutations in key virulence and resistance genes. Phylogenetic comparison was performed to determine strain relatedness and clonality. Genomic data and phylogenetic analysis revealed that three of the nine C. parapsilosis isolates were misidentified; two as C. orthopsilosis and C. metapsilosis belonging to the C. parapsilosis complex, while the third was C. albicans. Moreover, several known and novel mutations in key drug resistance and virulence genes were identified such as ERG11, ERG3, ERG6, CDR1, and FAS2. Phylogenetic analysis revealed a high degree of relatedness and clonality within our C. parapsilosis isolates. Our results showed that resistant isolates had no increased ergosterol content, no statistically significant difference in virulence, but exhibited an increase in biofilm content compared to the sensitive isolates. In conclusion, our study, the first of its kind in Lebanon, suggests several mechanisms of antifungal drug resistance in C. parapsilosis hospital isolates.

Inhibitory mechanism and application of cinnamon essential oil against Aspergillus flavus

Aspergillus flavus produces highly toxic and carcinogenic aflatoxins that cause severe economic losses and pose a serious threat to public health. Cinnamon essential oils (CEO) have recently attracted attention as promising alternative antifungal agents. In this study, the inhibition mechanism of CEO on A. flavus was investigated, and its application in the prevention of A. flavus in coix seeds was explored for the first time. The results showed that mycelial growth, spore germination, and aflatoxin synthesis of A. flavus were completely inhibited after treatment with 0.5 μL/mL of CEO. Morphological, physiological, and biochemical changes after CEO treatment were also analyzed to elucidate the inhibitory mechanism. Transcriptomic analysis showed that some genes involved in conidiophore development (flbA and abaA), ergosterol synthesis (CYP51, Erg1, Erg3, Erg7, Erg9, Erg24, and Erg25), aflatoxin biosynthesis (aflR, aflO, aflT, aflJ, aflW, and aflY), and oxidative stress (Pbs2, Ste20, CAT, atfB, mtfA, PacC, ppoC, and gprM) were significantly regulated. Moreover, A. flavus infection in coix seeds was effectively inhibited by 20.0 μL/g of CEO. This study shed important light on the processes underlying the antifungal mechanism of CEO and affirmed the possibility of using CEO as a green anti-aflatoxigenic agent in the preservation of coix seed.

Ergosterol promotes aggregation of natamycin in the yeast plasma membrane

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability became compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and caused a reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogues of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. Importantly, binding and membrane aggregation of natamycin was paralleled by a decrease of the dipole potential in the PM, and this effect was enhanced in the presence of myriocin. We conclude that ergosterol promotes binding and aggregation of natamycin in the yeast PM, which can be synergistically enhanced by inhibitors of sphingolipid synthesis.

Antifungal activity of bio-active cell-free culture extracts and volatile organic compounds (VOCs) synthesised by endophytic fungal isolates of Garden Nasturtium

Antimicrobial resistance in fungal pathogens (both human and plant) is increasing alarmingly, leading to massive economic crises. The existing anti-fungal agents are becoming ineffective, and the situation worsens on a logarithmic scale. Novel antifungals from unique natural sources are highly sought to cope sustainably with the situation. Metabolites from endophytic microbes are the best-fitted alternatives in this case. Endophytes are the untapped sources of ‘plants’ internal microbial population’ and are promising sources of effective bio-therapeutic agents. Fungal endophytes were isolated from Tropaeolum majus and checked for antifungal activity against selected plant and human pathogens. Bioactive metabolites were identified through chromatographic techniques. The mode of action of those metabolites was evaluated through various spectroscopic techniques. The production of antifungal metabolite was optimized also. In particular VOCs (volatile organic compounds) of TML9 were tested in vitro for their anti-phytopathogenic activity. Ethyl acetate (EA) extract of cell-free culture components of Colletotrichum aenigma TML3 exhibited broad-spectrum antifungal activity against four species of Candida and the major constituents reported were 6-pentyl-2H-pyran-2-one, 2-Nonanone, 1 propanol 2-amino. The volatile metabolites, trans-ocimene, geraniol, and 4-terpinyl acetate, produced from Curvularia lunata TML9, inhibited the growth of some selected phyto pathogens. EA extract hampered the biofilm formation, minimised the haemolytic effect, and blocked the transformation of Candida albicans (MTCC 4748) from yeast to hyphal form with a Minimum Fungicidal Concentration (MFC) of 200–600 µg mL−1. Central carbohydrate metabolism, ergosterol synthesis, and membrane permeability were adversely affected and caused the lethal leakage of necessary macromolecules of C. albicans. Volatile metabolites inhibited the growth of phytopathogens i.e., Rhizoctonia solani, Alternaria alternata, Botrytis cinerea, Cercospora beticola, Penicillium digitatum, Aspergillus fumigatus, Ceratocystis ulmi, Pythium ultimum up to 89% with an IC50 value of 21.3–69.6 µL 50 mL−1 and caused leakage of soluble proteins and other intracellular molecules. Citrusy sweet odor volatiles of TML9 cultured in wheat-husk minimised the infections of Penicillium digitatum (green mold), in VOC-exposed sweet oranges (Citrus sinensis). Volatile and non-volatile antifungal metabolites of these two T. majus endophytes hold agricultural and pharmaceutical interests. Metabolites of TML3 have strong anti-Candida activity and require further assessment for therapeutic applications. Also, volatile metabolites of TML9 can be further studied as a source of antifungals. The present investigational outcomes bio-prospects the efficacy of fungal endophytes of Garden Nasturtium.

Biofilm-associated metabolism via ERG251 in Candida albicans.

Biofilm formation by the fungal pathogen Candida albicans is the basis for its ability to infect medical devices. The metabolic gene ERG251 has been identified as a target of biofilm transcriptional regulator Efg1, and here we report that ERG251 is required for biofilm formation but not conventional free-living planktonic growth. An erg251Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo catheter infection model. In both in vitro and in vivo biofilm contexts, cell number is reduced and hyphal length is limited. To determine whether the mutant defect is in growth or some other aspect of biofilm development, we examined planktonic cell features in a biofilm-like environment, which was approximated with sealed unshaken cultures. Under those conditions, the erg251Δ/Δ mutation causes defects in growth and hyphal extension. Overexpression in the erg251Δ/Δ mutant of the paralog ERG25, which is normally expressed more weakly than ERG251, partially improves biofilm formation and biofilm hyphal content, as well as growth and hyphal extension in a biofilm-like environment. GC-MS analysis shows that the erg251Δ/Δ mutation causes a defect in ergosterol accumulation when cells are cultivated under biofilm-like conditions, but not under conventional planktonic conditions. Overexpression of ERG25 in the erg251Δ/Δ mutant causes some increase in ergosterol levels. Finally, the hypersensitivity of efg1Δ/Δ mutants to the ergosterol inhibitor fluconazole is reversed by ERG251 overexpression, arguing that reduced ERG251 expression contributes to this efg1Δ/Δ phenotype. Our results indicate that ERG251 is required for biofilm formation because its high expression levels are necessary for ergosterol synthesis in a biofilm-like environment.