Dimorphic Transition Research Articles

Mucor germinans, a novel dimorphic species resembling Paracoccidioides in a clinical sample: questions on ecological strategy.

Dimorphism is known among the etiologic agents of endemic mycoses as well as in filamentous Mucorales. Under appropriate thermal conditions, mononuclear yeast forms alternate with multi-nucleate hyphae. Here, we describe a dimorphic mucoralean fungus obtained from the sputum of a patient with Burkitt lymphoma and ongoing graft-versus-host reactions. The fungus is described as Mucor germinans sp. nov. Laboratory studies were performed to simulate temperature-dependent dimorphism, with two environmental strains Mucor circinelloides and Mucor kunryangriensis as controls. Both strains could be induced to form multinucleate arthrospores and subsequent yeast-like cells in vitro. Multilateral yeast cells emerge in all three Mucor species at elevated temperatures. This morphological transformation appears to occur at body temperature since the yeast-like cells were observed in the lungs of our immunocompromised patient. The microscopic appearance of the yeast-like cells in the clinical samples is easily confused with that of Paracoccidioides. The ecological role of yeast forms in Mucorales is discussed.IMPORTANCEMucormycosis is a devastating disease with high morbidity and mortality in susceptible patients. Accurate diagnosis is required for timely clinical management since antifungal susceptibility differs between species. Irregular hyphal elements are usually taken as the hallmark of mucormycosis, but here, we show that some species may also produce yeast-like cells, potentially being mistaken for Candida or Paracoccidioides. We demonstrate that the dimorphic transition is common in Mucor species and can be driven by many factors. The multi-nucleate yeast-like cells provide an effective parameter to distinguish mucoralean infections from similar yeast-like species in clinical samples.

Comparative proteomics of sugarcane smut fungus - Sporisorium scitamineum unravels dynamic proteomic alterations during the dimorphic transition

Life cycle of the dimorphic sugarcane smut fungi, Sporisorium scitamineum, involves recognition and mating of compatible saprophytic yeast-like haploid sporidia (MAT-1 and MAT-2) that upon fusion, develop into infective dikaryotic mycelia. Although the dimorphic transition is intrinsically linked with the pathogenicity and virulence of S. scitamineum, it has never been studied using a proteomic approach. In the present study, an iTRAQ-based comparative proteomic analysis of three distinct stages was carried out. The stages were: the dimorphic transition period - haploid sporidial stage (MAT-1 and MAT-2); the transition phase (24 h post co-culturing (hpc)) and the dikaryotic mycelial stage (48 hpc). Functional categorization of differentially abundant proteins showed that the most altered biological processes were energy production, primary metabolism, especially, carbohydrate, amino acid, fatty acid, followed by translation, post-translation and protein turnover. Several differentially abundant proteins (DAPs), especially in the dikaryotic mycelial stage were predicted as effectors. Taken together, key molecular mechanisms underpinning the dimorphic transition in S. scitamineum at the proteome level were highlighted. The catalogue of stage-specific and dimorphic transition-associated-proteins and potential effectors identified herein represents a list of potential candidates for defective mutant screening to elucidate their functional role in the dimorphic transition and pathogenicity in S. scitamineum. Biological significanceBeing the first comparative proteomics analysis of S. scitamineum, this study comprehensively examined three pivotal life cycle stages of the pathogen: the non-pathogenic haploid phase, the transition phase, and the pathogenic dikaryotic mycelial stage. While previous studies have reported the sugarcane and S. scitamineum interactions, this study endeavored to specifically identify the proteins responsible for pathogenicity. By analyzing the proteomic alterations between the haploid and dikaryotic mycelial phases, the study revealed significant changes in metabolic pathway-associated proteins linked to energy production, notably oxidative phosphorylation, and the citrate cycle. Furthermore, this study successfully identified key metabolic pathways that undergo reprogramming during the transition from the non-pathogenic to the pathogenic stage. The study also deciphered the underlying mechanisms driving the morphological and physiological alterations crucial for the S. scitamineum virulence. By studying its life cycle stages, identifying the key metabolic pathways and stage-specific proteins, it provides unprecedented insights into the pathogenicity and potential avenues for intervention. As proteomics continues to advance, such studies pave the way for a deeper understanding of plant-pathogen interactions and the development of innovative strategies to mitigate the impact of devastating pathogens like S. scitamineum.

Essential roles of Rad6 in conidial property, stress tolerance, and pathogenicity of Beauveria bassiana

ABSTRACT Rad6 functions as a ubiquitin-conjugating protein that regulates cellular processes in many fungal species. However, its role in filamentous entomopathogenic fungi remains poorly understood. This study characterizes Rad6 in Beauveria bassiana, a filamentous fungus widely employed as a critical fungicide globally. The results demonstrate a significant association between Rad6 and conidial properties, heat shock response, and UV-B tolerance. Concurrently, the mutant strain exhibited heightened sensitivity to oxidative stress, cell wall interfering agents, DNA damage stress, and prolonged heat shock. Furthermore, the absence of Rad6 significantly extended the median lethal time (LT50) of Galleria mellonella infected by B. bassiana. This delay could be attributed to reduced Pr1 proteases and extracellular cuticle-degrading enzymes, diminished dimorphic transition rates, and dysregulated antioxidant enzymes. Additionally, the absence of Rad6 had a more pronounced effect on genetic information processing, metabolism, and cellular processes under normal conditions. However, its impact was limited to metabolism in oxidative stress. This study offers a comprehensive understanding of the pivotal roles of Rad6 in conidial and hyphal stress tolerance, environmental adaptation, and the pathogenesis of Beauveria bassiana.

Fungal burden, dimorphic transition and candidalysin: Role in Candida albicans-induced vaginal cell damage and mitochondrial activation in vitro.

Candida albicans (C. albicans) can behave as a commensal yeast colonizing the vaginal mucosa, and in this condition is tolerated by the epithelium. When the epithelial tolerance breaks down, due to C. albicans overgrowth and hyphae formation, the generated inflammatory response and cell damage lead to vulvovaginal candidiasis (VVC) symptoms. Here, we focused on the induction of mitochondrial reactive oxygen species (mtROS) in vaginal epithelial cells after C. albicans infection and the involvement of fungal burden, morphogenesis and candidalysin (CL) production in such induction. Bioluminescent (BLI) C. albicans, C. albicans PCA-2 and C. albicans 529L strains were employed in an in vitro infection model including reconstituted vaginal epithelium cells (RVE), produced starting from A-431 cell line. The production of mtROS was kinetically measured by using MitoSOX™ Red probe. The potency of C. albicans to induced cell damage to RVE and C. albicans proliferation have also been evaluated. C. albicans induces a rapid mtROS release from vaginal epithelial cells, in parallel with an increase of the fungal load and hyphal formation. Under the same experimental conditions, the 529L C. albicans strain, known to be defective in CL production, induced a minor mtROS release showing the key role of CL in causing epithelial mithocondrial activation. C. albicans PCA-2, unable to form hyphae, induced comparable but slower mtROS production as compared to BLI C. albicans yeasts. By reducing mtROS through a ROS scavenger, an increased fungal burden was observed during RVE infection but not in fungal cultures grown on abiotic surface. Collectively, we conclude that CL, more than fungal load and hyphae formation, seems to play a key role in the rapid activation of mtROS by epithelial cells and in the induction of cell-damage and that mtROS are key elements in the vaginal epithelial cells response to C. albicans.

Inhibition of Candida albicans Biofilm Formation and Attenuation of Its Virulence by Liriope muscari.

(1) Background: Although Candida albicans accounts for the majority of fungal infections, therapeutic options are limited and require alternative antifungal agents with new targets; (2) Methods: A biofilm formation assay with RPMI1640 medium was performed with Liriope muscari extract. A combination antifungal assay, dimorphic transition assay, and adhesion assay were performed under the biofilm formation condition to determine the anti-biofilm formation effect. qRT-PCR analysis was accomplished to confirm changes in gene expression; (3) Results: L. muscari extract significantly reduces biofilm formation by 51.65% at 1.56 μg/mL use and therefore increases susceptibility to miconazole. L. muscari extract also inhibited the dimorphic transition of Candida; nearly 50% of the transition was inhibited when 1.56 μg/mL of the extract was treated. The extract of L. muscari inhibited the expression of genes related to hyphal development and extracellular matrix of 34.4% and 36.0%, respectively, as well as genes within the Ras1-cAMP-PKA, Cph2-Tec1, and MAP kinase signaling pathways of 25.58%, 7.1% and 15.8%, respectively, at 1.56 μg/mL of L. muscari extract treatment; (4) Conclusions: L. muscari extract significantly reduced Candida biofilm formation, which lead to induced antifungal susceptibility to miconazole. It suggests that L. muscari extract is a promising anti-biofilm candidate of Candida albicans since the biofilm formation of Candida albicans is an excellent target for candidiasis regulation.

Unraveling the dynamic transcriptomic changes during the dimorphic transition of Talaromyces marneffei through time-course analysis.

Systemic dimorphic fungi pose a significant public health challenge, causing over one million new infections annually. The dimorphic transition between saprophytic mycelia and pathogenic yeasts is strongly associated with the pathogenesis of dimorphic fungi. However, despite the dynamic nature of dimorphic transition, the current omics studies focused on dimorphic transition primarily employ static strategies, partly due to the lack of suitable dynamic analytical methods. We conducted time-course transcriptional profiling during the dimorphic transition of Talaromyces marneffei, a model organism for thermally dimorphic fungi. To capture non-uniform and nonlinear transcriptional changes, we developed DyGAM-NS (dynamic optimized generalized additive model with natural cubic smoothing). The performance of DyGAM-NS was evaluated by comparison with seven other commonly used time-course analysis methods. Based on dimorphic transition induced genes (DTIGs) identified by DyGAM-NS, cluster analysis was utilized to discern distinct gene expression patterns throughout dimorphic transitions of T. marneffei. Simultaneously, a gene expression regulatory network was constructed to probe pivotal regulatory elements governing the dimorphic transitions. By using DyGAM-NS, model, we identified 5,223 DTIGs of T. marneffei. Notably, the DyGAM-NS model showcases performance on par with or superior to other commonly used models, achieving the highest F1 score in our assessment. Moreover, the DyGAM-NS model also demonstrates potential in predicting gene expression levels throughout temporal processes. The cluster analysis of DTIGs suggests divergent gene expression patterns between mycelium-to-yeast and yeast-to-mycelium transitions, indicating the asymmetrical nature of two transition directions. Additionally, leveraging the identified DTIGs, we constructed a regulatory network for the dimorphic transition and identified two zinc finger-containing transcription factors that potentially regulate dimorphic transition in T. marneffei. Our study elucidates the dynamic transcriptional profile changes during the dimorphic transition of T. marneffei. Furthermore, it offers a novel perspective for unraveling the underlying mechanisms of fungal dimorphism, emphasizing the importance of dynamic analytical methods in understanding complex biological processes.

Redundant and Distinct Roles of Two 14-3-3 Proteins in Fusarium sacchari, Pathogen of Sugarcane Pokkah Boeng Disease.

Fusarium sacchari, a key pathogen of sugarcane, is responsible for the Pokkah boeng disease (PBD) in China. The 14-3-3 proteins have been implicated in critical developmental processes, including dimorphic transition, signal transduction, and carbon metabolism in various phytopathogenic fungi. However, their roles are poorly understood in F. sacchari. This study focused on the characterization of two 14-3-3 protein-encoding genes, FsBmh1 and FsBmh2, within F. sacchari. Both genes were found to be expressed during the vegetative growth stage, yet FsBmh1 was repressed at the sporulation stage in vitro. To elucidate the functions of these genes, the deletion mutants ΔFsBmh1 and ΔFsBmh2 were generated. The ΔFsBmh2 exhibited more pronounced phenotypic defects, such as impaired hyphal branching, septation, conidiation, spore germination, and colony growth, compared to the ΔFsBmh1. Notably, both knockout mutants showed a reduction in virulence, with transcriptome analysis revealing changes associated with the observed phenotypes. To further investigate the functional interplay between FsBmh1 and FsBmh2, we constructed and analyzed mutants with combined deletion and silencing (ΔFsBmh/siFsBmh) as well as overexpression (O-FsBmh). The combinations of ΔFsBmh1/siFsBmh2 or ΔFsBmh2/siFsBmh1 displayed more severe phenotypes than those with single allele deletions, suggesting a functional redundancy between the two 14-3-3 proteins. Yeast two-hybrid (Y2H) assays identified 20 proteins with pivotal roles in primary metabolism or diverse biological functions, 12 of which interacted with both FsBmh1 and FsBmh2. Three proteins were specifically associated with FsBmh1, while five interacted exclusively with FsBmh2. In summary, this research provides novel insights into the roles of FsBmh1 and FsBmh2 in F. sacchari and highlights potential targets for PBD management through the modulation of FsBmh functions.

Regulation of morphogenesis and pathogenicity by OsMep2, OsCph1, and OsPes1 in dimorphic entomopathogenic fungus Ophiocordyceps sinensis (Hypocreales: Ophiocordycipitaceae).

Polarized growth plays a key role in all domains of their biology, including morphogenesis and pathogenicity of filamentous fungi. However, little information is available about the determinants of polarized growth. The fungal Mep2, Pes1, and Cph1 proteins were identified to be involved in the dimorphic transition between yeast and hyphal forms in Candida albicans. In this study, evidence that the dimorphic fungal entomopathogen Ophiocordyceps sinensis Mep2, Pes1, and Cph1 proteins are involved in polarized growth is presented. OsMep2 was significantly upregulated at aerial hyphae and conidia germination stages. OsCph1 was significantly upregulated at aerial hyphae, conidia initiation, and conidia germination stages, and OsPes1 was significantly upregulated at the conidia germination stage. Deletions of OsMep2, OsCph1, and OsPes1 provoked defects in the polarized growth. The abilities of hyphal formation and the yields of blastospores and conidia for the ∆ OsMep2, ∆OsCph1, and ∆ OsPes1 mutants were significantly reduced. The conidia yields of the ΔOsMep2, ΔOsCph1, and ΔOsPes1 mutants were decreased by 69.17%, 60.90%, and 75.82%, respectively. Moreover, the pathogenicity of the ∆ OsMep2, ∆OsCph1, and ∆ OsPes1 mutants against Thitarodes xiaojinensis was significantly reduced. The mummification rate caused by wide type and ΔOsMep2, ΔOsCph1, and ΔOsPes1 mutants were 36.98% ± 8.52%, 0.31% ± 0.63%, 1.15% ± 1.57%, and 19.69% ± 5.6%, respectively. These results indicated that OsMep2, OsCph1, and OsPes1 are involved in the regulation of hyphal formation, sporulation, and pathogenicity of O. sinensis. This study provided a basis for the understanding of the fungal dimorphic development and improving the efficiency of artificial cultivation of O. sinensis.

A Cyclam Salt as an Antifungal Agent: Interference with Candida spp. and Cryptococcus neoformans Mechanisms of Virulence.

The importance of fungal infections, particularly those caused by yeasts, is increasing among the medical community. Candida albicans and Cryptococcus neoformans are amongst the high-priority fungal species identified by the World Health Organization (WHO) and are considered in the critical group, while Candida krusei is included in the medium-priority group. The cyclam salt H4[H2(4-CF3PhCH2)2Cyclam]Cl4 proved to be active against the growth of these three yeasts, and the aim of this work was to verify its interference with their virulence mechanisms, whether shared or unique. H4[H2(4-CF3PhCH2)2Cyclam]Cl4 significantly inhibited biofilm production and catalase activity, being able to interfere with C. albicans dimorphic transition and C. neoformans melanin production. At the minimal inhibitory concentration (MIC) values, H4[H2(4-CF3PhCH2)2Cyclam]Cl4 had no antioxidant effect, as determined by the DPPH method. When using the RAW264.7 macrophage cell line, H4[H2(4-CF3PhCH2)2Cyclam]Cl4 reduced nitric oxide (NO) detection (the Griess reaction), but this effect was associated with a significant toxic effect on the cells.

A Novel Approach: Targeting Virulence Factors of Candida albicans with Essential Oil Components.

Focusing on phytochemicals to target the virulence factors of Candida albicans is a promising avenue for novel antifungal compounds. Given the limited prior research on essential oil (EO) components and their specific effects on C. albicans virulence, our study aimed to explore their impact and uncover the underlying mechanisms. We examined the effects on viability, dimorphic transition, biofilm formation, and changes in the expression of critical virulence-related genes. The results showed that Dehydrocostus Lactone, displayed the most potent growth-inhibiting activity with the lowest MIC value, followed by Thymol, and Costunolide. A substantial, dose-dependent decrease in germ tube formation occurred after exposure to sub-inhibitory concentrations of the EO components, with Carvacrol, Dehydrocostus Lactone, and Thymol exerting the most potent inhibitory effects. Across sub-inhibitory concentrations, Alpha Bisabolol consistently showcased the most potent antibiofilm activity followed by lower but significant inhibitory effects with Dehydrocostus Lactone, Thymol, Alpha Pinene, Costunolide, Carvone, and Carvacrol. Alpha Bisabolol, Alpha Pinene and Dehydrocostus Lactone caused almost total downregulation of ACT1 whilst minimal changes occurred in expression of HWP1, SAP4, ALS3 and ECE1. Considering that actin is essential for various cellular processes, including budding, cell shape maintenance, and the formation of filaments in C. albicans, it is a plausible hypothesis that inhibiting ACT1 or disturbing actin's normal functioning could potentially affect the fungus's virulence, which warrants additional research and exploration. This study underscores the potent antifungal and anti-virulence properties of various EO components which effectively cripple C. albicans and reduce its disease-causing ability. This innovative approach holds promise for effective clinical therapies.

Knockdown of Thitarodes host genes influences dimorphic transition of Ophiocordyceps sinensis in the host hemolymph.

The Chinese cordyceps, a unique parasitic complex of Thitarodes/Hepialus ghost moths and Ophiocordyceps sinensis fungus in the Tibetan Plateau, is a highly valuable biological resource for medicine and health foods in Asian countries. Efficient system for artificial cultivation of Chinese cordyceps relies on understanding the gene functions involved in the induction of growing blastospores into hyphae in the larval hemolymph of insect host, during O. sinensis infection. Transcriptome analysis and ribonucleic acid interference (RNA interference) method were employed to identify the key differentially expressed genes and to demonstrate their functions in Thitarodes xiaojinensis. Key larval genes critical for O. sinensis blastospore development or filamentation were identified. Nine of the 20 top upregulated genes encoded cuticles proteins, indicating that these proteins highly activated when the larval hemolymph was full of blastospores. Small interfering RNA (siRNA) knockdown of five larval genes such as Flightin, larval cuticle protein LCP-30, 26-hydroxylase (CYP18A1), cuticle protein 18.6, isoform B, and probable chitinase 3 significantly stimulated the dimorphic transition from blastospores to prehyphae in O. sinensis in the larval hemolymph after 120 h after injection. The expressions of these genes determined by quantitative real-time PCR were suppressed in various levels from 38.64% to 91.54%, compared to the controls. These results demonstrated that injection of the siRNAs of key upregulated genes into the larval hemolymph containing high load of blastospores caused the gene silence in T. xiaojinensis larvae and induced the fungal transition from blastospores to prehyphae, providing novel knowledge on the regulation of O. sinensis fungal dimorphism by Thitarodes host and cues for further study of Thitarodes biology and commercial cultivation of Chinese cordyceps.

Multi-omics analysis reveals the mechanism underlying the dimorphic formation of Saccharomycopsis fibuligera during dough fermentation

To unravel the mechanism underlying the dimorphic formation of Saccharomycopsis fibuligera during dough fermentation, we assembled a high-quality chromosome-scale genome and conducted comparative genomic analysis. Furthermore, we examined the differences between the yeast-like cells and hyphae at both transcriptome and metabolome levels. The completed S. fibuligera ACX0001 genome of 19.29 Mb comprised seven chromosomes and contained 6115 predicted protein-coding genes. Comparative genomic analysis revealed that S. fibuligera possessed 51 unique gene families comprising 85 annotated genes. The advantageous genes related to carbon metabolism pathway were accumulated in S. fibuligera during evolution, including the unique genes encoding glycosyl hydrolases such as glucan endo-1,3-β glucosidase, which distinguished it from the other yeasts. Transcriptomic and metabolomic analysis identified 568 differentially expressed genes and 109 differentially abundant metabolites between the yeast-like cells and hyphae, respectively. In response to the dough environment, S. fibuligera activated the expression of the glucan endo-1,3-β glucosidase, which can soften the cell wall and promote germination of yeast-like cells. Simultaneously, the flux of acetyl-CoA was redirected towards fatty acid and steroid biosynthesis in S. fibuligera, which influenced the production of quorum-sensing molecules, thereby contributing to the dimorphic transition. Therefore, the dimorphic growth of S. fibuligera was collectively governed by the glycosyl hydrolase family genes, central carbon metabolism, and quorum-sensing molecules. This study provides a theoretical foundation for the potential application of S. fibuligera in food industry.

Volatile Fingerprint Mediates Yeast-to-Mycelial Conversion in Two Strains of Beauveria bassiana Exhibiting Varied Virulence.

Beauveria bassiana is a dimorphic and entomopathogenic fungus with different ecological roles in nature. In pathogenic fungi, yeast-to-mycelial conversion, which is controlled by environmental factors, is required for virulence. Here, we studied the effects of different stimuli on the morphology of two B. bassiana strains and compared the toxicities of culture filtrates. In addition, we explored the role of volatiles as quorum sensing-like signals during dimorphic transition. The killing assays in Caenorhabditis elegans (Nematoda: Rhabditidae) showed that strain AI2 isolated from a mycosed insect cadaver had higher toxicity than strain AS5 isolated from soil. Furthermore, AI2 showed earlier yeast-to-mycelial switching than AS5. However, an increase in inoculum size induced faster yeast-to-mycelium conversion in AS5 cells, suggesting a cell-density-dependent phenomenon. Gas chromatography-mass spectrometry (GC-MS) analyses showed that the fingerprint of the volatiles was strain-specific; however, during the morphological switching, an inverse relationship between the abundance of total terpenes and 3-methylbutanol was observed in both strains. Fungal exposure to 3-methylbutanol retarded the yeast-to-mycelium transition. Hence, this study provides evidence that volatile compounds are associated with critical events in the life cycle of B. bassiana.

Fungal co-expression network analyses identify pathogen gene modules associated with host insect invasion.

The broad host range fungal insect pathogen, Beauveria bassiana, has been commercialized as an alternative to chemical insecticides for pest control worldwide. B. bassiana represents a unique model system with which to examine host-pathogen interactions, and a wide range of genes and processes have been studied. However, significant aspects of virulence, particularly on the genomic scale, remain poorly studied. Here, we have combined available transcriptomes with three newly generated data sets for a combined total analysis of 76 deep-sequenced samples covering growth, development, stress responses, and infection during the life cycle of B. bassiana. Co-expression network analyses resulted in the identification of gene modules enriched during two critical stages of the infection process, namely (i) cuticle penetration and (ii) in vivo hyphal body (dimorphic transition) growth capable of avoiding innate and humoral immune defenses. These analyses identify unique signatures of metabolism, signaling, secondary metabolite production, host defense suppression, membrane reorganization, effector production, and secretion for each stage, including genetic regulators and epigenetic patterns. These data provide a comprehensive framework for understanding and probing fungal adaptations to its pathogenic life cycle and expand the candidate repertoire for continued dissection of the host-pathogen interaction. IMPORTANCE Insect fungal pathogens have evolved unique strategies for overcoming host structural and immunological defenses that span from the sclerotized cuticle to innate and humoral cellular responses. Two critical stages of the infection process involve (i) cuticle penetration and (ii) immune evasion within the insect hemocoel. A set of 76 global transcriptomic data for B. bassiana that include the cuticle penetration and hemocoel growth stages were analyzed for patterns (gene modules) of expression, yielding unique insights into these different life stages. These analyses integrate gene networks involved in fungal development, stress response and pathogenesis to further the systematic understanding of the global processes integral to the unique adaptation employed by fungal pathogens of insects.

Mutation in yl-HOG1 represses the filament-to-yeast transition in the dimorphic yeast Yarrowia lipolytica

BackgroundYarrowia lipolytica is a dimorphic fungus, which switches from yeast to filament form in response to environmental conditions. For industrial purposes it is important to lock cells in the yeast or filamentous form depending on the fermentation process. yl-Hog1 kinase is a key component of the HOG signaling pathway, responsible for activating the osmotic stress response. Additionally, deletion of yl-Hog1 leads to increased filamentation in Yarrowia lipolytica, but causes significant sensitivity to osmotic stress induced by a high concentration of a carbon source.ResultsIn this study, we tested the effect of point mutations on the function of yl-Hog1 protein kinase. The targets of modification were the phosphorylation sites (T171A-Y173A) and the active center (K49R). Introduction of the variant HOG1-49 into the hog1∆ strain partially improved growth under osmotic stress, but did not recover the yeast-like shape of the cells. The HOG1-171/173 variant was not functional, and its introduction further weakened the growth of hog1∆ strains in hyperosmotic conditions. To verify a genetic modification in filament form, we developed a new system based on green fluorescent protein (GFP) for easier screening of proper mutants.ConclusionsThese results provide new insights into the functions of yl-Hog1 protein in dimorphic transition and constitute a good starting point for further genetic modification of Y. lipolytica in filament form.

Host switching by an ambrosia beetle fungal mutualist: Mycangial colonization of indigenous beetles by the invasive laurel wilt fungal pathogen.

Ambrosia beetles require their fungal symbiotic partner as their cultivated (farmed) food source in tree galleries. While most fungal-beetle partners do not kill the host trees they inhabit, since their introduction (invasion) into the United states around ~2002, the invasive beetle Xyleborus glabratus has vectored its mutualist partner (but plant pathogenic) fungus, Harringtonia lauricola, resulting in the deaths of over 300 million trees. Concerningly, indigenous beetles have been caught bearing H. lauricola. Here, we show colonization of the mycangia of the indigenous X. affinis ambrosia beetle by H. lauricola. Mycangial colonization occurred within 1 h of feeding, with similar levels seen for H. lauricola as found for the native X. affinis-R. arxii fungal partner. Fungal mycangial occupancy was stable over time and after removal of the fungal source, but showed rapid turnover when additional fungal cells were available. Microscopic visualization revealed two pre-oral mycangial pouches of ~100-200 × 25-50 μm/each, with narrow entry channels of 25-50 × 3-10 μm. Fungi within the mycangia underwent a dimorphic transition from filamentous/blastospore growth to yeast-like budding with alterations to membrane structures. These data identify the characteristics of ambrosia beetle mycangial colonization, implicating turnover as a mechanism for host switching of H. lauricola to other ambrosia beetle species.

Ada2 and Ada3 Regulate Hyphal Growth, Asexual Development, and Pathogenicity in Beauveria bassiana by Maintaining Gcn5 Acetyltransferase Activity.

The histone acetyltransferase (HAT) Gcn5 ortholog is essential for a variety of fungi. Here, we characterize the roles of Ada2 and Ada3, which are functionally linked to Gcn5, in the insect-pathogenic fungus Beauveria bassiana. Loss of Ada2 and Ada3 led to severe hyphal growth defects on rich and minimal media and drastic decreases in blastospore yield and conidiation capacity, with abnormal conidia-producing structures. ΔAda2 and ΔAda3 exhibited a delay in conidial germination and increased sensitivity to multiple chemical stresses and heat shock. Nearly all their pathogenicity was lost, and their ability to secrete extracellular enzymes, Pr1 proteases and chitinases for cuticle degradation was reduced. A yeast two-hybrid assay demonstrated that Ada2 binds to Ada3 and directly interacts with Gcn5, confirming the existence of a yeast-like Ada3-Ada2-Gcn5 HAT complex in this fungus. Additionally, deletion of the Ada genes reduced the activity of Gcn5, especially in the ΔAda2 strain, which was consistent with the acetylation level of histone H3 determined by Western blotting. These results illustrate the dependence of Gcn5 enzyme activity on Ada2 and Ada3 in fungal hyphal growth, asexual development, multiple stress responses, and pathogenicity in B. bassiana. IMPORTANCE The histone acetyltransferase Gcn5 ortholog contributes significantly to the growth and development of various fungi. In this study, we found that Ada2 and Ada3 have critical regulatory effects on Gcn5 enzyme activity and influence the acetylation of histone H3. Deletion of Ada2 or Ada3 decreased the fungal growth rate and asexual conidial yield and increased susceptibility to multiple stresses in Beauveria bassiana. Importantly, Ada genes are vital virulence factors, and their deletion caused the most virulence loss, mainly by inhibiting the activity of a series of hydrolytic enzymes and the dimorphic transition ability. These findings provide a new perspective on the function of the Gcn5 acetyltransferase complex in pathogens.

Analysis of gene expression related to polyamine concentration and dimorphism induced in ornithine decarboxylase (odc) and spermidine synthase (spd) Ustilago maydis mutants

Polyamines are ubiquitous small organic cations, and their roles as regulators of several cellular processes are widely recognized. They are implicated in the key stages of the fungal life cycle. Ustilago maydis is a phytopathogenic fungus, the causal agent of common smut of maize and a model system to understand dimorphism and virulence. U. maydis grows in yeast form at pH 7 and it can develop its mycelial form in vitro at pH 3. Δodc mutants that are unable to synthesize polyamines, grow as yeast at pH 3 with a low putrescine concentration, and to complete its dimorphic transition high putrescine concentration is require. Δspd mutants require spermidine to grow and cannot form mycelium at pH 3. In this work, the increased expression of the mating genes, mfa1 and mfa2, on Δodc mutants, was related to high putrescine concentration. Global gene expression analysis comparisons of Δodc and Δspd U. maydis mutants indicated that 2,959 genes were differentially expressed in the presence of exogenous putrescine at pH 7 and 475 genes at pH 3. While, in Δspd mutant, the expression of 1,426 genes was affected by exogenous spermine concentration at pH 7 and 11 genes at pH 3. Additionally, we identified 28 transcriptional modules with correlated expression during seven tested conditions: mutant genotype, morphology (yeast, and mycelium), pH, and putrescine or spermidine concentration. Furthermore, significant differences in transcript levels were noted for genes in modules relating to pH and genotype genes involved in ribosome biogenesis, mitochondrial oxidative phosphorylation, N-glycan synthesis, and Glycosylphosphatidylinositol (GPI)-anchor. In summary, our results offer a valuable tool for the identification of potential factors involved in phenomena related to polyamines and dimorphism.

Copper Ion Mediates Yeast-to-Hypha Transition in Yarrowia lipolytica.

Copper is an essential element that maintains yeast physiological function at low concentrations, but is toxic in excess. This study reported that Cu(II) significantly promoted the yeast-to-hypha transition of Yarrowia lipolytica in dose-dependent manner. Strikingly, the intracellular Cu(II) accumulation was drastically reduced upon hyphae formation. Moreover, we investigated the effect of Cu(II) on the physiological function of Y. lipolytica during the dimorphic transition and found that cellular viability and thermomyces lanuginosus lipase (TLL) were both influenced by the Cu(II)-induced yeast-to-hypha transition. Overall, hyphal cells survived better than yeast-form cells with copper ions. Furthermore, transcriptional analysis of the Cu(II)-induced Y. lipolytica before and after hyphae formation revealed a transition state between them. The results showed multiple differentially expressed genes (DEGs) were turned over between the yeast-to-transition and the transition-to-hyphae processes. Furthermore, gene set enrichment analysis (GSEA) identified that multiple KEGG pathways, including signaling, ion transport, carbon and lipid metabolism, ribosomal, and other biological processes, were highly involved in the dimorphic transition. Importantly, overexpression screening of more than thirty DEGs further found four novel genes, which are encoded by YALI1_B07500g, YALI1_C12900g, YALI1_E04033g, and YALI1_F29317g, were essential regulators in Cu-induced dimorphic transition. Overexpression of each of them will turn on the yeast-to-hypha transition without Cu(II) induction. Taken together, these results provide new insight to explore further the regulatory mechanism of dimorphic transition in Y. lipolytica.

Chemical Profile, Anti-Microbial and Anti-Inflammaging Activities of Santolina rosmarinifolia L. Essential Oil from Portugal.

Fungal infections and the accompanying inflammatory responses are associated with great morbidity and mortality due to the frequent relapses triggered by an increased resistance to antifungal agents. Furthermore, this inflammatory state can be exacerbated during inflammaging and cellular senescence. Essential oils (EO) are receiving increasing interest in the field of drug discovery due to their lipophilic nature and complex composition, making them suitable candidates in the development of new antifungal drugs and modulators of numerous molecular targets. This work chemically characterized the EO from Santolina rosmarinifolia L., collected in Setúbal (Portugal), and assessed its antifungal potential by determining its minimum inhibitory (MIC) and minimum lethal (MLC) concentration in accordance with the Clinical Laboratory Standard Guidelines (CLSI) guidelines, as well as its effect on several Candida albicans virulence factors. The anti-inflammatory effect was unveiled using lipopolysaccharide (LPS)-stimulated macrophages by assessing several pro-inflammatory mediators. The wound healing and anti-senescence potential of the EO was also disclosed. The EO was mainly characterized by β-pinene (29.6%), borneol (16.9%), myrcene (15.4%) and limonene (5.7%). It showed a strong antifungal effect against yeasts and filamentous fungi (MIC = 0.07-0.29 mg/mL). Furthermore, it inhibited dimorphic transition (MIC/16), decreased biofilm formation with a preeminent effect after 24 h (MIC/2) and disrupted preformed biofilms in C. albicans. Additionally, the EO decreased nitric oxide (NO) release (IC50 = 0.52 mg/mL) and pro-IL-1β and inducible nitric oxide synthase (iNOS) expression in LPS-stimulated macrophages, promoted wound healing (91% vs. 81% closed wound) and reduced cellular senescence (53% vs. 73% β-galactosidase-positive cells). Overall, this study highlights the relevant pharmacological properties of S. rosmarinifolia, opening new avenues for its industrial exploitation.