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.

The vacuolar fusion regulated by HOPS complex promotes hyphal initiation and penetration in Candida albicans.

The transition between yeast and hyphae is crucial for regulating the commensalism and pathogenicity in Candida albicans. The mechanisms that affect the invasion of hyphae in solid media, whose deficiency is more related to the pathogenicity of C. albicans, have not been elucidated. Here, we found that the disruption of VAM6 or VPS41 which are components of the homotypic vacuolar fusion and protein sorting (HOPS) complex, or the Rab GTPase YPT72, all responsible for vacuole fusion, led to defects in hyphal growth in both liquid and solid media, but more pronounced on solid agar. The phenotypes of vac8Δ/Δ and GTR1OE-vam6Δ/Δ mutants indicated that these deficiencies are mainly caused by the reduced mechanical forces that drive agar and organs penetration, and confirmed that large vacuoles are required for hyphal mechanical penetration. In summary, our study revealed that large vacuoles generated by vacuolar fusion support hyphal penetration and provided a perspective to refocus attention on the role of solid agar in evaluating C. albicans invasion.

A highly conserved tRNA modification contributes to C. albicans filamentation and virulence.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis, differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections.

Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.

High levels of short-chain fatty acids secreted by Candida albicans hyphae induce neutrophil chemotaxis via free fatty acid receptor 2.

Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.

Significant variation of filamentation phenotypes in clinical Candida albicans strains.

Introduction: Candida albicans is an opportunistic human pathogen that typically resides as part of the microbiome in the gastrointestinal and genitourinary tracts of a large portion of the human population. This fungus lacks a true sexual cycle and evolves in a largely clonal pattern. The ability to cause disease is consistent across the species as strains causing systemic infections appear across the known C. albicans intra-species clades. Methods: In this work, strains collected from patients with systemic C. albicans infections isolated at the Nebraska Medicine clinical laboratory were typed by MLST analysis. Since the ability to form filaments has been linked to pathogenesis in C. albicans, these clinical strains, as well as a previously genotyped set of clinical strains, were tested for their ability to filament across a variety of inducing conditions. Results: Genotyping of the clinical strains demonstrated that the strains isolated at one of the major medical centers in our region were as diverse as strains collected across the United States. We demonstrated that clinical strains exhibit a variety of filamentation patterns across differing inducing conditions. The only consistent pattern observed in the entire set of clinical strains tested was an almost universal inability to filament in standard solid inducing conditions used throughout the C. albicans field. A different solid filamentation assay that produces more robust filamentation profiles from clinical strains is proposed in this study, although not all strains expected to filament in vivo were filamentous in this assay. Discussion: Our data supports growing evidence that broad phenotypic diversity exists between the C. albicans type strain and clinical strains, suggesting that the type strain poorly represents filamentation patterns observed in most clinical isolates. These data further highlight the need to use diverse clinical strains in pathogenesis assays.

Strain variation in gene expression impact of hyphal cyclin Hgc1 in Candida albicans.

Formation of hyphae is a key virulence trait of the fungal pathogen Candida albicans. Hypha morphogenesis depends upon the cyclin Hgc1, which acts together with cyclin-dependent protein kinase Cdc28 to phosphorylate effectors that drive polarized growth. Hgc1 has also been implicated in gene regulation through its effects on 2 transcription factors, Efg1 and Ume6. Here, we report RNA-sequencing (RNA-seq) analysis of 2 pairs of hgc1Δ/Δ mutants and their respective wild-type strains, which lie in 2 different genetic backgrounds. We find that hgc1Δ/Δ mutations alter expression of 271 genes in both genetic backgrounds and 266 of those genes respond consistently with regard to up- or down-regulation. Consistency is similar to what has been observed with efg1Δ/Δ mutations and greater than observed with nrg1Δ/Δ mutations in these 2 backgrounds. The gene expression response includes genes under Efg1 control, as expected from prior studies. Hgc1-responsive genes also include ergosterol biosynthetic genes and bud neck-related genes, which may reflect interactions between Hgc1 and additional transcription factors as well as effects of Hgc1 on cellular length-to-width ratios.

Longevity of Fungal Mycelia and Nuclear Quality Checks: a New Hypothesis for the Role of Clamp Connections in Dikaryons.

This paper addresses the stability of mycelial growth in fungi and differences between ascomycetes and basidiomycetes. Starting with general evolutionary theories of multicellularity and the role of sex, we then discuss individuality in fungi. Recent research has demonstrated the deleterious consequences of nucleus-level selection in fungal mycelia, favoring cheaters with a nucleus-level benefit during spore formation but a negative effect on mycelium-level fitness. Cheaters appear to generally be loss-of-fusion (LOF) mutants, with a higher propensity to form aerial hyphae developing into asexual spores. Since LOF mutants rely on heterokaryosis with wild-type nuclei, we argue that regular single-spore bottlenecks can efficiently select against such cheater mutants. We then zoom in on ecological differences between ascomycetes being typically fast-growing but short-lived with frequent asexual-spore bottlenecks and basidiomycetes being generally slow-growing but long-lived and usually without asexual-spore bottlenecks. We argue that these life history differences have coevolved with stricter nuclear quality checks in basidiomycetes. Specifically, we propose a new function for clamp connections, structures formed during the sexual stage in ascomycetes and basidiomycetes but during somatic growth only in basidiomycete dikaryons. During dikaryon cell division, the two haploid nuclei temporarily enter a monokaryotic phase, by alternatingly entering a retrograde-growing clamp cell, which subsequently fuses with the subapical cell to recover the dikaryotic cell. We hypothesize that clamp connections act as screening devices for nuclear quality, with both nuclei continuously testing each other for fusion ability, a test that LOF mutants will fail. By linking differences in longevity of the mycelial phase to ecology and stringency of nuclear quality checks, we propose that mycelia have a constant and low lifetime cheating risk, irrespective of their size and longevity.

Cellular differentiation into hyphae and spores in halophilic archaea.

Several groups of bacteria have complex life cycles involving cellular differentiation and multicellular structures. For example, actinobacteria of the genus Streptomyces form multicellular vegetative hyphae, aerial hyphae, and spores. However, similar life cycles have not yet been described for archaea. Here, we show that several haloarchaea of the family Halobacteriaceae display a life cycle resembling that of Streptomyces bacteria. Strain YIM 93972 (isolated from a salt marsh) undergoes cellular differentiation into mycelia and spores. Other closely related strains are also able to form mycelia, and comparative genomic analyses point to gene signatures (apparent gain or loss of certain genes) that are shared by members of this clade within the Halobacteriaceae. Genomic, transcriptomic and proteomic analyses of non-differentiating mutants suggest that a Cdc48-family ATPase might be involved in cellular differentiation in strain YIM 93972. Additionally, a gene encoding a putative oligopeptide transporter from YIM 93972 can restore the ability to form hyphae in a Streptomyces coelicolor mutant that carries a deletion in a homologous gene cluster (bldKA-bldKE), suggesting functional equivalence. We propose strain YIM 93972 as representative of a new species in a new genus within the family Halobacteriaceae, for which the name Actinoarchaeum halophilum gen. nov., sp. nov. is herewith proposed. Our demonstration of a complex life cycle in a group of haloarchaea adds a new dimension to our understanding of the biological diversity and environmental adaptation of archaea.

Features of disruption mutants of genes encoding for hydrophobin Vmh2 and Vmh3 in mycelial formation and resistance to environmental stress in Pleurotus ostreatus.

Hydrophobins, which are small-secreted proteins with both hydrophobic and hydrophilic parts, can self-assemble into an amphiphilic film at the air-water interface, helping the fungus to form aerial hyphae. In the agaricomycete Pleurotus ostreatus, more than 20 putative hydrophobin genes have been predicted. Of these, two hydrophobin genes, vmh2 and vmh3, are predominantly expressed in the vegetative mycelium. In this study, we focused on the functions of Vmh2 and Vmh3 in vegetative mycelia. Based on the observation of the mycelial cross-section by transmission electron microscopy and the disappearance time of water droplets on the mycelial surface, Vmh2 and Vmh3 were considered essential for the maintenance of the surface hydrophobicity of the mycelium. The Δvmh3 and Δvmh2Δvmh3 strains exhibited relatively slower aerial mycelia formation on a liquid medium, and no significant alteration was observed in Δvmh2 strains. Only the Δvmh3 and Δvmh2Δvmh3 strains grew slower than the wild-type strain under stress conditions involving SDS and H2O2 on agar plates. This study revealed possible distinct roles for these hydrophobins in stress resistance. These results suggest that Agaricomycetes, including P. ostreatus, have evolved to possess multiple different hydrophobins as a means of adapting to various environments.

Apoptotic Factors, CaNma111 and CaYbh3, Function in Candida albicans Filamentation by Regulating the Hyphal Suppressors, Nrg1 and Tup1.

The morphological switch from the yeast to hyphal form is a key virulence attribute of the opportunistic fungal pathogen, Candida albicans. Our recent report showed that deletion of the newly identified apoptotic factor, CaNma111 or CaYbh3, leads to hyperfilamentation and increased virulence in a mouse infection model. CaNma111 and CaYbh3 are homologs of the pro-apoptotic protease, HtrA2/Omi, and BH3-only protein, respectively. In this study, we examined the effects of CaNMA111 and CaYBH3 deletion mutations on the expression levels of the hypha-specific transcription factors, Cph1 (a hyphal activator), Nrg1 (a hyphal repressor), and Tup1 (a hyphal repressor). The protein levels of Nrg1 were decreased in Caybh3/Caybh3 cells while those of Tup1 were decreased in both Canma111/Canma111 and Caybh3/Caybh3 cells. These effects on Nrg1 and Tup1 proteins were retained during serum-induced filamentation and appear to explain the hyperfilamentation phenotypes of the CaNMA111 and CaYBH3 deletion mutants. Treatment with the apoptosis-inducing dose of farnesol decreased the Nrg1 protein levels in the wild-type strain and more evidently in Canma111/Canma111 and Caybh3/Caybh3 mutant strains. Together, our results suggest that CaNma111 and CaYbh3 are key regulators of Nrg1 and Tup1 protein levels in C. albicans.

Functional Dichotomy for a Hyphal Repressor in Candida albicans.

Nrg1 is a repressor of hypha formation and hypha-associated gene expression in the fungal pathogen Candida albicans. It has been well studied in the genetic background of the type strain SC5314. Here, we tested Nrg1 function in four other diverse clinical isolates through an analysis of nrg1Δ/Δ mutants, with SC5314 included as a control. In three strains, nrg1Δ/Δ mutants unexpectedly produced aberrant hyphae under inducing conditions, as assayed by microscopic observation and endothelial cell damage. The nrg1Δ/Δ mutant of strain P57055 had the most severe defect. We examined gene expression features under hypha-inducing conditions by RNA-sequencing (RNA-Seq) for the SC5314 and P57055 backgrounds. The SC5314 nrg1Δ/Δ mutant expressed six hypha-associated genes at reduced levels compared with wild-type SC5314. The P57055 nrg1Δ/Δ mutant expressed 17 hypha-associated genes at reduced levels compared with wild-type P57055, including IRF1, RAS2, and ECE1. These findings indicate that Nrg1 has a positive role in hypha-associated gene expression and that this role is magnified in strain P57055. Remarkably, the same hypha-associated genes affected by the nrg1Δ/Δ mutation in strain P57055 were also naturally expressed at lower levels in wild-type P57055 than those in wild-type SC5314. Our results suggest that strain P57055 is defective in a pathway that acts in parallel with Nrg1 to upregulate the expression of several hypha-associated genes. IMPORTANCE Hypha formation is a central virulence trait of the fungal pathogen Candida albicans. Control of hypha formation has been studied in detail in the type strain but not in other diverse C. albicans clinical isolates. Here, we show that the hyphal repressor Nrg1 has an unexpected positive role in hypha formation and hypha-associated gene expression, as revealed by the sensitized P57055 strain background. Our findings indicate that reliance on a single type strain limits understanding of gene function and illustrate that strain diversity is a valuable resource for C. albicans molecular genetic analysis.

Morphogenic plasticity: the pathogenic attribute of Candida albicans.

Candida albicans is a commensal organism of the human gastrointestinal tract and a prevalent opportunistic pathogen. It exhibits different morphogenic forms to survive in different host niches with distinct environmental conditions (pH, temperature, oxidative stress, nutrients, serum, chemicals, radiation, etc.) and genetic factors (transcription factors and genes). The different morphogenic forms of C. albicans are yeast, hyphal, pseudohyphal, white, opaque, and transient gray cells, planktonic and biofilm forms of cells. These forms differ in the parameters like cellular phenotype, colony morphology, adhesion to solid surfaces, gene expression profile, and the virulent traits. Each form is functionally distinct and responds discretely to the host immune system and antifungal drugs. Hence, morphogenic plasticity is the key to virulence. In this review, we address the characteristics, the pathogenic potential of the different morphogenic forms and the conditions required for morphogenic transitions.

Hgc1 Independence of Biofilm Hyphae in Candida albicans.

Biofilm and hypha formation are central to virulence of the fungal pathogen Candida albicans. The G1 cyclin gene HGC1 is required for hypha formation under diverse in vitro and in vivo growth conditions. Hgc1 is required for disseminated infection and is a linchpin in the argument that hyphal morphogenesis itself is required for pathogenicity. We report here that HGC1 is dispensable for hypha formation during biofilm formation both in vitro, under strong inducing conditions, and in vivo, in a mouse oropharyngeal candidiasis model. These findings are validated with two or more C. albicans isolates. Systematic screening of overexpressed cyclin genes indicates that CCN1 and CLN3 can compensate partially for Hgc1 function during biofilm growth. This conclusion is also supported by the severity of the hgc1Δ/Δ ccn1Δ/Δ double mutant biofilm defect. Our results suggest that hypha formation in biofilm is accomplished by combined action of multiple cyclins, not solely by Hgc1. IMPORTANCE The HGC1 gene encodes a cyclin that is required for virulence of the fungal pathogen Candida albicans. It is required to produce the elongated hyphal filaments of free-living planktonic cells that are associated with virulence. Here, we show that HGC1 is not required to produce hyphae in the alternative growth form of a biofilm community. We observe Hgc1-independent hyphae in two infection-relevant situations, biofilm growth in vitro and biofilm-like oropharyngeal infection. Our analysis suggests that hypha formation in the biofilm state reflects combined action of multiple cyclins.

Intravital imaging-based genetic screen reveals the transcriptional network governing Candida albicans filamentation during mammalian infection.

Candida albicans is one of the most common human fungal pathogens. C. albicans pathogenesis is tightly linked to its ability to under a morphogenetic transition from typically budding yeast to filamentous forms of hyphae and pseudohyphae. Filamentous morphogenesis is the most intensively studied C. albicans virulence traits; however, nearly all of these studies have been based on in vitro induction of filamentation. Using an intravital imaging assay of filamentation during mammalian (mouse) infection, we have screened a library of transcription factor mutants to identify those that modulate both the initiation and maintenance of filamentation in vivo. We coupled this initial screen with genetic interaction analysis and in vivo transcription profiling to characterize the transcription factor network governing filamentation in infected mammalian tissue. Three core positive (Efg1, Brg1, and Rob1) and two core negative regulators (Nrg1 and Tup1) of filament initiation were identified. No previous systematic analysis of genes affecting the elongation step has been reported and we found that large set of transcription factors affect filament elongation in vivo including four (Hms1, Lys14, War1, Dal81) with no effect on in vitro elongation. We also show that the gene targets of initiation and elongation regulators are distinct. Genetic interaction analysis of the core positive and negative regulators revealed that the master regulator Efg1 primarily functions to mediate relief of Nrg1 repression and is dispensable for expression of hypha-associated genes in vitro and in vivo. Thus, our analysis not only provide the first characterization of the transcriptional network governing C. albicans filamentation in vivo but also revealed a fundamentally new mode of function for Efg1, one of the most widely studied C. albicans transcription factors.

Reinforcement amid genetic diversity in the Candida albicans biofilm regulatory network.

Biofilms of the fungal pathogen Candida albicans include abundant long filaments called hyphae. These cells express hypha-associated genes, which specify diverse virulence functions including surface adhesins that ensure biofilm integrity. Biofilm formation, virulence, and hypha-associated gene expression all depend upon the transcription factor Efg1. This transcription factor has been characterized extensively in the C. albicans type strain SC5314 and derivatives, but only recently has its function been explored in other clinical isolates. Here we define a principal set of Efg1-responsive genes whose expression is significantly altered by an efg1Δ/Δ mutation across 17 clinical isolates. This principal gene set includes 68 direct Efg1 targets, whose 5' regions are bound by Efg1 in five clinical isolates, and 42 indirect Efg1 targets, whose 5' regions are not detectably bound by Efg1. Three direct Efg1 target genes encode transcription factors-BRG1, UME6, and WOR3 -whose increased expression in an efg1Δ/Δ mutant restores expression of multiple indirect and direct principal targets, as well as biofilm formation ability. Although BRG1 and UME6 are well known positive regulators of hypha-associated genes and biofilm formation, WOR3 is best known as an antagonist of Efg1 in the sexual mating pathway. We confirm the positive role of WOR3 in biofilm formation with the finding that a wor3Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo biofilm model. Positive control of Efg1 direct target genes by other Efg1 direct target genes-BRG1, UME6, and WOR3 -may buffer principal Efg1-responsive gene expression against the impact of genetic variation in the C. albicans species.

Comparative transcriptomics of growth metabolism and virulence reveal distinct morphogenic profiles of yeast-like cells and hyphae of the fungus Metarhizium rileyi.

Metarhizium rileyiis an entomopathogenic fungus with a narrow host range which distinguishes it from other Metarhiziumspecies with broad host ranges. This species is also unique because the initial yeast-like growth on solid media is only observed in liquid culture in other Metharizium species. A lack of knowledge about the metabolism and genetic signatures of M. rileyiduring this yeast-like phase on solid and in liquid media is a bottleneck for its large-scale production as a commercial biocontrol agent.In this study wefound that M. rileyiyeast-like cells produced on solid medium infected and killed the important insect pest Spodoptera frugiperda with comparable efficiency as yeast-like cells grown in liquid medium. Secondly, we used comparative transcriptomic analysis to investigate theactive genes and genomic signatures of the M. rileyi yeast-like morphotypes produced on solid and in liquid media. Yeast-like cells grown in liquid medium had upregulated genes relating specifically to signal transduction andparticular membrane transporters. Thirdly, we compared the transcriptomic profiles of yeast-like phases of M. rileyi with those of M. anisopliae. The yeast-like phase of M. rileyi grown on solid medium upregulated unique genes not found in otherMetarhiziumspecies including specific membrane proteins and several virulence factors. Orthologous genes associated with heat shock protein, iron permease, membrane proteins and key virulence traits (e.g. collagen-like protein Mcl1) were upregulated in both species. Comparative transcriptome analyses of gene expression showed more differences than similarities between M. anisopliae and M. rileyi yeast-like cells.

Morphological and transcriptomic analysis revealing morphological variations and genetic clues in one Lentinula edodes abnormal browning strain.

Strain abnormal browning is a common problem during cultivation of Lentinula edodes. In this study, the L. edodes strain mycelia isolated from Le-WB and cultured on MYG (Le-WP) isolated from an abnormal browning bag was compared with its normal control mycelia isolated from Le-BB and cultured on MYG (Le-BP). The aerial hyphae of Le-WP were white, and the hyphal growth was significantly reduced. Morphological observation of Le-WP under scanning electron microscope (SEM) and transmission electron microscopy (TEM) revealed abnormal organelle structures. Through transcriptomic analysis, more differentially expressed genes (DEGs) were expressed in the metabolic process and catalytic activity in Le-WP than Le-BP. Two Kyoto encyclopedia of genes and genomes (KEGG) pathways named pentose and glucorunate interconversions, and starch and sucrose metabolism were found to be enriched in Le-WP. The gene expression profiles involved in these two pathways were further analyzed and 12 key genes were selected to be verified by quantitative real-time PCR (qRT-PCR), and the results showed that most of these genes were upregulated in Le-WP. Additionally, the content of 1,3-beta-glucan in Le-WP was also significantly higher than in other samples. This research suggests that abnormal strains may be related to the abnormal synthesis of 1,3-beta-glucan, and it needs further research. This research exhibits possible morphological and genetic clues of Le-WP and lays the foundation for understanding the degeneration of L. edodes strains.

Fungal hyphae regulate bacterial diversity and plasmid-mediated functional novelty during range expansion.

The amount of bacterial diversity present on many surfaces is enormous; however, how these levels of diversity persist in the face of the purifying processes that occur as bacterial communities expand across space (referred to here as range expansion) remains enigmatic. We shed light on this apparent paradox by providing mechanistic evidence for a strong role of fungal hyphae-mediated dispersal on regulating bacterial diversity during range expansion. Using pairs of fluorescently labeled bacterial strains and a hyphae-forming fungal strain that expand together across a nutrient-amended surface, we show that a hyphal network increases the spatial intermixing and extent of range expansion of the bacterial strains. This is true regardless of the type of interaction (competition or resource cross-feeding) imposed between the bacterial strains. We further show that the underlying cause is that flagellar motility drives bacterial dispersal along the hyphal network, which counteracts the purifying effects of ecological drift at the expansion frontier. We finally demonstrate that hyphae-mediated spatial intermixing increases the conjugation-mediated spread of plasmid-encoded antibiotic resistance. In conclusion, fungal hyphae are important regulators of bacterial diversity and promote plasmid-mediated functional novelty during range expansion in an interaction-independent manner.

Hydroquinones Including Tetrachlorohydroquinone Inhibit Candida albicans Biofilm Formation by Repressing Hyphae-Related Genes.

Candida albicans is an opportunistic pathogenic fungus responsible for candidiasis. The pathogen readily forms antifungal agent-resistant biofilms on implanted medical devices or human tissue. Morphologic transition from yeast to filamentous cells and subsequent biofilm formation is a key virulence factor and a prerequisite for biofilm development by C. albicans. We investigated the antibiofilm and antifungal activities of 18 hydroquinones against fluconazole-resistant C. albicans. Tetrachlorohydroquinone (TCHQ) at subinhibitory concentrations (2 to 10 µg/mL) significantly inhibited C. albicans biofilm formation with an MIC of 50 µg/mL, whereas the backbone hydroquinone did not (MIC > 400 µg/mL), and it markedly inhibited cell aggregation and hyphal formation. Transcriptomic analyses showed that TCHQ downregulated the expressions of several hyphae-forming and biofilm-related genes (ALS3, ECE1, HWP1, RBT5, and UME6) but upregulated hyphae- and biofilm-inhibitory genes (IFD6 and YWP1). Furthermore, it prevented C. albicans biofilm development on porcine skin and at concentrations of 20 to 50 µg/mL was nontoxic to the nematode Caenorhabditis elegans and did not adversely affect Brassica rapa seed germination and growth. This study indicates that hydroquinones, particularly TCHQ, diminish the virulence, biofilm formation, and animal tissue adhesion of C. albicans, which suggests hydroquinones should be considered potential candidate antifungal agents against drug-resistant C. albicans strains. IMPORTANCE Persistence in chronic infections by Candida albicans is due to its ability of biofilm formation that endures conventional antifungals and host immune systems. Hence, the inhibition of biofilm formation and virulence characteristics is another mean of addressing infections. This study is a distinctive one since 18 hydroquinone analogues were screened and TCHQ efficiently inhibited the biofilm formation by C. albicans with significantly changed expressional profile of hyphae-forming and biofilm-related genes. The antibiofilm efficacy was confirmed using a porcine skin model and chemical toxicity was investigated using plant seed germination and nematode models. Our findings reveal that TCHQ can efficiently control the C. albicans biofilms and virulence characteristics.