Fungal Membrane Permeability Research Articles

Antifungal potential, mechanism of action, and toxicity of 1,4-naphthoquinone derivatives.

The rising prevalence of fungal infections and challenges such as adverse effects and resistance against existing antifungal agents have driven the exploration of new antifungal substances. We specifically investigated naphthoquinones, known for their broad biological activities and promising antifungal capabilities. It specifically examined the effects of a particular naphthoquinone on the cellular components of Candida albicans ATCC 60193. The study also assessed cytotoxicity in MRC-5 cells, Artemia salina, and the seeds of tomatoes and arugula. Among four tested naphthoquinones, 2,3-DBNQ (2,3-dibromonaphthalene-1,4-dione) was identified as highly effective, showing potent antifungal activity at concentrations between 1.56 and 6.25 μg mL-1. However, its cytotoxicity in MRC-5 cells (IC50 = 15.44 µM), complete mortality in A. salina at 50 μg mL-1, and significant seed germination inhibition suggest limitations for its clinical use. The findings indicate that primary antifungal mechanism of 2,3-DBNQ might involve disrupting fungal membrane permeability, which leads to increased nucleotide leakage. This insight underscores the need for further research to enhance the selectivity and safety of naphthoquinones for potential therapeutic applications.

Antifungal and Antivirulence Activity of Vanillin and Tannic Acid Against Aspergillus fumigatus and Fusarium solani.

Aspergillus fumigatus and Fusarium solani infections have become severe health threat; both pathogens are considered a priority due to the increasing emergence of antifungal-resistant strains and high mortality rates. Therefore, the discovery of new therapeutic strategies has become crucial. In this study, we evaluated the antifungal and antivirulence effects of vanillin and tannic acid against Aspergillus fumigatus and Fusarium solani. The minimum inhibitory concentrations of the compounds were determined by the microdilution method in RPMI broth in 96-well microplates according to CLSI. Conidial germination, protease production, biofilm formation, and in vivo therapeutic efficacy assays were performed. The results demonstrated that vanillin and tannic acid had antifungal activity against Aspergillus fumigatus, while tannic acid only exhibited antifungal activity against Fusarium solani. We found that vanillin and tannic acid inhibited conidial germination and secreted protease production and biofilm formation of the fungal pathogens using sub-inhibitory concentrations. Besides, vanillin and tannic acid altered the fungal membrane permeability, and both compounds showed therapeutic effect against aspergillosis and fusariosis in an infection model in Galleria mellonella larvae. Our results highlight the antivirulence effect of vanillin and tannic acid against priority pathogenic fungi as a possible therapeutic alternative for human fungal infections.

The Combined Effects of Azoxystrobin and the Biosurfactant-Producing Bacillus sp. Kol B3 against the Phytopathogenic Fungus Fusarium sambucinum IM 6525.

This study aimed to evaluate how the combined presence of the synthetic fungicide azoxystrobin (AZ) and the biosurfactant-producing Bacillus sp. Kol B3 influences the growth of the phytopathogenic fungus Fusarium sambucinum IM 6525. The results showed a noticeable increase in antifungal effectiveness when biotic and abiotic agents were combined. This effect manifested across diverse parameters, including fungal growth inhibition, changes in hyphae morphology, fungal membrane permeability and levels of intracellular reactive oxygen species (ROS). In response to the presence of Fusarium and AZ in the culture, the bacteria changed the proportions of biosurfactants (surfactin and iturin) produced. The presence of both AZ and/or Fusarium resulted in an increase in iturin biosynthesis. Only in 72 h old bacterial-fungal co-culture a 20% removal of AZ was noted. In the fungal cultures (with and without the addition of the bacteria), the presence of an AZ metabolite named azoxystrobin free acid was detected in the 48th and 72nd hours of the process. The possible involvement of increased iturin and ROS content in antifungal activity of Bacillus sp. and AZ when used together are also discussed. Biosurfactants were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Microscopy techniques and biochemical assays were also used.

TPP crosslinking influence on the effect of sprayed dried microparticles based on industry waste sourced-chitosan over crop pathogens

The use of fishing industry waste residues for biotechnological developments is blossoming in the last decade to improve the management of marine resources while reducing the environmental impact of the fisheries sector. Chitosan (CS), is a natural and nontoxic biopolymer that exerts great antimicrobial properties and is being considered as GRAS by the United States Food and Drug Administration. In this study, we aimed to produce at a gram scale high molecular weight CS microparticles (CS-MP) from a low quality commercial raw material intended to develop an environmental friendly antimicrobial. Thus, we aimed to test the effect of sodium tripolyphosphate (TPP) used as a crosslinker on the antimicrobial performance of the CS-MP. Hence, we synthesized CS using shrimp fishing industry waste from Argentinean Patagonia at a pilot scale in order to be able to provide a cost-effective second life to the industry waste. By varying the ratio polymer/crosslinker, we studied its influence on the final physicochemical properties as well as their performance against representative crop pathogens: Pseudomonas syringae pv. tomato DC 3000 and Fusarium solani f. sp. eumartii. Our results showed a correlation between the surface charge exhibited by CS-MPs and their antimicrobial properties, through an imbalance on fungal membrane permeability. Thus, given the difference in the CS-MPs performance, it is clear that the electrostatic interaction of the particles with the negatively charged surface of the microorganism plays a key role in determining their ultimate activity. The CS-MPs exhibited great potential against crop pathogens providing a second life to fishing industry waste by developing an eco-friendly plant protectant from a valuable synergy of the national industry and academics.

Arthrocolins Synergizing with Fluconazole Inhibit Fluconazole-Resistant Candida albicans by Increasing Riboflavin Metabolism and Causing Mitochondrial Dysfunction and Autophagy.

Our previous study reported that seminaturally occurring arthrocolins A to C with unprecedented carbon skeletons could restore the antifungal activity of fluconazole against fluconazole-resistant Candida albicans. Here, we showed that arthrocolins synergized with fluconazole, reducing the fluconazole minimum and dramatically augmenting the survivals of 293T human cells and nematode Caenorhabditis elegans infected with fluconazole-resistant C. albicans. Mechanistically, fluconazole can induce fungal membrane permeability to arthrocolins, leading to the intracellular arthrocolins that were critical to the antifungal activity of the combination therapy by inducing abnormal cell membranes and mitochondrial dysfunctions in the fungus. Transcriptomics and reverse transcription-quantitative PCR (qRT-PCR) analysis indicated that the intracellular arthrocolins induced the strongest upregulated genes that were involved in membrane transports while the downregulated genes were responsible for fungal pathogenesis. Moreover, riboflavin metabolism and proteasomes were the most upregulated pathways, which were accompanied by inhibition of protein biosynthesis and increased levels of reactive oxygen species (ROS), lipids, and autophagy. Our results suggested that arthrocolins should be a novel class of synergistic antifungal compounds by inducing mitochondrial dysfunctions in combination with fluconazole and provided a new perspective for the design of new bioactive antifungal compounds with potential pharmacological properties. IMPORTANCE The prevalence of antifungal-resistant Candida albicans, which is a common human fungal pathogen causing life-threatening systemic infections, has become a challenge in the treatment of fungal infections. Arthrocolins are a new type of xanthene obtained from Escherichia coli fed with a key fungal precursor toluquinol. Different from those artificially synthesized xanthenes used as important medications, arthrocolins can synergize with fluconazole against fluconazole-resistant Candida albicans. Fluconazole can induce the fungal permeability of arthrocolins into fungal cells, and then the intracellular arthrocolins exerted detrimental effects on the fungus by inducing fungal mitochondrial dysfunctions, leading to dramatically reduced fungal pathogenicity. Importantly, the combination of arthrocolins and fluconazole are effective against C. albicans in two models, including human cell line 293T and nematode Caenorhabditis elegans. Arthrocolins should be a novel class of antifungal compounds with potential pharmacological properties.

The Water-Soluble Chitosan Derivative, N-Methylene Phosphonic Chitosan, Is an Effective Fungicide against the Phytopathogen Fusarium eumartii.

Chitosan has been considered an environmental-friendly polymer. However, its use in agriculture has not been extended yet due to its relatively low solubility in water. N-Methylene phosphonic chitosan (NMPC) is a water-soluble derivative prepared by adding a phosphonic group to chitosan. This study demonstrates that NMPC has a fungicidal effect on the phytopathogenic fungus Fusarium solani f. sp. eumartii (F. eumartii) judged by the inhibition of F. eumartti mycelial growth and spore germination. NMPC affected fungal membrane permeability, reactive oxygen species production, and cell death. Also, this chitosan-derivative exerted antifungal effects against two other phytopathogens, Botrytis cinerea, and Phytophthora infestans. NMPC did not affect tomato cell viability at the same doses applied to these phytopathogens to exert fungicide action. In addition to water solubility, the selective biological cytotoxicity of NMPC adds value in its application as an antimicrobial agent in agriculture.

Production of cationic antifungal peptides from kenaf seed protein as natural bio preservatives to prolong the shelf-life of tomato puree

This study determined the favourable fermentation conditions for the production of antifungal peptides from kenaf seeds and their effectiveness in extending the shelf-life of tomato puree. The optimum fermentation conditions for the maximum activity of the antifungal peptides were 8.4% (w/v), 7 days and 3.7% for substrate/water ratio, fermentation time and glucose concentration, respectively. Eight cationic peptides of low molecular weight ranging from 840 to 1876 Da were identified in kenaf seed peptides mixture (KSPM). The minimum inhibitory concentration and minimum fungicidal concentration of KSPM against Fusarium sp. were 0.18 mg/mL and 0.70 mg/mL, respectively, while those for Aspergillus niger were 1.41 mg/mL and 2.81 mg/mL respectively. KSPM exhibited a fungicidal effect and a prolonged lag phase, with increased fungal membrane permeability as the concentration of KSPM increased, as evidenced by the release of intracellular constituents. The treatment of tomato puree with 1000 mg/kg KSPM delayed fungal growth for up to 14 and 23 days at 25 °C and 4 °C respectively, significantly reducing Aspergillus niger and Fusarium sp. counts. In conclusion, kenaf seed peptides prepared by lacto-fermentation possess antifungal activity, hence can be applied as natural bio preservatives to extend the shelf-life of food products such as tomato puree.

Combining Colistin and Fluconazole Synergistically Increases Fungal Membrane Permeability and Antifungal Cidality.

The increasing emergence of drug-resistant fungal pathogens, together with the limited number of available antifungal drugs, presents serious clinical challenges to treating systemic, life-threatening infections. Repurposing existing drugs to augment the antifungal activity of well-tolerated antifungals is a promising antifungal strategy with the potential to be implemented rapidly. Here, we explored the mechanism by which colistin, a positively charged lipopeptide antibiotic, enhances the antifungal activity of fluconazole, the most widely used orally available antifungal. In a range of susceptible and drug-resistant isolates and species, colistin was primarily effective at reducing fluconazole tolerance, a property of subpopulations of cells that grow slowly in the presence of a drug and may promote the emergence of persistent infections and resistance. Clinically relevant concentrations of colistin synergized with fluconazole, reducing fluconazole minimum inhibitory concentration 4-fold. Combining fluconazole and colistin also increased survival in a C. albicans Galleria mellonella infection, especially for a highly fluconazole-tolerant isolate. Mechanistically, colistin increased permeability to fluorescent antifungal azole probes and to intracellular dyes, accompanied by an increase in cell death that was dependent upon pharmacological or genetic inhibition of the ergosterol biosynthesis pathway. The positive charge of colistin is critical to its antifungal, and antibacterial, activity: colistin directly binds to several eukaryotic membrane lipids (i.e., l-α-phosphatidylinositol, l-α-phosphatidyl-l-serine, and l-α-phosphatidylethanolamine) that are enriched in the membranes of ergosterol-depleted cells. These results support the idea that colistin binds to fungal membrane lipids and permeabilizes fungal cells in a manner that depends upon the degree of ergosterol depletion.

Antifungal screening and in silico mechanistic studies of an in-house azole library.

Systemic Candida infections pose a serious public health problem with high morbidity and mortality. C.albicans is the major pathogen identified in candidiasis; however, non-albicans Candida spp. with antifungal resistance are now more prevalent. Azoles are first-choice antifungal drugs for candidiasis; however, they are ineffective for certain infections caused by the resistant strains. Azoles block ergosterol synthesis by inhibiting fungal CYP51, which leads to disruption of fungal membrane permeability. In this study, we screened for antifungal activity of an in-house azole library of 65 compounds to identify hit matter followed by a molecular modeling study for their CYP51 inhibition mechanism. Antifungal susceptibility tests against standard Candida spp. including C.albicans revealed derivatives 12 and 13 as highly active. Furthermore, they showed potent antibiofilm activity as well as neglectable cytotoxicity in a mouse fibroblast assay. According to molecular docking studies, 12 and 13 have the necessary binding characteristics for effective inhibition of CYP51. Finally, molecular dynamics simulations of the C.albicans CYP51 (CACYP51) homology model's catalytic site complexed with 13 were stable demonstrating excellent binding.

Antifungal mode of action of macrocarpal C extracted from Eucalyptus globulus Labill (Lan An) towards the dermatophyte Trichophyton mentagrophytes.

BackgroundThe fresh leaves of Eucalyptus globulus Labill. (Lan An) have been used in Chinese medicine for many years to treat dermatomycosis. Macrocarpal C was isolated from this herb and identified as its major antifungal component by bioassay-guided purification. This study aims to investigate the antifungal activity of macrocarpal C against Trichophyton mentagrophytes, which can cause tinea pedis.MethodsFresh leaves of E. globulus were extracted with 95 % ethanol, and the resulting ethanolic extracts were dried before being partitioned with n-hexane. The n-hexane layer was then subjected to chromatographic purification to give macrocarpal C. The antifungal minimum inhibitory concentration (MIC) of macrocarpal C was determined using the standard M38-A2 method described by the Clinical Laboratory Standards Institute (CLSI). The mode of action of macrocarpal C was elucidated using three in vitro assays, including (1) a fungal membrane permeability test using SYTOX® Green; (2) a reactive oxygen species (ROS) production test using 5-(and-6)-carboxy-2′,7′-dihydrodichlorofluorescein diacetate as a cell-permeable fluorogenic probe; and (3) a DNA fragmentation test based on terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) detection. Terbinafine hydrochloride and nystatin were used as positive controls.ResultsThe suppression in the growth of T. mentagrophytes following its treatment with macrocarpal C was associated with an increase in the permeability of the fungal membrane (P = 0.0043 when compared to control); an increase in the production of intracellular ROS (P = 0.0063); and the induction of apoptosis as a consequence of DNA fragmentation (P = 0.0007).ConclusionThis study demonstrated that the antifungal action of macrocarpal C was associated with increases of membrane permeability, intracellular ROS and DNA fragmentation.

Anti-dermatophytic activity of bakuchiol: In vitro mechanistic studies and in vivo tinea pedis-inhibiting activity in a guinea pig model

Bakuchiol was an active antifungal compound isolated from Psoraleae Fructus by means of bioassay-guided fractionation in our previous study. The present work aimed to investigate the underlying mechanisms and the therapeutic effect of bakuchiol in Trichophyton mentagrophytes-induced tinea pedis. After exposure to bakuchiol at 0.25-fold, 0.5-fold and 1-fold of minimum inhibitory concentration (MIC) (3.91μg/ml) for 24h, the fungal conidia of T. mentagrophytes demonstrated a significant dose-dependent increase in membrane permeability. Moreover, bakuchiol at 1-fold MIC elicited a 187% elevation in reactive oxygen species (ROS) level in fungal cells after a 3-h incubation. However, bakuchiol did not induce DNA fragmentation. In a guinea pig model of tinea pedis, bakuchiol at 1%, 5% or 10% (w/w) concentration in aqueous cream could significantly reduce the fungal burden of infected feet (p<0.01–0.05). In conclusion, this is the first report to demonstrate that bakuchiol is effective in relieving tinea pedis and in inhibiting the growth of the dermatophyte T. mentagrophytes by increasing fungal membrane permeability and ROS generation, but not via induction of DNA fragmentation.

In planta transformation of rice (Oryza sativa) using thaumatin-like protein gene for enhancing resistance to sheath blight

Among the fungal diseases, sheath blight caused by Rhizoctonia solani Kuhn is one of the most important wide spread diseases found in all the rice plants ( Oryza sativa ) growing countries. The control of the disease with agricultural alternation and traditional breeding has not been successful so far. Application of fungicide is not recommended. Resistant genes which can cause immunity to the disease have not been found. D34 is a thaumatin-like protein (TLP) belonging to the group 5 of pathogen-related proteins (PRs). These proteins can probably change permeability of fungal membrane. In this research, an attempt was made to enhance the resistance to the sheath blight fungus on rice plant through the expression of TLP gene under the control of a CaMV35 promoter. To achieve this goal, rice plants ( O. sativa ) were transformed by the pAJ21-CaMV35S-tlpD34 construct via in planta method using Agrobacterium tumefaciens EHA101. The transformed plants were confirmed using polymerase chain reaction (PCR) amplifying a 710 bp fragment of the cloned gene. Keywords: In planta , pathogenesis-related protein, Rhizoctonia solani , rice, sheath blight, thaumatin-like protein, transformation

Preventive and post‐infection control of Botrytis cinerea in tomato plants by hexanoic acid

The antifungal activity of hexanoic acid on the phytopathogen Botrytis cinerea was studied. This chemical inhibited both spore germination and mycelial growth in vitro in a concentration‐ and pH‐dependent manner, and stopped spore germination at a very early stage, preventing germ‐tube development. The minimum fungicidal concentration (MFC) for in vitro spore germination was 16 mm. Hexanoic acid also inhibited in vitro mycelial growth of germinated spores at an MFC of 12 mm. Studies performed to characterize the mechanisms underlying the antimicrobial effect of hexanoic acid showed that it alters fungal membrane permeability. In addition, hexanoic acid treatment increased the levels of spermine, spermidine, putrescine and cadaverine in B. cinerea mycelia. Spray application of hexanoic acid at fungicidal concentrations on 4‐week‐old tomato plants prior to fungal inoculation reduced necrosis diameter by approximately 60%. Application of the same hexanoic acid concentrations on previously infected plants reduced further necrosis expansion by around 30%. The results suggest that this chemical acts as a preventive and curative fungicide. Interestingly, treatments with hexanoic acid at concentrations below the MFC in hydroponic solution prior to fungal inoculation significantly reduced necrosis area. These results suggest an inducer effect of plant responses for hexanoic acid treatments at these concentrations. Hexanoic acid is a good candidate for safe antifungal treatments for the control of B. cinerea, which is responsible for many economic losses on fruits, vegetables and flowers.

リポソーマル アムホテリシンＢ

Liposomal amphotericin B (AmBisome) is a DDS (drug delivery system) formulation of amphotericin B (AMPH-B), and has been developed in an attempt to reduce the toxicity of AMPH-B while retaining its therapeutic efficacy. AMPH-B has been the "gold standard" of antifungal therapy over the past four decades. It has a broad spectrum of fungicidal activity against a number of clinically important pathogens including Aspergillus and Candida. The mechanism of action of AMPH-B involves binding to ergosterol, the principal sterol in fungal cell membranes. Binding to ergosterol causes an increase in fungal membrane permeability, electrolyte leakage, and cell death. AMPH-B has affinity for cholesterol in mammalian membranes, which leads to severe side-effects including kidney damage. AmBisome is a unilamellar vesicle composed of AMPH-B and phospholipid. Upon administration, AmBisome remains intact in the blood and distributes to the tissues where fungal infection may occur, and is disrupted after attachment to the outside of fungal cells, resulting in fungal cell death. AmBisome and AMPH-B show similar in vitro and in vivo antifungal activity and clinical efficacy. However, AmBisome has less infusion-related toxicity and nephrotoxicity than AMPH-B.

Mistletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi.

Viscotoxins (Vts) are basic peptides expressed in mistletoe leaves, seeds and stems which have been shown to be cytotoxic to mammalian cells. The aim of this study was to analyse whether Vts were able to control and/or inhibit the growth of phytopathogenic fungi to obtain a clue to their biological function. Incubation of two Vt isoforms, VtA(3) and VtB, at a final concentration of 10 micro M resulted in a complete blockage of the germination of spores from three different pathogenic fungi. It was also shown that lower concentrations than 10 micro M of VtA(3) and VtB inhibit their mycelial growth in a dose-dependent manner. The protein dose required to inhibit the growth of Fusarium solani and Sclerotinia sclerotiorum to a 50% was between 1.5 and 3.75 micro M, which represents a potent activity. No significant differences in the antifungal potency for each Vt isoform, either VtA(3) and VtB, were observed, although they have been shown to exert differential cytotoxicity on mammalian cells. It was also demonstrated that Vts act as fungicidal compounds. To explore the basis of the antifungal activity the ability of VtA(3) to induce changes in membrane permeability and on the oxidative status of F. solani spores was analysed. By using a specific fluorescent probe on intact spores, it was demonstrated that VtA(3) produces rapid changes in fungal membrane permeability. It also induces H(2)O(2) production verified by a histochemical staining. The data presented in this study support a direct role of Vts in the plant defence determined by their lethal effect on fungal pathogens.

Effect of nystatin on the release of glycerol from salt-stressed cells of the salt-tolerant yeast Zygosaccharomyces rouxii.

The polyene antibiotic nystatin, which affects fungal membrane permeability, inhibited the growth of Zygosaccharomyces rouxii grown in medium containing 15% (w/v) NaCl, whereas yeast grown in medium without NaCl were only slightly inhibited. Nystatin caused salt-stressed cells to release large amounts of glycerol and inhibited their growth, but amino acids and materials with an absorbance at 260 nm were not released from the cells. The leakage was increased by the addition of glucose, and more than 90% of the intracellular glycerol was released into the medium during a 2-h incubation with 0.11 microM nystatin and 2% (w/v) glucose. Glycerol was indispensable for the growth of Z. rouxii grown in culture medium containing 15% NaCl.

白癬菌に対する化学療法 (第2報)

Antifungal action, in vitro of various surface active agents against Trichophyton was tested by which cationic agent, trimethylcetylammonium halide, and anionic agent, sodium p-laurylphenylsulfonate, were found to possess excellent properties. Nonionic surface active agents were found to be practically devoid of antifungal activities. The increase in the efficacy of surface active agents by the concurrent use of 2-dimethylamino 6-(β-diethylaminoethoxy)-benzothiazole dihydrochloride (Asterol), a chemotherapeutic for Trichophyton, was found to be 3-30 times, and the surface active agents that failed to show any efficacy by themselves were also found to have acquired such efficacy. This was assumed to be due to the increase of permeability of fungal membrane of Trichophyton by surface active agents.