The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway.

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.

Fangchinoline inhibits growth and biofilm of Candida albicans by inducing ROS overproduction.

Infections caused by Candida species, especially Candida albicans, threaten the public health and create economic burden. Shortage of antifungals and emergence of drug resistance call for new antifungal therapies while natural products were attractive sources for developing new drugs. In our study, fangchinoline, a bis-benzylisoquinoline alkaloid from Chinese herb Stephania tetrandra S. Moore, exerted antifungal effects on planktonic growth of several Candida species including C. albicans, with MIC no more than 50 µg/mL. In addition, results from microscopic, MTT and XTT reduction assays showed that fangchinoline had inhibitory activities against the multiple virulence factors of C. albicans, such as adhesion, hyphal growth and biofilm formation. Furthermore, this compound could also suppress the metabolic activity of preformed C. albicans biofilms. PI staining, followed by confocal laser scanning microscope (CLSM) analysis showed that fangchinoline can elevate permeability of cell membrane. DCFH-DA staining suggested its anti-Candida mechanism also involved overproduction of intracellular ROS, which was further confirmed by N-acetyl-cysteine rescue tests. Moreover, fangchinoline showed synergy with three antifungal drugs (amphotericin B, fluconazole and caspofungin), further indicating its potential use in treating C. albicans infections. Therefore, these results indicated that fangchinoline could be a potential candidate for developing anti-Candida therapies.

Inhibitory activity of bacterial lipopeptides against Fusarium oxysporum f.sp. Strigae.

This study investigated the influence of bacterial cyclic lipopeptides (LP; surfactins, iturins, fengycins) on microbial interactions. The objective was to investigate whether the presence of bacteria inhibits fungal growth and whether this inhibition is due to the release of bacterial metabolites, particularly LP. Selected endophytic bacterial strains with known plant-growth promoting potential were cultured in the presence of Fusarium oxysporum f.sp. strigae (Fos), which was applied as model fungal organism. The extracellular metabolome of tested bacteria, with a focus on LP, was characterized, and the inhibitory effect of bacterial LP on fungal growth was investigated. The results showed that Bacillus velezensis GB03 and FZB42, as well as B. subtilis BSn5 exhibited the strongest antagonism against Fos. Paraburkholderia phytofirmans PsJN, on the other hand, tended to have a slight, though non-significant growth promotion effect. Crude LP from strains GB03 and FZB42 had the strongest inhibitory effect on Fos, with a significant inhibition of spore germination and damage of the hyphal structure. Liquid chromatography tandem mass spectrometry revealed the production of several variants of iturin, fengycin, and surfactin LP families from strains GB03, FZB42, and BSn5, with varying intensity. Using plate cultures, bacillomycin D fractions were detected in higher abundance in strains GB03, FZB42, and BSn5 in the presence of Fos. Additionally, the presence of Fos in dual plate culture triggered an increase in bacillomycin D production from the Bacillus strains. The study demonstrated the potent antagonistic effect of certain Bacillus strains (i.e., GB03, FZB42, BSn5) on Fos development. Our findings emphasize the crucial role of microbial interactions in shaping the co-existence of microbial assemblages.

Enhanced control efficacy of Bacillus subtilis NM4 via integration of chlorothalonil on potato early blight caused by Alternaria solani.

Early blight caused by Alternaria solani is a common foliar disease of potato around the world, and serious infections result in reduced yields and marketability due to infected tubers. The major aim of this study is to figure out the synergistic effect between microorganism and fungicides and to evaluate the effectiveness of Bacillus subtilis NM4 in the control of early blight in potato. Based on its colonial morphology and a 16S rRNA analysis, a bacterial antagonist isolated from kimchi was identified as B. subtilis NM4 and it has strong antifungal and anti-oomycete activity against several phytopathogenic fungi and oomycetes. The culture filtrate of strain NM4 with the fungicide effectively suppressed the mycelial growth of A. solani, with the highest growth inhibition rate of 83.48%. Although exposure to culture filtrate prompted hyphal alterations in A. solani, including bulging, combining it with the fungicide caused more severe hyphal damage with continuous bulging. Surfactins and fengycins, two lipopeptide groups, were isolated and identified as the main compounds in two fractions using LC-ESI-MS. Although the surfactin-containing fraction failed to inhibit growth, the fengycin-containing fraction, alone and in combination with chlorothalonil, restricted mycelial development, producing severe hyphal deformations with formation of chlamydospores. A pot experiment combining strain NM4, applied as a broth culture, with fungicide, at half the recommended concentration, resulted in a significant reduction in potato early blight severity. Our results indicate the feasibility of an integrated approach for the management of early blight in potato that can reduce fungicide application rates, promoting a healthy ecosystem in agriculture.

Inhibition of hyphal formation together with biochar addition promotes erythritol production by Yarrowia lipolytica.

This study aimed to investigate the effect of hyphal formation in Yarrowia lipolytica and biochar addition on erythritol production by submerged fermentation. Hyphal formation significantly inhibited erythritol production by Y. lipolytica. Transcriptome analysis suggested that the impaired erythritol synthesis of hyphal cells was associated with the differential expression of genes involved in amino acid metabolism, lipid metabolism, and cell wall stability. Deletion of RAS2 responsible for yeast-to-hypha transition and EYD1 included in erythritol degradation blocked hyphal formation and improved erythritol production. Biochar prepared from corncob, sugarcane bagasse (SB), corn straw, peanut shell, coconut shell, and walnut shell (WS) had a positive effect on erythritol production, of which WS pyrolyzed at 500°C (WSc) performed the best in flask fermentation. In a 3.7 L bioreactor, 220.20 ± 10 g/L erythritol with a productivity of 2.30 ± 0.10 g/L/h was obtained in the presence of 1.4% (w/v) WSc and 0.7% SBc (SB pyrolyzed at 500°C) within 96 h. These results suggest that inhibition of hyphal formation together with biochar addition is an efficient way to promote erythritol production.

Butyl isothiocyanate exhibits antifungal and anti-biofilm activity against Candida albicans by targeting cell membrane integrity, cell cycle progression and oxidative stress.

The prevalence of Candida albicans infection has increased during the past few years, which contributes to the need for new, effective treatments due to the increasing concerns regarding antifungal drug toxicity and multidrug resistance. Butyl isothiocyanate (butylITC) is a glucosinolate derivative, and has shown a significant antifungal effect contrary to Candida albicans. Additionally, how butylITC affects the virulence traits of C. albicans and molecular mode of actions are not well known. Present study shows that at 17.36 mM concentration butylITC inhibit planktonic growth. butylITC initially slowed the hyphal transition at 0.542 mM concentration. butylITC hampered biofilm development, and inhibits biofilm formation at 17.36 mM concentration which was analysed using metabolic assay (XTT assay) and Scanning Electron Microscopy (SEM). In addition, it was noted that butylITC inhibits ergosterol biosynthesis. The permeability of cell membranes was enhanced by butylITC treatment. Moreover, butylITC arrests cells at S-phase and induces intracellular Reactive Oxygen Species (ROS) accumulation in C. albicans. The results suggest that butylITC may have a dual mode of action, inhibit virulence factors and modulate cellular processes like inhibit ergosterol biosynthesis, cell cycle arrest, induces ROS production which leads to cell death in C. albicans.

Membrane protein Bcsdr2 mediates biofilm integrity, hyphal growth and virulence of Botrytis cinerea.

Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal plant pathogen. Membrane proteins are important targets of fungicides and hotspots in the research and development of fungicide products. Wuyiencin affects the permeability and pathogenicity of B. cinerea, parallel reaction monitoring revealed the association of membrane protein Bcsdr2, and the bacteriostatic mechanism of wuyiencin was elucidated. In the present work, we generated and characterised ΔBcsdr2 deletion and complemented mutant B. cinerea strains. The ΔBcsdr2 deletion mutants exhibited biofilm loss and dissolution, and their functional activity was illustrated by reduced necrotic colonisation on strawberry and grape fruits. Targeted deletion of Bcsdr2 also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted gene complementation. The roles of Bcsdr2 in biofilms and pathogenicity were also supported by quantitative real-time RT-PCR results showing that phosphatidylserine decarboxylase synthesis gene Bcpsd and chitin synthase gene BcCHSV II were downregulated in the early stages of infection for the ΔBcsdr2 strain. The results suggest that Bcsdr2 plays important roles in regulating various cellular processes in B. cinerea. KEY POINTS: • The mechanism of wuyiencin inhibits B. cinerea is closely associated with membrane proteins. • Wuyiencin can downregulate the expression of the membrane protein Bcsdr2 in B. cinerea. • Bcsdr2 is involved in regulating B. cinerea virulence, growth and development.

Tacrolimus inhibits stress responses and hyphal formation via the calcineurin signaling pathway in Trichosporon asahii.

The pathogenic fungus Trichosporon asahii causes fatal deep-seated mycosis in immunocompromised patients. Calcineurin, which is widely conserved in eukaryotes, regulates cell growth and various stress responses in fungi. Tacrolimus (FK506), a calcineurin inhibitor, induces sensitivity to compounds that cause stress on the cell membrane and cell wall integrity. In this study, we demonstrated that FK506 affects stress responses and hyphal formation in T. asahii. In silico structural analysis revealed that amino acid residues in the binding site of the calcineurin-FKBP12 complex that interact with FK506 are conserved in T. asahii. The growth of T. asahii was delayed by FK506 in the presence of SDS or Congo red but not in the presence of calcium chloride. FK506 also inhibited hyphal formation in T. asahii. A mutant deficient of the cnb gene, which encodes the regulatory subunit B of calcineurin, exhibited stress sensitivities on exposure to SDS and Congo red and reduced the hyphal forming ability of T. asahii. In the cnb-deficient mutant, FK506 did not increase the stress sensitivity or reduce hyphal forming ability. These results suggest that FK506 affects stress responses and hyphal formation in T. asahii via the calcineurin signaling pathway.

Plasma membrane architecture protects Candida albicans from killing by copper.

The ability to resist copper toxicity is important for microbial pathogens to survive attack by innate immune cells. A sur7Δ mutant of the fungal pathogen Candida albicans exhibits decreased virulence that correlates with increased sensitivity to copper, as well as defects in other stress responses and morphogenesis. Previous studies indicated that copper kills sur7Δ cells by a mechanism distinct from the known resistance pathways involving the Crp1 copper exporter or the Cup1 metallothionein. Since Sur7 resides in punctate plasma membrane domains known as MCC/eisosomes, we examined overexpression of SUR7 and found that it rescued the copper sensitivity of a mutant that fails to form MCC/eisosomes (pil1Δ lsp1Δ), indicating that these domains act to facilitate Sur7 function. Genetic screening identified new copper-sensitive mutants, the strongest of which were similar to sur7Δ in having altered plasma membranes due to defects in membrane trafficking, cortical actin, and morphogenesis (rvs161Δ, rvs167Δ, and arp2Δ arp3Δ). Consistent with the mutants having altered plasma membrane organization, they were all more readily permeabilized by copper, which is known to bind phosphatidylserine and phosphatidylethanolamine and cause membrane damage. Although these phospholipids are normally localized to the intracellular leaflet of the plasma membrane, their exposure on the surface of the copper-sensitive mutants was indicated by increased susceptibility to membrane damaging agents that bind to these phospholipids. Increased copper sensitivity was also detected for a drs2Δ mutant, which lacks a phospholipid flippase that is involved in maintaining phospholipid asymmetry. Copper binds phosphatidylserine with very high affinity, and deleting CHO1 to prevent phosphatidylserine synthesis rescued the copper sensitivity of sur7Δ cells, confirming a major role for phosphatidylserine in copper sensitivity. These results highlight how proper plasma membrane architecture protects fungal pathogens from copper and attack by the immune system, thereby opening up new avenues for therapeutic intervention.

Design, Synthesis and Antifungal/Nematicidal Activity of Novel 1,2,4-Oxadiazole Derivatives Containing Amide Fragments.

Plant diseases that are caused by fungi and nematodes have become increasingly serious in recent years. However, there are few pesticide chemicals that can be used for the joint control of fungi and nematodes on the market. To solve this problem, a series of novel 1,2,4-oxadiazole derivatives containing amide fragments were designed and synthesized. Additionally, the bioassays revealed that the compound F15 demonstrated excellent antifungal activity against Sclerotinia sclerotiorum (S. sclerotiorum) in vitro, and the EC50 value of that was 2.9 µg/mL, which is comparable with commonly used fungicides thifluzamide and fluopyram. Meanwhile, F15 demonstrated excellent curative and protective activity against S. sclerotiorum-infected cole in vivo. The scanning electron microscopy results showed that the hyphae of S. sclerotiorum treated with F15 became abnormally collapsed and shriveled, thereby inhibiting the growth of the hyphae. Furthermore, F15 exhibited favorable inhibition against the succinate dehydrogenase (SDH) of the S. sclerotiorum (IC50 = 12.5 µg/mL), and the combination mode and binding ability between compound F15 and SDH were confirmed by molecular docking. In addition, compound F11 showed excellent nematicidal activity against Meloidogyne incognita at 200 µg/mL, the corrected mortality rate was 93.2%, which is higher than that of tioxazafen.

Mutanobactin D from the Human Microbiome: Total Synthesis, Configurational Assignment, and Biological Evaluation.

Mutanobactin D is a non-ribosomal, cyclic peptide isolated from Streptococcus mutans and shows activity reducing yeast-to-hyphae transition as well as biofilm formation of the pathogenic yeast Candida albicans. We report the first total synthesis of this natural product, which relies on enantioselective, zinc-mediated 1,3-dipolar cycloaddition and a sequence of cascading reactions, providing the key lipidated γ-amino acid found in mutanobactin D. The synthesis enables configurational assignment, determination of the dominant solution-state structure, and studies to assess the stability of the lipopeptide substructure found in the natural product. The information stored in the fingerprint region of the IR spectra in combination with quantum chemical calculations proved key to distinguishing between epimers of the α-substituted ß-keto amide. Synthetic mutanobactin D drives discovery and analysis of its effect on growth of other members of the human oral consortium. Our results showcase how total synthesis is central for elucidating the complex network of interspecies communications of human colonizers.

Oxidative stress induced by piperine leads to apoptosis in Candida albicans.

Candida albicans is a member of pathogens with potential drug resistance threat that needs novel chemotherapeutic strategies. Considering the multifarious biological activities including bioenhancer activity, anti-Candida potential of piperine was evaluated against planktonic/biofilm and hyphal growth of C. albicans alone or in combination as a synergistic agent with fluconazole. Piperine inhibits planktonic growth at or less than 15 µg/ml, hyphae induction at 5 µg/ml concentration, and exhibits stage-dependent activity against biofilm growth of a fluconazole-resistant strain of C. albicans (ATCC10231). Though piperine couldn't kill inoculum completely at minimum inhibitory concentration (MIC), it is fungicidal at higher concentrations, as shown in apoptosis assay. FIC index values indicate that piperine exhibits excellent synergistic activity with fluconazole against planktonic (0.123) and biofilm (0.215) growth of an FLC resistant strain. Mode of anti-Candida activity was studied by identifying piperine responsive proteins wherein the abundance of 25 proteins involved in stress response, signal transduction and cell cycle were modulated (22 up and 3 down-regulated) significantly in response to piperine (MIC50). Modulation of the proteins involved suggests that piperine affects membrane integrity leading to oxidative stress followed by cell cycle arrest and apoptosis in C. albicans. Flow cytometry-based mitochondrial membrane potential (MMP), cell cycle and apoptosis assay, as well as real-time quantitative polymerase chain reaction analysis of selected genes, confirms piperine induced oxidative stress (TRR1), cell cycle arrest and apoptosis (CaMCA1). Based on our results, we conclude that piperine inhibits planktonic and difficult-to treat-biofilm growth of C. albicans by affecting membrane integrity thereby inducing oxidative stress and apoptosis. LAY ABSTRACT: Piperine inhibit Candida albicans growth (planktonic and biofilm) significantly in our study. Piperine exhibits excellent synergistic potential with fluconazole The proteome analysis suggests that piperine induced membrane damage leads to oxidative stress followed by cell cycle arrest and apoptosis.

Inhibitory effects of essential oils from Asteraceae plant against pathogenic fungi of Panax notoginseng.

AIMS: Diseases caused by pathogenic fungi was a major constrain in increasing productivity and improving quality of Panax notoginseng. The aim of this research was to evaluate the inhibitory activity of essential oils (EOs) from Asteraceae family, Chrysanthemum indicum and Laggera pterodonta, against pathogenic fungi of P. notoginseng. METHODS AND RESULTS: The antifungal activity was investigated using multiple methods, disclosing that the EOs from C. indicum and L. pterodonta are active against hypha growth of different fungi but with different degrees of potency. Checkerboard testing indicated that the combination of EOs with hymexazol had synergistic effect against Pythium aphanidermatum, and exhibited additive effects against bulk of targeted pathogenic fungi. Besides, we found that the baseline sensitivity of Fusarium oxysporum to L. pterodonta EOs was higher than those of C. indicum by means of mycelium growth rate method. Finally, the practicability of those EOs as plant pesticide was confirmed by in vivo model showing that EOs can significantly inhibit the occurrence of root rot of P. notoginseng caused by F. oxysporum. CONCLUSION: Those studies suggest that the EOs from C. indicum and L. pterodonta had the potential to develop into new pollution-free pesticides for the protection of precious Chinese herbal medicines. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided a new way of biological control for overcoming the frequent diseases occurrence of P. notoginseng.

Enhanced in vitro antimicrobial activity of amphotericin B with berberine against dual-species biofilms of Candida albicans and Staphylococcus aureus.

AIMS: Multi-species biofilms formed by fungi and bacteria are clinically common and confer the commensal micro-organisms with protection against antimicrobial therapies. Previously, the plant alkaloid berberine was reported to show antimicrobial efficacy to eliminate bacterial and fungal biofilms. In this study, the combination of berberine and amphotericin B, an antifungal agent, was evaluated against dual-species Candida albicans/Staphylococcus aureus biofilms. METHODS AND RESULTS: Combinatorial treatment by berberine and amphotericin B significantly reduced the biomass and viability of residing species in biofilms. Moreover, morphological examination revealed hyphal filamentation of C. albicans and coadhesion between C. albicans/S. aureus were considerably impaired by the treatment. These effects coincided with the reduced expression of cell surface components and quorum-sensing-related genes in both C. albicans and S. aureus. Additionally, in C. albicans, the core transcription factors for controlling biofilm formation together with a crucial component of dual-species biofilms were also downregulated. CONCLUSIONS: These results demonstrated synergistic effects of berberine and amphotericin B against C. albicans/S. aureus dual-species biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the potential of berberine and amphotericin B for treating the C. albicans/S. aureus biofilms related infections and reveals molecular basis for the efficacy of combinatorial treatment.

Antifungal activity screening for mint and thyme essential oils against Rhizopus stolonifer and their application in postharvest preservation of strawberry and peach fruits.

AIMS: With a goal to identify specific essential oils that can control postharvest Rhizopus rot on strawberry and peach fruits, we performed screening for 26 essential oils based on their antifungal activity against Rhizopus stolonifer in vitro and investigated the underlying mechanism. METHODS AND RESULTS: Mentha spicata (Ms), Mentha piperita (Mp), Thymus vulgaris CT carvacrol (Tc) and Thymus vulgaris CT thymol (Tt) exhibited strong inhibition on R. stolonifer growth in the screening. These essential oils increased plasma membrane permeability of R. stolonifer and resulted in the outflow of intercellular electrolyte, nucleic acid, protein and soluble sugar. Morphology of R. stolonifer mycelia was greatly altered by these essential oils. Hyphae treated by these essential oils exhibited high accumulation of superoxide anion and malonaldehyde. Combination of these essential oils in commercial package reduced Rhizopus rot on strawberry and peach fruits, with Mp showing the strongest efficiency. CONCLUSION: Ms, Mp, Tc and Tt essential oils inhibited R. stolonifer growth by targeting its plasma membrane and reduced Rhizopus rot on strawberry and peach fruits. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides potential applications of natural plant extract, as alternatives to tradition fungicides, to control postharvest decay on fruits and vegetables.

Antifungal mechanism of Streptomyces ma. FS-4 on fusarium wilt of banana.

AIM: Research on prevention and cure of banana wilt is important to ensure the healthy development of the banana industry. In this study, antifungal mechanism of Streptomyces ma. FS-4 on fusarium wilt of banana was investigated. METHODS AND RESULTS: The physiological strain of banana fusarium pathogen Fusarium oxysporum f. sp. cubense Race 4 (FOC.4) was used as the target fungus, and the antifungal mechanism of the crude extract of Streptomyces ma. FS-4 was investigated. Eighteen different compounds identified by gas chromatography-mass spectrometry were composed of aldehydes, methyl, hydrocarbons, amides, esters and acids. FS-4 significantly inhibited the spore germination of the target fungi, with an EC50 of 22·78 µg ml-1 . After treatment with 100 µg ml-1 FS-4 crude extract, the N-acetylglucosamine content in the mycelium increased 1·95-fold. However, the extract had no significant effect on ß-1,3-glucanase. At the FS-4 crude extract dose of 100 µg ml-1 , the total sugar and protein contents decreased by 28·6 and 29·1% respectively, and the fat content was 41·3%. FS-4 significantly inhibited the activity of the mitochondrial complex III of Foc4, which was reduced by 52·45%. Moreover FS-4 reduced the activity of succinate dehydrogenase, a key enzyme in the Krebs cycle, by 60·2%. However, FS-4 had no significant effect on malate dehydrogenase. The membrane potential on the mitochondrial inner membrane was significantly reduced at the test concentration of 100 µg ml-1 . ROS gradually accumulated in the Foc4 hypha, and the burst was 3·97 times higher than the control. CONCLUSIONS: This study demonstrated that the antifungal mechanism of Streptomyces ma. FS-4 against Foc4 includes the destruction of the plasma membrane and mitochondrial dysfunction and finally induction of cell apoptosis. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may indicate the prevention and control of banana wilt, which is of great significance to the healthy development of banana industry system.

Different susceptibility of spores and hyphae of Trichophyton rubrum to methylene blue mediated photodynamic treatment in vitro.

BACKGROUND: In recent years, methylene blue mediated-photodynamic therapy (MB-PDT) has proved to be an effective inhibitor to a variety of microorganisms, including Trichophyton rubrum, the most common dermatophyte worldwide. However, previous studies mainly focused on the spore form of T rubrum, but rarely on its hyphal form, although the latter is the main pathogenic form of T rubrum in vivo. OBJECTIVE: To investigate the inhibitory effect of MB-PDT on T rubrum in different growth phases in vitro. METHODS: The suspensions of spores and hyphae obtained from T rubrum (ATCC28188) were prepared, respectively, incubated with MB solution (0.15-40 µg/mL) and irradiated with 635 nm red light. Varied light energy and MB concentration were used. The specimen in the absence of light exposure or/and MB served as controls. MIC determination, colony counts and MTT assay were employed to evaluate the antifungal effect of MB-PDT. RESULTS: The MICs of MB-PDT for hyphae and spores of T. rubrum were 6.300 ± 1.072 µg/mL and 1.984 ± 1.072 µg/mL, respectively, at a fixed light dose of 60 J/cm2 . CFU counts gave the minimum critical combinations of MB concentration and light dose to achieve 100% inhibitory rate. For hyphae, they were 5 µg/mL + 100 J/cm2 or 10 µg/mL + 60 J/cm2 . For spores, they were 1.25 µg/mL + 40 J/cm2 or 5 µg/mL + 20 J/cm2 . The outcomes of MTT assay were consistent with those of CFU counts, but less accurate. CONCLUSION: MB-PDT is a potent inhibitor to both spores and hyphae of T. rubrum in vitro, and the spores are more sensitive to it. Its antifungal efficacy is positively correlated with the concentration of MB and light dose.

Nicotinamide Mononucleotide Potentiates Resistance to Biotrophic Invasion of Fungal Pathogens in Barley.

Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), induces disease resistance to the Fusarium head blight fungus Fusarium graminearum in Arabidopsis and barley, but it is unknown at which stage of the infection it acts. Since the rate of haustorial formation of an obligate biotrophic barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was significantly reduced in NMN-treated coleoptile epidermal cells, the possibility that NMN induces resistance to the biotrophic stage of F. graminearum was investigated. The results show that NMN treatment caused the wandering of hyphal growth and suppressed the formation of appressoria-like structures. Furthermore, we developed an experimental system to monitor the early stage of infection in real-time and analyzed the infection behavior. We observed that the hyphae elongated windingly by NMN treatment. These results suggest that NMN potentiates resistance to the biotrophic invasion of F. graminearum as well as Bgh.

Adenophora triphylla var. japonica Inhibits Candida Biofilm Formation, Increases Susceptibility to Antifungal Agents and Reduces Infection.

(1) Background: Candida is the most common cause of fungal infections worldwide, but due to the limited option of antifungal therapies, alternative strategies are required. (2) Methods: Adenophora triphylla var. japonica extract was used for the biofilm formation assay using RPMI1640. The combinatorial antifungal assay, the dimorphic transition assay, and the adherence assay were done to see the influence of inhibition of biofilm formation. qRT-PCR analysis were performed to check the gene expression. (3) Results: Adenophora triphylla var. japonica extract inhibited the Candida biofilm formation. Treatment of extract increased the antifungal susceptibility of miconazole from a 37% reduction in fungal growth to 99.05%, and also dose-dependently reduced the dimorphic transition of Candida and the attachment of Candida to HaCaT cells. The extract blocked the expression of hyphal-related genes, extracellular matrix genes, Ras1-cAMP-PKA pathway genes, Cph2-Tec1 pathway gene, and MAP kinase pathway gene. (4) Conclusions: In this study, the treatment of Adenophora triphylla var. japonica extract showed inhibition of fungal biofilm formation, activation of antifungal susceptibility, and reduction of infection. These results suggest that fungal biofilm formation is a good target for the development of antifungal adjuvants, and Adenophora triphylla var. japonica extract should be a good candidate for biofilm-associated fungal infections.

Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species.

The increasing rate of fungal infections causes global problems not only in human healthcare but agriculture as well. To combat fungal pathogens limited numbers of antifungal agents are available therefore alternative drugs are needed. Antimicrobial peptides are potent candidates because of their broad activity spectrum and their diverse mode of actions. The model legume Medicago truncatula produces >700 nodule specific cysteine-rich (NCR) peptides in symbiosis and many of them have in vitro antimicrobial activities without considerable toxicity on human cells. In this work we demonstrate the anticandidal activity of the NCR335 and NCR169 peptide derivatives against five Candida species by using the micro-dilution method, measuring inhibition of biofilm formation with the XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, and assessing the morphological change of dimorphic Candida species by microscopy. We show that both the N- and C-terminal regions of NCR335 possess anticandidal activity as well as the C-terminal sequence of NCR169. The active peptides inhibit biofilm formation and the yeast-hypha transformation. Combined treatment of C. auris with peptides and fluconazole revealed synergistic interactions and reduced 2-8-fold the minimal inhibitory concentrations. Our results demonstrate that shortening NCR peptides can even enhance and broaden their anticandidal activity and therapeutic potential.