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.

The sterol C-24 methyltransferase encoding gene, erg6, is essential for viability of Aspergillus species.

Triazoles, the most widely used class of antifungal drugs, inhibit the biosynthesis of ergosterol, a crucial component of the fungal plasma membrane. Inhibition of a separate ergosterol biosynthetic step, catalyzed by the sterol C-24 methyltransferase Erg6, reduces the virulence of pathogenic yeasts, but its effects on filamentous fungal pathogens like Aspergillus fumigatus remain unexplored. Here, we show that the lipid droplet-associated enzyme Erg6 is essential for the viability of A. fumigatus and other Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Downregulation of erg6 causes loss of sterol-rich membrane domains required for apical extension of hyphae, as well as altered sterol profiles consistent with the Erg6 enzyme functioning upstream of the triazole drug target, Cyp51A/Cyp51B. Unexpectedly, erg6-repressed strains display wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, we show that erg6 repression results in significant reduction in mortality in a murine model of invasive aspergillosis. Taken together with recent studies, our work supports Erg6 as a potentially pan-fungal drug target.

Sensitivity of Globisporangium ultimum to the fungicide metalaxyl is enhanced by the infection with a toti-like mycovirus.

In recent years, numerous oomycete mycoviruses have been discovered; however, very few studies have focused on their effects on the host oomycete phenotype. In this study, we investigated the impact of toti-like Pythium ultimum RNA virus 2 (PuRV2) infection on the phytopathogenic soil-borne oomycete Globisporangium ultimum, which serves as a model species for Globisporangium and Pythium, specifically the UOP226 isolate in Japan. We generated a PuRV2-free isogenic line through hyphal tip isolation using high-temperature culture and subsequently compared the phenotypic characteristics and gene expression profiles of UOP226 and the PuRV2-free isogenic line. Our findings revealed that the metalaxyl sensitivity of UOP226 was greater than that of the PuRV2-free isogenic line, whereas the mycelial growth rate and colony morphology remained unchanged in the absence of the fungicide. Furthermore, transcriptome analyses using RNA-seq revealed significant downregulation of ABC-type transporter genes, which are involved in fungicide sensitivity, in UOP226. Our results suggest that PuRV2 infection influences the ecology of G. ultimum in agricultural ecosystems where metalaxyl is applied.

An oxylipin signal confers protection against antifungal echinocandins in pathogenic aspergilli.

Aspergillus fumigatus is the leading causative agent of life-threatening invasive aspergillosis in immunocompromised individuals. One antifungal class used to treat Aspergillus infections is the fungistatic echinocandins, semisynthetic drugs derived from naturally occurring fungal lipopeptides. By inhibiting beta-1,3-glucan synthesis, echinocandins cause both fungistatic stunting of hyphal growth and repeated fungicidal lysis of apical tip compartments. Here, we uncover an endogenous mechanism of echinocandin tolerance in A. fumigatus whereby the inducible oxylipin signal 5,8-diHODE confers protection against tip lysis via the transcription factor ZfpA. Treatment of A. fumigatus with echinocandins induces 5,8-diHODE synthesis by the fungal oxygenase PpoA in a ZfpA dependent manner resulting in a positive feedback loop. This protective 5,8-diHODE/ZfpA signaling relay is conserved among diverse isolates of A. fumigatus and in two other Aspergillus pathogens. Our findings reveal an oxylipin-directed growth program-possibly arisen through natural encounters with native echinocandin producing fungi-that enables echinocandin tolerance in pathogenic aspergilli.

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.

Ca2+ affects the hyphal differentiation to sclerotia formation of Athelia rolfsii.

RNA-Sequencing (RNA-Seq) and transcriptomic analyses have become powerful tools to study the developmental stages of fungal structures scuh as sclerotia. While RNA-Seq experiments have been set up for many important sclerotia- and microsclerotia-forming fungi, it has not been implemented to study Athelia rolfsii, which is one of the earliest fungi used in literature to uncover the roles of reactive oxygen species (ROS) in stimulating sclerotia formation. This study applied RNA-Seq to profile gene expression in four developmental stages of A. rolfsii sclerotia. Surprisingly, gene ontology and expression patterns suggested that most ROS-scavenging genes were not up-regulated in the stages from hyphal differentiation to the initial sclerotia stage. Using antioxidant and oxidant-amended culture assay, the results suggested none of the ascorbic acid, dithiothreitol (DTT), H2O2, or superoxide dismutase inhibitors [diethyldithiocarbamate (DETC), NaN3, and sodium dodecyl sulfate] affected the sclerotia number. Instead, only glutathione reduced the sclerotia number. Because glutathione has also been suggested to facilitate Ca2+ influx, therefore, glutathione culture assays with the combination of CaCl2, Ca2+-chelator egtazic acid, DETC, and H2O2 were tested on A. rolfsii, as well as two other fungi (Sclerotinia sclerotiorum and Macrophomina phaseolina) for comparison. Although the addition of CaCl2 caused sclerotia or microsclerotia reduction for all three fungi, the CaCl2-ROS interaction was only observed for S. sclerotiorum and M. phaseolina, but not A. rolfsi. Collectively, this study not only pointed out a conserved function of Ca2+ in suppressing fungal sclerotia and microsclerotia formation but also highlighted sclerotia formation of A. rolfsii being only sensitive to Ca2+ and independent of ROS stimuli.IMPORTANCEManagement for plant diseases caused by soil-borne fungal pathogens is challenging because many soil-borne fungal pathogens form sclerotia for long-term survival. Advanced understanding of the molecular and cellular mechanisms of sclerotia formation may provide novel insights to prevent these fungal residues in fields. This study discovered that Ca2+ acts as a negative signal cue to suppress sclerotia and microsclerotia formation in three economically important fungal pathogens. Moreover, the southern blight fungus Athelia rolfsii appears to be only regulated by Ca2+ but not reactive oxygen species. Accordingly, A. rolfsii can be a useful system for studying the detailed mechanism of Ca2+, and the applicability of Ca2+ in reducing sclerotia could be further assessed for disease management.

Multifactorial inhibition of Candida albicans by combinations of lactobacilli and probiotic Saccharomyces cerevisiae CNCM I-3856.

Strategies against the opportunistic fungal pathogen Candida albicans based on probiotic microorganisms represent a promising alternative to traditional antifungals. Here, we investigated the effects of Lactobacillaceae isolates from fermented foods or the human vagina, alone or in combination with the probiotic yeast Saccharomyces cerevisiae CNCM I-3856, against C. albicans in vitro. Nine out of nineteen tested strains of Lactobacillaceae inhibited growth of C. albicans with inhibition zones of 1-3 mm in spot assays. Five out of nineteen lactobacilli tested as such or in combination with S. cerevisiae CNCM I-3856 also significantly inhibited C. albicans hyphae formation, including Limosilactobacillus fermentum LS4 and L. fermentum LS5 resulting in respectively 62% and 78% hyphae inhibition compared to the control. Thirteen of the tested nineteen lactobacilli aggregated with the yeast form of C. albicans, with Lactiplantibacillus carotarum AMBF275 showing the strongest aggregation. The aggregation was enhanced when lactobacilli were combined with S. cerevisiae CNCM I-3856. No significant antagonistic effects were observed between the tested lactobacilli and S. cerevisiae CNCM I-3856. The multifactorial activity of Lactobacillaceae strains alone or combined with the probiotic S. cerevisiae CNCM I-3856 against C. albicans without antagonistic effects between the beneficial strains, paves the way for developing consortium probiotics for in vivo applications.

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.

Regulatory features of Candida albicans hemin-induced filamentation.

Candida albicans is a prominent fungal pathogen that can infect the bloodstream and deep tissues. One key pathogenicity trait is the ability to transition between yeast and hyphal growth. Hyphae are critical for the formation of biofilms, which in turn enable device-associated infection. Among signals that drive hypha formation is the presence of hemin, an oxidized Fe(III)-containing heme derivative found in blood. In this study, we asked 4 questions. First, how uniform is the filamentation response to hemin among C. albicans strains? We tested 26 diverse isolates and found that the strength of a strain's filamentation response to hemin reflected its filamentation level in the absence of hemin. Second, does hemin induce biofilm formation? Hemin biofilm induction was evident in 5 out of 10 isolates tested, including most of the weaker biofilm formers tested. Third, what is the gene expression response to hemin? We compared RNA-seq data for type strain SC5314 grown in pH 5.5 minimal media with or without hemin. We also compared that response to SC5314 grown in pH 7.0 minimal media, where it undergoes well-studied pH-dependent filamentation. We found a common set of 72 genes with upregulated RNA levels in response to both signals, including many known hypha-associated genes. Surprisingly, overlap among those 72 genes with 2 recent consensus definitions of hypha-associated genes was limited to only 16 genes. Fourth, which regulators govern hemin-induced filamentation? A mutant survey indicated that the response depends upon filamentation regulators Efg1, Brg1, and Rim101, but not upon heme acquisition regulator Hap1 or its target genes HMX1, RBT5, PGA10, PGA7, and CSA2. These findings argue that hemin induces hypha formation independently of its utilization.

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.

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.

Aspergillus nidulans Septa Are Indispensable for Surviving Cell Wall Stress.

Septation in filamentous fungi is a normal part of development, which involves the formation of cross-hyphal bulkheads, typically containing pores, allowing cytoplasmic streaming between compartments. Based on previous findings regarding septa and cell wall stress, we hypothesized that septa are critical for survival during cell wall stress. To test this hypothesis, we used known Aspergillus nidulans septation-deficient mutants (ΔsepH, Δbud3, Δbud4, and Δrho4) and six antifungal compounds. Three of these compounds (micafungin, Congo red, and calcofluor white) are known cell wall stressors which activate the cell wall integrity signaling pathway (CWIS), while the three others (cycloheximide, miconazole, and 2,3-butanedione monoxime) perturb specific cellular processes not explicitly related to the cell wall. Our results show that deficiencies in septation lead to fungi which are more susceptible to cell wall-perturbing compounds but are no more susceptible to other antifungal compounds than a control. This implies that septa play a critical role in surviving cell wall stress. IMPORTANCE The ability to compartmentalize potentially lethal damage via septation appears to provide filamentous fungi with a facile means to tolerate diverse forms of stress. However, it remains unknown whether this mechanism is deployed in response to all forms of stress or is limited to specific perturbations. Our results support the latter possibility by showing that presence of septa promotes survival in response to cell wall damage but plays no apparent role in coping with other unrelated forms of stress. Given that cell wall damage is a primary effect caused by exposure to the echinocandin class of antifungal agents, our results emphasize the important role that septa might play in enabling resistance to these drugs. Accordingly, the inhibition of septum formation could conceivably represent an attractive approach to potentiating the effects of echinocandins and mitigating resistance in human fungal pathogens.

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.

Quantitative Monitoring of Mycelial Growth of Aspergillus fumigatus in Liquid Culture by Optical Density.

Filamentous fungi form multicellular hyphae, which generally form pellets in liquid shake cultures, during the vegetative growth stage. Because of these characteristics, growth-monitoring methods commonly used in bacteria and yeast have not been applied to filamentous fungi. We have recently revealed that the cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) contribute to hyphal aggregation in Aspergillus oryzae. Here, we tested whether Aspergillus fumigatus shows dispersed growth in liquid media that can be quantitatively monitored, similar to that of yeasts. We constructed a double disruptant mutant of both the primary α-1,3-glucan synthase gene ags1 and the putative GAG synthase gene gtb3 in A. fumigatus AfS35 and found that the hyphae of this mutant were fully dispersed. Although the mutant lost α-1,3-glucan and GAG, its growth and susceptibility to antifungal agents were not different from those of the parental strain. Mycelial weight of the mutant in shake-flask cultures was proportional to optical density for at least 18 h. We were also able to quantify the dose response of hyphal growth to antifungal agents by measuring optical density. Overall, we established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly used for screening for novel antifungals against Aspergillus species. IMPORTANCE Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi so that we may apply the methods used for unicellular organisms such as yeast and bacteria. In the present study, by using the fungal pathogen Aspergillus fumigatus strain with modified hyphal surface polysaccharides, we succeeded in monitoring the hyphal growth quantitatively by optical density. The principle of this easy measurement by optical density could lead to a novel standard of hyphal quantification such as those that have been used for yeasts and bacteria. Dose response of hyphal growth by antifungal agents could also be monitored. This method could be useful for screening for novel antifungal reagents against Aspergillus species.

Presence of Malassezia Hyphae Is Correlated with Pathogenesis of Seborrheic Dermatitis.

Seborrheic dermatitis (SD) is a common, chronic, and relapsing skin disease. The roles of Malassezia spp. in the pathogenesis of SD are still not clear due to the lack of direct evidence for the existence of hyphae within affected skin tissues. We set out to elucidate if Malassezia mycelium contributes to the onset and development of SD and if Malassezia mycelium is correlated with the clinical severity of SD patients. We detected Malassezia hyphae in patients with SD using potassium hydroxide (KOH) and calcofluor white (CFW) staining. Fluorescent microscopy was performed for the analysis of fungal cell wall and morphological characteristics of Malassezia under CFW staining. Culture growth in modified Dixon agar was used for DNA extraction and sequencing, and Malassezia species were confirmed by a sequencing data BLAST search against the NCBI database. We demonstrated that Malassezia hyphae were positively correlated with the clinical severity of SD patients (P = 3.1738 × 10-11). All the patients responded well to antifungal treatment. There is no significant difference for species dominance across the variant groups. However, the exact molecular mechanisms of how Malassezia spp. affect SD need to be further explored. The results show that Malassezia spp. in the hyphal stage are restricted to SD patients compared with healthy controls, suggesting that the presence of Malassezia hyphae contributes to the pathogenesis of SD. The results highlight the importance of the antifungal therapy for the future treatment of SD patients. IMPORTANCE Our results support the proposal that the hyphal form of Malassezia could be one of the pathogenic factors that contribute to SD, which has been previously less well studied. This clinical observation paves the way for further investigations of the molecular mechanisms of Malassezia hyphal pathogenicity in SD.

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.

Superoxide Initiates the Hyphal Differentiation to Microsclerotia Formation of Macrophomina phaseolina.

The infection of Macrophomina phaseolina often results in a grayish appearance with numerous survival structures, microsclerotia, on the plant surface. Past works have studied the development of fungal survival structures, sclerotia and microsclerotia, in the Leotiomycetes and Sordariomycetes. However, M. phaseolina belongs to the Dothideomycetes, and it remains unclear whether the mechanism of microsclerotia formation remains conserved among these phylogenetic clades. This study applied RNA-sequencing (RNA-Seq) to profile gene expressions at four stages of microsclerotia formation, and the results suggested that reactive oxygen species (ROS)-related functions were significantly different between the microsclerotia stages and the hyphal stage. Microsclerotia formation was reduced in the plates amended with antioxidants such as ascorbic acid, dithiothreitol (DTT), and glutathione. Surprisingly, DTT drastically scavenged H2O2, but the microsclerotia amount remained similar to the treatment of ascorbic acid and glutathione that both did not completely eliminate H2O2. This observation suggested the importance of [Formula: see text] over H2O2 in initiating microsclerotia formation. To further validate this hypothesis, the superoxide dismutase 1 (SOD1) inhibitor diethyldithiocarbamate trihydrate (DETC) and H2O2 were tested. The addition of DETC resulted in the accumulation of endogenous [Formula: see text] and more microsclerotia formation, but the treatment of H2O2 did not. The expression of SOD1 genes were also found to be upregulated in the hyphae to the microsclerotia stage, which suggested a higher endogenous [Formula: see text] stress presented in these stages. In summary, this study not only showed that the ROS stimulation remained conserved for initiating microsclerotia formation of M. phaseolina but also highlighted the importance of [Formula: see text] in initiating the hyphal differentiation to microsclerotia formation. IMPORTANCE Reactive oxygen species (ROS) have been proposed as the key stimulus for sclerotia development by studying fungal systems such as Sclerotinia sclerotiorum, and the theory has been adapted for microsclerotia development in Verticillium dahliae and Nomuraea rileyi. While many studies agreed on the association between (micro)sclerotia development and the ROS pathway, which ROS type, superoxide ([Formula: see text]) or hydrogen peroxide (H2O2), plays a major role in initiating hyphal differentiation to the (micro)sclerotia formation remains controversial, and literature supporting either [Formula: see text] or H2O2 can be found. This study confirmed the association between ROS and microsclerotia formation for the charcoal rot fungus Macrophomina phaseolina. Moreover, the accumulation of [Formula: see text] but not H2O2 was found to induce higher density of microsclerotia. By integrating transcriptomic and phenotypic assays, this study presented the first conclusive case for M. phaseolina that [Formula: see text] is the main ROS stimulus in determining the amount of microsclerotia formation.

Vascular pythiosis caused by Pythium aphanidermatum: the first case report in Asia.

BACKGROUND: Pythium, soil-borne plant pathogens, are in the class Oomycetes. They are not true fungi, but are related to diatom and algae. There are two human pathogens including P. insidiosum and P. aphanidermatum. To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia. CASE PRESENTATION: A 47-year-old Thai woman, living in North Thailand, with ß thalassemia/hemoglobin E presented with acute recurrent arterial insufficiency of both legs. Emergent embolectomy with clot removal was performed. The pathology of the clot exhibited noncaseous granulomatous inflammation with many fungal hyphal elements. PCR identified P. aphanidermatum with 100% identity. Final diagnosis is vascular pythiosis. Unfortunately, the patient eventually expired after treatment with itraconazole, terbinafine, azithromycin, and doxycycline. CONCLUSIONS: To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia.

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.

A small molecule produced by Lactobacillus species blocks Candida albicans filamentation by inhibiting a DYRK1-family kinase.

The fungus Candida albicans is an opportunistic pathogen that can exploit imbalances in microbiome composition to invade its human host, causing pathologies ranging from vaginal candidiasis to fungal sepsis. Bacteria of the genus Lactobacillus are colonizers of human mucosa and can produce compounds with bioactivity against C. albicans. Here, we show that some Lactobacillus species produce a small molecule under laboratory conditions that blocks the C. albicans yeast-to-filament transition, an important virulence trait. It remains unexplored whether the compound is produced in the context of the human host. Bioassay-guided fractionation of Lactobacillus-conditioned medium linked this activity to 1-acetyl-ß-carboline (1-ABC). We use genetic approaches to show that filamentation inhibition by 1-ABC requires Yak1, a DYRK1-family kinase. Additional biochemical characterization of structurally related 1-ethoxycarbonyl-ß-carboline confirms that it inhibits Yak1 and blocks C. albicans biofilm formation. Thus, our findings reveal Lactobacillus-produced 1-ABC can prevent the yeast-to-filament transition in C. albicans through inhibition of Yak1.