Deciphering the salt induced morphogenesis and functional potentials of Hortaea werneckii; a black pigmented halotolerant yeast isolated from solar saltern.

An intense black pigmented halotolerant yeast GUBPC1, was obtained from the solar salterns of Nerul, Goa-India. The isolate could tolerate 0 to 20 % NaCl. FE-SEM analysis revealed its polymorphic nature, exhibiting oval cells at higher salt concentrations and filamentous spindle like shapes at lower concentrations. Initially, the cells appear oval, yeast like in shape but gradually after 15 days of incubation, it becomes elongated and undergoes budding, exhibiting various budding patterns, from single polar bud to bipolar buds with annellidic ring, to lateral buds and eventually forming filamentous hyphae. The intracellular black pigment was identified as melanin based on ultraviolet-visible spectroscopy analysis. The molecular identification of the culture showed closest similarity with Hortaea werneckii. Plant polymer-degrading enzymatic activities such as cellulase, laccase, chitinase, xylanase, pectinase, amylase and protease were exhibited by the isolate GUBPC1. To further understand and explore its biotechnological potential, we performed whole-genome sequencing and analysis. The obtained genome size was 26.93 Mb with 686 contigs and a GC content of 53.24 %. We identified 9383 protein-coding genes, and their functional annotation revealed the presence of 435 CAZyme genes and 16 functional genes involved in secondary metabolite synthesis, thus providing a basis for its potential value in various biotechnological applications.

Hyphal swelling induced in the phagosome of macrophages.

Macrophages play critical protective roles as sentinels of the innate immune system against fungal infection. It is therefore important to understand the dynamics of the interaction between these phagocytes and their fungal prey. We show here that many of the hyphal apices formed by Candida albicans within the macrophage ceased elongating, and apical and sub-apical hyphal compartments became swollen. Swollen hyphal cell compartments assimilated less Lysotracker-Red than non-swollen compartments, suggesting they had enhanced viability. Staining with florescent dyes suggested that there were higher levels of ß-glucan and chitin in internalized fungal filaments compared to non-internalized hyphae, suggesting active cell wall remodelling within macrophages. These observations suggest that the stresses imposed by macrophages upon the fungus lead to changes in cell wall composition, inhibition of polarised growth and the induction of swelling in hyphal compartments, and that this can prevent or delay loss of viability of hyphal cells within the phagocyte.

Histone-like transcription factor Hfl1p in Candida albicans harmonizes nuclear and mitochondrial genomic network in regulation of energy metabolism and filamentation development.

Candida albicans is an opportunistic fungal pathogen known for surviving in various nutrient-limited conditions within the host and causing infections. Our prior research revealed that Hfl1p, an archaeal histone-like or Hap5-like protein, is linked to mitochondrial ATP generation and yeast-hyphae morphogenesis. However, the specific roles of Hfl1p in these virulence behaviours, through its function in the CBF/NF-Y complex or as a DNA polymerase II subunit, remain unclear. This study explores Hfl1p's diverse functions in energy metabolism and morphogenesis. By combining proteomic analysis and phenotypic evaluations of the hfl1Δ/hfl1Δ mutant with ChIP data, we found that Hfl1p significantly impacts mitochondrial DNA-encoded CI subunits, the tricarboxylic acid (TCA) cycle, and morphogenetic pathways. This influence occurs either independently or alongside other transcription factors recognizing a conserved DNA motif (TAXXTAATTA). These findings emphasize Hfl1p's critical role in linking carbon metabolism and mitochondrial respiration to the yeast-to-filamentous form transition, enhancing our understanding of C. albicans' metabolic adaptability during morphological transition, an important pathogenic trait of this fungus. This could help identify therapeutic targets by disrupting the relationship between energy metabolism and cell morphology in C. albicans.

Revealing real-time 3D in vivo pathogen dynamics in plants by label-free optical coherence tomography.

Microscopic imaging for studying plant-pathogen interactions is limited by its reliance on invasive histological techniques, like clearing and staining, or, for in vivo imaging, on complicated generation of transgenic pathogens. We present real-time 3D in vivo visualization of pathogen dynamics with label-free optical coherence tomography. Based on intrinsic signal fluctuations as tissue contrast we image filamentous pathogens and a nematode in vivo in 3D in plant tissue. We analyze 3D images of lettuce downy mildew infection (Bremia lactucae) to obtain hyphal volume and length in three different lettuce genotypes with different resistance levels showing the ability for precise (micro) phenotyping and quantification of the infection level. In addition, we demonstrate in vivo longitudinal imaging of the growth of individual pathogen (sub)structures with functional contrast on the pathogen micro-activity revealing pathogen vitality thereby opening a window on the underlying molecular processes.

[Aspergillus Cell Surface Structural Analysis and Its Applications to Industrial and Medical Use].

The hyphal surface of cells of filamentous fungi is covered with cell wall, which is mainly composed of polysaccharides. Since the cell wall is the first structure to come in contact with the infection host, the environment, and the fungus itself, the elucidation of the cell wall structure and biogenesis is essential for understanding fungal ecology. Among filamentous fungi, the genus Aspergillus is an important group in the industrial, food, and medical fields. It is known that Aspergillus species form hyphal pellets in shake liquid culture. The authors previously found the role of α-1,3-glucan in hyphal aggregation in Aspergillus species. In addition, extracellular polysaccharide galactosaminogalactan contributed to hyphal aggregation as well, and dual disruption of biosynthesis genes of α-1,3-glucan and galactosaminogalactan resulted in complete hyphal dispersion in shake liquid culture. The characteristic of mycelia to form pellets under liquid culture conditions was the main reason why the growth measurement methods used for unicellular organisms could not be applied. We reported that hyphal growth of the dual disruption mutant could be measured by optical density. A real-time plate reader could be used to determine the growth curve of the mycelial growth of the dual disruption mutant. This measurement approach not only provides basic microbiological insights in filamentous fungi, but also has the potential to be applied to high-throughput screening of anti-Aspergillus drugs.

Antifungal activities of Equol against Candida albicans in vitro and in vivo.

Candida albicans is an opportunistic fungal pathogen that can cause systemic infections in immunocompromised individuals. Morphological transition and biofilm formation are major virulence factors of C. albicans. Moreover, biofilm enhances resistance to antifungal agents. Therefore, it is urgent to identify new and effective compounds to target the biofilm of C. albicans. In the present study, the antifungal activities of equol against C. albicans were investigated. In vitro, the microdilution analysis and spot assay result showed that equol exhibited potent inhibitory activities against C. albicans. Further investigations confirmed that the antifungal effects of equol involved interference with the transition from yeast to hypha and biofilm formation of C. albicans. In addition, transcriptome sequencing and reverse transcription-quantitative PCR (qRT-PCR) analysis showed that equol significantly downregulated the expression of several genes in the Ras1-cAMP-PKA pathway related to hyphae and biofilm formation and significantly upregulated the expression of the negative transcriptional repressors RFG1 and TUP1. Moreover, equol effectively reduced the production of cAMP, a key messenger in the Ras1-cAMP-PKA pathway, while supplementation with cAMP partly rescued the equol-induced defects in hyphal development. Furthermore, in a mouse model of systemic candidiasis (SC), equol treatment significantly decreased the fungal burden (liver, kidneys, and lung) in mice and local tissue damage, while enhancing the production of interleukin-10 (IL-10). Together, these findings confirm that equol is a potentially effective agent for treatment of SC.

Stealth strategies of Candida albicans to evade host immunity.

During opportunistic pathogenic episodes, Candida albicans employs classical strategies such as the yeast-to-hyphae transition and immunogenic masking. In this issue of Cell Host & Microbe, Luo et al. unveil that the effector protein Cmi1 can be translocated into host cells and targets TBK1, thereby negatively regulating the host's antifungal immune responses.

Replicating biological 3D root and hyphal networks in transparent glass chips.

Replicating the complex 3D microvascular architectures found in biological systems is a critical challenge in tissue engineering and other fields requiring efficient mass transport. Conventional microfabrication techniques often face limitations in creating extensive hierarchical networks, especially within bulk materials. Here, we report a versatile bioinspired approach to generate optimized 3D microvascular networks within transparent glass matrix by transcribing the natural growth patterns of plants and fungi. Plant seeds or fungal spores are first cultivated on nanoparticle-based culture media. Subsequent heat treatment removes the biological species while sintering the surrounding compound into a solidified chip with replica root/hyphal architectures as open microchannels. A diverse range of architectures, including the hierarchical branching of plant roots and the intricate networks formed by fungal hyphae, can be faithfully replicated. The resultant glass microvascular networks exhibit high chemical and thermal stability, enabling applications under harsh conditions. Fluid flow experiments validate the functionalities of the fabricated channels. By co-cultivating plants and fungi, hierarchical multi-scale architectures mimicking natural vascular systems are achieved. This bioinspired manufacturing technique leverages autonomous biological growth for architectural optimization, offering a complementary approach to existing microfabrication methods. The transparent nature of the glass chips allows for direct optical inspection, potentially facilitating integration with imaging components. This versatile platform holds promise for various engineering applications, such as microreactors, heat exchangers, and advanced filtration systems.

Ctr9 promotes virulence of Candida albicans by regulating methionine metabolism.

Candida albicans, a part of normal flora, is an opportunistic fungal pathogen and causes severe health issues in immunocompromised patients. Its pathogenicity is intricately linked to the transcriptional regulation of its metabolic pathways. Paf1 complex (Paf1C) is a crucial transcriptional regulator that is highly conserved in eukaryotes. The objective of this study was to explore the role of Paf1C in the metabolic pathways and how it influences the pathogenicity of C. albicans. Paf1C knockout mutant strains of C. albicans (ctr9Δ/Δ, leo1Δ/Δ, and cdc73Δ/Δ) were generated using the CRISPR-Cas9 system. To investigate the effect of Paf1C on pathogenicity, macrophage interaction assays and mouse survival tests were conducted. The growth patterns of the Paf1C knockout mutants were analyzed through spotting assays and growth curve measurements. Transcriptome analysis was conducted under yeast conditions (30°C without serum) and hyphal conditions (37°C with 10% FBS), to further elucidate the role of Paf1C in the pathogenicity of C. albicans. CTR9 deletion resulted in the attenuation of C. albicans virulence, in macrophage and mouse models. Furthermore, we confirmed that the reduced virulence of the ctr9Δ/Δ mutant can be attributed to a decrease in C. albicans cell abundance. Moreover, transcriptome analysis revealed that metabolic processes required for cell proliferation are impaired in ctr9Δ/Δ mutant. Notably, CTR9 deletion led to the downregulation of methionine biosynthetic genes and the cAMP-PKA signaling pathway-related hypha essential genes, which are pivotal for virulence. Our results suggest that Ctr9-regulated methionine metabolism is a crucial factor for determining C. albicans pathogenicity.

Glucocorticoids Suppress NF-κB-Mediated Neutrophil Control of Aspergillus fumigatus Hyphal Growth.

Glucocorticoids are a major class of therapeutic anti-inflammatory and immunosuppressive drugs prescribed to patients with inflammatory diseases, to avoid transplant rejection, and as part of cancer chemotherapy. However, exposure to these drugs increases the risk of opportunistic infections such as with the fungus Aspergillus fumigatus, which causes mortality in >50% of infected patients. The mechanisms by which glucocorticoids increase susceptibility to A. fumigatus are poorly understood. In this article, we used a zebrafish larva Aspergillus infection model to identify innate immune mechanisms altered by glucocorticoid treatment. Infected larvae exposed to dexamethasone succumb to infection at a significantly higher rate than control larvae. However, both macrophages and neutrophils are still recruited to the site of infection, and dexamethasone treatment does not significantly affect fungal spore killing. Instead, the primary effect of dexamethasone manifests later in infection with treated larvae exhibiting increased invasive hyphal growth. In line with this, dexamethasone predominantly inhibits neutrophil function rather than macrophage function. Dexamethasone-induced mortality also depends on the glucocorticoid receptor. Dexamethasone partially suppresses NF-κB activation at the infection site by inducing the transcription of IκB via the glucocorticoid receptor. Independent CRISPR/Cas9 targeting of IKKγ to prevent NF-κB activation also increases invasive A. fumigatus growth and larval mortality. However, dexamethasone treatment of IKKγ crispant larvae further increases invasive hyphal growth and host mortality, suggesting that dexamethasone may suppress other pathways in addition to NF-κB to promote host susceptibility. Collectively, we find that dexamethasone acts through the glucocorticoid receptor to suppress NF-κB-mediated neutrophil control of A. fumigatus hyphae in zebrafish larvae.

Evaluation of antifungal spectrum of Cupferron against Candida albicans.

Candida albicans is an opportunistic yeast accounting for about 50-90 % of all cases of candidiasis in humans, ranging from superficial to systemic potentially life-threatening infections. The presence of several virulence factors, including biofilm, hyphal transition, and proteolytic enzymes production, worsens the fungal infections burden on healthcare system resources. Hence, developing new bioactive compounds with antifungal activity is a pressing urgence for the scientific community. In this perspective, we evaluated the anti-Candida potential of the N-Nitroso-N-phenylhydroxylamine ammonium salt (cupferron) against standard and clinical C. albicans strains. Firstly, the in vitro cytotoxicity of cupferron was checked in the range 400-12.5 µg/mL against human microglial cells (HMC-3). Secondly, its antifungal spectrum was explored via disk diffusion test, broth-microdilution method, and time-killing curve analysis, validating the obtained results through scanning electron microscopy (SEM) observations. Additionally, we evaluated the cupferron impact on the main virulence determinants of Candida albicans. At non-toxic concentrations (100-12.5 µg/mL), the compound exerted interesting anti-Candida activity, registering a minimum inhibitory concentration (MIC) between 50 and 100 µg/mL against the tested strains, with a fungistatic effect until 100 µg/mL. Furthermore, cupferron was able to counteract fungal virulence at MIC and sub-MIC values (50-12.5 µg/mL). These findings may propose cupferron as a new potential antifungal option for the treatment of Candida albicans infections.

Discovery of petroselinic acid with in vitro and in vivo antifungal activity by targeting fructose-1,6-bisphosphate aldolase.

BACKGROUND: The incidence of invasive fungal diseases (IFDs), represented by Candida albicans infection, is increasing year by year. However, clinically available antifungal drugs are very limited and encounter challenges such as limited efficacy, drug resistance, high toxicity, and exorbitant cost. Therefore, there is an urgent need for new antifungal drugs. PURPOSE: This study aims to find new antifungal compounds from plants, preferably those with good activity and low toxicity, and reveal their antifungal targets. METHODS: In vitro antifungal activities of compounds were investigated using broth microdilution method, spot assay, hyphal growth assay and biofilm formation assay. Synergistic effects were assessed using broth microdilution checkerboard technique. In vivo antifungal activities were evaluated using Galleria mellonella and murine candidiasis models. Cytotoxicity of compounds was investigated using Cell Counting Kit-8 (CCK-8). Discovery and validation of antifungal targets of compounds were conducted by using monoallelic knockout library of C. albicans, haploinsufficiency profiling (HIP), thermal shift assay (TSA), enzyme inhibitory effect assay, molecular docking, and in vitro and in vivo antifungal studies. RESULTS: 814 plant products were screened, among which petroselinic acid (PeAc) was found as an antifungal molecule. As a rare fatty acid isolated from coriander (Coriandrum sativum), carrot (Daucus carota) and other plants of the Apiaceae family, PeAc had not previously been found to have antifungal effects. In this study, PeAc was revealed to inhibit the growth of various pathogenic fungi, exhibited synergistic effects with fluconazole (FLC), inhibited the formation of C. albicans hyphae and biofilms, and showed antifungal effects in vivo. PeAc was less toxic to mammalian cells. Fructose-1,6-bisphosphate aldolase (Fba1p) was identified as a target of PeAc by using HIP, TSA, enzyme inhibitory effect assay and molecular docking methods. PeAc exerted antifungal effects more effectively on fba1Δ/FBA1 than wild-type (WT) strain both in vitro and in vivo. CONCLUSIONS: PeAc is an effective and low toxic antifungal compound. The target of PeAc is Fba1p. Fba1p is a promising target for antifungal drug development.

Mechanistic insights into antifungal potential of Alexidine dihydrochloride and hexachlorophene in Candida albicans: a drug repurposing approach.

Candida albicans has been listed in the critical priority group by the WHO in 2022 depending upon its contribution in invasive candidiasis and increased resistance to conventional drugs. Drug repurposing offers an efficient, rapid, and cost-effective solution to develop alternative therapeutics against pathogenic microbes. Alexidine dihydrochloride (AXD) and hexachlorophene (HCP) are FDA approved anti-cancer and anti-septic drugs, respectively. In this study, we have shown antifungal properties of AXD and HCP against the wild type (reference strain) and clinical isolates of C. albicans. The minimum inhibitory concentrations (MIC50) of AXD and HCP against C. albicans ranged between 0.34 and 0.69 µM and 19.66-24.58 µM, respectively. The biofilm inhibitory and eradication concentration of AXD was reported comparatively lower than that of HCP for the strains used in the study. Further investigations were performed to understand the antifungal mode of action of AXD and HCP by studying virulence features like cell surface hydrophobicity, adhesion, and yeast to hyphae transition, were also reduced upon exposure to both the drugs. Ergosterol content in cell membrane of the wild type strain was upregulated on exposure to AXD and HCP both. Biochemical analyses of the exposed biofilm indicated reduced contents of carbohydrate, protein, and e-DNA in the extracellular matrix of the biofilm when compared to the untreated control biofilm. AXD exposure downregulated activity of tissue invading enzyme, phospholipase in the reference strain. In wild type strain, ROS level, and activities of antioxidant enzymes were found elevated upon exposure to both drugs. FESEM analysis of the drug treated biofilms revealed degraded biofilm. This study has indicated mode of action of antifungal potential of alexidine dihydrochloride and hexachlorophene in C. albicans.

Biochemical Characterization of Recombinant Enterococcus faecalis EntV Peptide to Elucidate Its Antihyphal and Antifungal Mechanisms against Candida albicans.

Candida albicans is a common opportunistic fungus in humans, whose morphological switch between yeast and hyphae forms represents a key virulence trait. Developing strategies to inhibit C. albicans hyphal growth may provide insights into designs of novel antivirulent therapeutics. Importantly, the gut commensal bacterium, Enterococcus faecalis, secretes a bacteriocin EntV which has potent antivirulent and antifungal effects against C. albicans in infection models; however, hampered by the challenges to access large quantities of bioactive EntV, the detailed understanding of its mechanisms on C. albicans has remained elusive. In this work, we biochemically reconstituted the proteolytic cleavage reaction to obtain recombinant EntV88-His6 on a large preparative scale, providing facile access to the C-terminal EntV construct. Under in vitro C. albicans hyphal assay with specific inducers, we demonstrated that EntV88-His6 exhibits potent bioactivity against GlcNAc-triggered hyphal growth. Moreover, with fluorescent FITC-EntV88-His6, we revealed that EntV88-His6 enters C. albicans via endocytosis and perturbs the proper localization of the polarisome scaffolding Spa2 protein. Our findings provide important clues on EntV's mechanism of action. Surprisingly, we showed that EntV88-His6 does not affect C. albicans yeast cell growth but potently exerts cytotoxicity against C. albicans under hyphal-inducing conditions in vitro. The combination of EntV88-His6 and GlcNAc displays rapid killing of C. albicans, rendering it a promising antivirulent and antifungal agent.

VmSom1 is essential for growth, development, maintenance of cell wall integrity and virulence in Valsa mali.

Apple Valsa canker disease, caused by Valsa mali Miyabe et Yamada, severely endangers the healthy growth of apple trees. The Som1, located downstream of the cyclic AMP-dependent protein kinase A (cAMP-PKA) pathway, plays crucial roles in the growth, development, morphological differentiation, and virulence of filamentous fungi. In this study, we identify and functionally characterize VmSom1, a homolog of Som1, in Valsa mali. The VmSom1 gene is located on chromosome 12, encoding an 824 amino acid protein. Phylogenetic analysis reveals VmSom1 as a fungal Som1 homolog. The VmSom1 deletion mutants exhibit slower growth rates and fail to produce pycnidia. Additionally, their hyphal growth is significantly inhibited on media containing Calcofluor White, Congo Red, NaCl, and sorbitol. The growth rate of VmSom1 deletion mutants is reduced on maltose, lactose, sucrose and fructose media but increases on glucose medium. Moreover, the mycelial growth rate of the VmSom1 deletion mutant is significantly lower than that of the wild-type strain in peptone, NH4SO4, NaNO3, and no nitrogen. Notably, the distances between the septa increase, and chitin concentration shifts to the hyphal tip in the VmSom1 deletion mutant. Furthermore, compared with the wild-type strain, the VmSom1 deletion mutant exhibits fewer diseased spots on apple fruit and branches. Overall, our findings demonstrate that VmSom1 is involved in regulating the growth and development, colony surface hydrophobicity, osmotic stress, cell wall integrity maintenance, carbon and nitrogen source utilization, septa formation, and virulence of V. mali.

Inhibitory effects and mode of antifungal action of isobavachalcone on Candida albicans growth and virulence factors.

The fungus Candida albicans causes various kinds of human infections, including oral thrush, vulvovaginitis and life-endangering bloodstream infections, the incidence of which are rising. Worsening this, the clinical antifungals are limited to a few, highlighting the necessity to develop novel antifungal therapies. In this study, the antifungal activities of isobavachalcone against C. albicans SC5314 and nine C. albicans clinical isolates were tested. The effects of isobavachalcone (IBC) on C. albicans virulence factors, such as hyphal formation, adhesion, biofilm formation and extracellular phospholipase production, as well as the underlying mechanism, were also evaluated. Antifungal susceptibility test revealed that IBC has significant anti-Candida activities, with both MIC and MFC being 4-5 µg/mL against all strains tested. Hyphal formation in RPMI-1640, Spider and GlcNAc medium, adhesion to abiotic polystyrene surfaces and surfaces of A549 cells, could be inhibited by IBC. Most important, IBC could inhibit the C. albicans biofilm formation and development. PI staining tests showed that IBC could increase the cell membrane permeability, suggesting the damages to the fungal cell membrane. IBC was further demonstrated to induce excessive ROS production in C. albicans planktonic cells and its mature biofilms, as revealed by DCFH fluorescence detection through flowcytometry and relative fluorescence intensity analysis (with a microplate reader). The roles of ROS in the antifungal activity of IBC were further confirmed through antioxidant rescue assays in MIC and biofilm formation tests. Compared to its antifungal activity, the cytotoxicity against mammalian cells was low, indicating its potential in developing antifungal therapies.

The ER-Resident Ras Inhibitor 1 (Eri1) of Candida albicans Inhibits Hyphal Morphogenesis via the Ras-Independent cAMP-PKA Pathway.

Ras signaling and glycosylphosphatidylinositol (GPI) biosynthesis are mutually inhibitory in S. cerevisiae (Sc). The inhibition is mediated via an interaction of yeast Ras2 with the Eri1 subunit of its GPI-N-acetylglucosaminyl transferase (GPI-GnT), the enzyme catalyzing the very first GPI biosynthetic step. In contrast, Ras signaling and GPI biosynthesis in C. albicans (Ca) are mutually activated and together control the virulence traits of the human fungal pathogen. What might be the role of Eri1 in this pathogen? The present manuscript addresses this question while simultaneously characterizing the cellular role of CaEri1. It is either nonessential or required at very low levels for cell viability in C. albicans. Severe depletion of CaEri1 results in reduced GPI biosynthesis and cell wall defects. It also produces hyperfilamentation phenotypes in Spider medium as well as in bicarbonate medium containing 5% CO2, suggesting that both the Ras-dependent and Ras-independent cAMP-PKA pathways for hyphal morphogenesis are activated in these cells. Pull-down and acceptor-photobleaching FRET experiments suggest that CaEri1 does not directly interact with CaRas1 but does so through CaGpi2, another GPI-GnT subunit. We showed previously that CaGpi2 is downstream of CaEri1 in cross talk with CaRas1 and for Ras-dependent hyphal morphogenesis. Here we show that CaEri1 is downstream of all GPI-GnT subunits in inhibiting Ras-independent filamentation. CaERI1 also participates in intersubunit transcriptional cross talk within the GPI-GnT, a feature unique to C. albicans. Virulence studies using G. mellonella larvae show that a heterozygous strain of CaERI1 is better cleared by the host and is attenuated in virulence.

Unusual plant-extract based media for the differentiation between species in the Candida albicans complex: A comparative study.

Candida albicans is the most common agent in human fungal infections; nevertheless, in the last decades, the closely related yeasts Candida dubliniensis and Candida africana have emerged as pathogens. The purpose of this study was to compare tobacco agar with another five agars prepared from plant extracts (Origanum vulgare, Rosmarinus officinalis, Solanum rudepannum, Solanum oblongifolium and Brugmansia arborea) on the differentiation of C. albicans complex. The hyphae and chlamyconidia formation and the color and margin of the colonies of 200 clinical isolates of C. albicans, C. dubliniensis and C. africana were evaluated. After seven days of incubation at 28 °C, Tobacco agar, S. rudepannum and B. arborea agars allowed the differentiation of 100 % C. dubliniensis. Additionally, 24% of C. africana isolates produced brownish colonies in the medium prepared from Rosmarinus officinalis (rosemary) extract. These results indicate that S. rudepannun, B. arborea and rosemary agar could be used as screening for the phenotypic differentiation between the species of C. albicans complex. Rosemary agar could be used to aid in the differentiation of C. albicans from C. africana. These culture media based on plants, could be used as simple and inexpensive screening methods in the phenotypic differentiation of C. dubliniensis and C. africana.

Insight into the nuclear distribution patterns of conidia and the asexual life cycle of Polyporus umbellatus.

P. umbellatus sclerotium is a traditional Chinese medicine that is widely utilized in China, Korea, Japan, and other countries due to its diverse medicinal activities, such as diuretic, antitumor, anticancer, and immune system enhancement effects. Conidia, which are common asexual spores in various fungi, are not universally present in Polyporus species. In this study, the asexual life cycle of P. umbellatus was elucidated. Conidia, i.e. arthorconidia, were produced by both dikaryotic and monokaryotic strains. In the dikaryotic strain, binucleate, uninucleate, and nuclei-free conidia were identified with proportions of 67.9 %, 12.4 %, and 19.7 %, respectively. Conversely, the monokaryotic strain did not produce binucleate conidia. This discrepancy suggests that binucleate spores are heterokaryons, while uninucleate spores are homokaryons. Clamp connections were observed in dikaryotic hyphae, but were absent in monokaryotic hyphae. Monokaryotic strains were obtained from conidia of the dikaryotic strain. Additionally, mating types were determined through pairing tests, and successful crossbreeding occurred between monokaryotic strains derived from conidia and basidiospores from different strains. This study introduced the first crossbreeding strategy for P. umbellatus.

A Brg1-Rme1 circuit in Candida albicans hyphal gene regulation.

Major Candida albicans virulence traits include its ability to make hyphae, to produce a biofilm, and to damage host cells. These traits depend upon expression of hypha-associated genes. A gene expression comparison among clinical isolates suggested that transcription factor Rme1, established by previous studies to be a positive regulator of chlamydospore formation, may also be a negative regulator of hypha-associated genes. Engineered RME1 overexpression supported this hypothesis, but no relevant rme1Δ/Δ mutant phenotype was detected. We reasoned that Rme1 may function within a specific regulatory pathway. This idea was supported by our finding that an rme1Δ/Δ mutation relieves the need for biofilm regulator Brg1 in biofilm formation. The impact of the rme1Δ/Δ mutation is most prominent under static or "biofilm-like" growth conditions. RNA sequencing (RNA-seq) of cells grown under biofilm-like conditions indicates that Brg1 activates hypha-associated genes indirectly via repression of RME1: hypha-associated gene expression levels are substantially reduced in a brg1Δ/Δ mutant and partially restored in a brg1Δ/Δ rme1Δ/Δ double mutant. An rme1Δ/Δ mutation does not simply bypass Brg1, because iron homeostasis genes depend upon Brg1 regardless of Rme1. Rme1 thus connects Brg1 to the targets relevant to hypha and biofilm formation under biofilm growth conditions.IMPORTANCECandida albicans is a major fungal pathogen of humans, and its ability to grow as a surface-associated biofilm on implanted devices is a common cause of infection. Here, we describe a new regulator of biofilm formation, RME1, whose activity is most prominent under biofilm-like growth conditions.