Genetic response analysis of Beauveria bassiana Z1 under high concentration Cd(II) stress.

Cadmium (Cd(II)) has carcinogenic and teratogenic toxicity, which can be accumulated in the human body through the food chain, endangering human health and life. In this study, a highly Cd(II)-tolerant fungus named Beauveria bassiana Z1 was studied, and its Cd(Ⅱ) removal efficiency was 71.2% when the Cd(II) concentration was 10 mM. Through bioanalysis and experimental verification of the transcriptome data, it was found that cadmium entered the cells through calcium ion channels, and then complexed with intracellular glutathione (GSH) and stored in vacuoles or excluded extracellular by ABC transporters. Cytochrome P450 was significantly upregulated in many pathways and actively participated in detoxification related reactions. The addition of cytochrome inhibitor taxifolin reduced the removal efficiency of Cd(II) by 45%. In the analysis, it demonstrated that ACOX1 gene and OPR gene of jasmonic acid (JA) synthesis pathway were significantly up-regulated, and were correlated with bZIP family transcription factors cpc-1_0 and pa p1_0. The results showed that exogenous JA could improve the removal efficiency of Cd(II) by strain Z1.

Impact of Beauveria bassiana on antioxidant enzyme activities and metabolomic profiles of Spodoptera frugiperda.

Spodoptera frugiperda is a pest that poses a serious threat to the production of food and crops. Entomopathogenic fungi, such as Beauveria bassiana, have shown potential for S. frugiperda control. However, the mechanism of this biological control of pathogens is not fully understood, such as how antioxidant enzyme activities and metabolic profiles in S. frugiperda larvae are affected when infected by entomopathogenic fungi. This study assessed the antioxidant enzyme activities and shift in metabolomic profile in the S. frugiperda larvae infected with B.bassiana. The results indicate a pattern of initial increase and subsequent decrease in the activities of superoxide dismutase, catalase, and peroxidase in the B.bassiana-infected larvae. And the enzyme activities at 60 h of infection ended significantly lower than those of the uninfected larvae. A total of 93 differential metabolites were identified in the B.bassiana-infected larvae, of which 41 metabolites were up-regulated and 52 were down-regulated. These metabolites mainly included amino acids, nucleotides, lipids, carbohydrates, and their derivatives. Among the changed metabolites, cystathionine, l-tyrosine, l-dopa, arginine, alpha-ketoglutaric acid, d-sedoheptulose-7-phosphate and citric acid were significantly decreased in B. bassiana-infected larvae. This indicated that the fungal infection might impair the ability of S. frugiperda larvae to cope with oxidative stress, leading to a negative impact of organism fitness. Further analyses of key metabolic pathways reveal that B. bassiana infection might affect purine metabolism, arginine biosynthesis, butanoate metabolism, and phenylalanine metabolism of S. frugiperda larvae. The findings from this study will contribute to our understanding of oxidative stress on immune defense in insects, and offer fundamental support for the biological control of S. frugiperda.

OTU7B Modulates the Mosquito Immune Response to Beauveria bassiana Infection via Deubiquitination of the Toll Adaptor TRAF4.

The Aedes aegypti mosquito transmits devastating flaviviruses, such as Zika, dengue, and yellow fever viruses. For more effective control of the vector, the pathogenicity of Beauveria bassiana, a fungus commonly used for biological control of pest insects, may be enhanced based on in-depth knowledge of molecular interactions between the pathogen and its host. Here, we identified a mechanism employed by B. bassiana, which efficiently blocks the Ae. aegypti antifungal immune response by a protease that contains an ovarian tumor (OTU) domain. RNA-sequencing analysis showed that the depletion of OTU7B significantly upregulates the mRNA level of immunity-related genes after a challenge of the fungus. CRISPR-Cas9 knockout of OTU7B conferred a higher resistance of mosquitoes to the fungus B. bassiana. OTU7B suppressed activation of the immune response by preventing nuclear translocation of the NF-κB transcription factor Rel1, a mosquito orthologue of Drosophila Dorsal. Further studies identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as an interacting protein of OTU7B. TRAF4-deficient mosquitoes were more sensitive to fungal infection, indicating TRAF4 to be the adaptor protein that activates the Toll pathway. TRAF4 is K63-link polyubiquitinated at K338 residue upon immune challenge. However, OTU7B inhibited the immune signaling by enzymatically removing the polyubiquitin chains of mosquito TRAF4. Thus, this study has uncovered a novel mechanism of fungal action against the host innate immunity, providing a platform for further improvement of fungal pathogen effectiveness. IMPORTANCE Insects use innate immunity to defend against microbial infection. The Toll pathway is a major immune signaling pathway that is associated with the antifungal immune response in mosquitoes. Our study identified a fungal-induced deubiquitinase, OTU7B, which, when knocked out, promotes the translocation of the NF-κB factor Rel1 into the nucleus and confers enhanced resistance to fungal infection. We further found the counterpart of OTU7B, TRAF4, which is a component of the Toll pathway and acts as an adaptor protein. OTU7B enzymatically removes K63-linked polyubiquitin chains from TRAF4. The immune response is suppressed, and mosquitoes become much more sensitive to the Beauveria bassiana infection. Our findings reveal a novel mechanism of fungal action against the host innate immunity.

Succinate Dehydrogenase Subunit C Contributes to Mycelial Growth and Development, Stress Response, and Virulence in the Insect Parasitic Fungus Beauveria bassiana.

Succinate dehydrogenase (SDH), also known as respiratory chain complex II, plays a crucial role in energy production in which SdhC functions as an anchored subunit in the inner membrane of mitochondria. In this study, domain annotation analyses revealed that two SdhC domain-containing proteins were present in the filamentous insect-pathogenic fungus Beauveria bassiana, and they were named BbSdhC1 and BbSdhC2, respectively. Only BbSdhC1 localized to mitochondria; hence, this protein is considered the ortholog of SdhC in B. bassiana. Ablation of BbSdhC1 led to significantly reduced vegetative growth on various nutrients. The ΔBbsdhc1 mutant displayed the significantly reduced ATP synthesis and abnormal differentiation under aerial and submerged conditions. Notably, the BbSdhC1 loss resulted in enhanced intracellular levels of reactive oxygen species (ROS) and impaired growth of mycelia under oxidative stress. Finally, insect bioassays (via cuticle and intrahemocoel injection infection) revealed that disruption of BbSdhC1 significantly attenuated fungal virulence against the insect hosts. These findings indicate that BbSdhC1 contributes to vegetative growth, resistance to oxidative stress, differentiation, and virulence of B. bassiana due to its roles in energy generation and maintaining the homeostasis of the intracellular ROS levels. IMPORTANCE The electron transport chain (ETC) is critical for energy supply by mediating the electron flow along the mitochondrial membrane. Succinate dehydrogenase (SDH) is also known as complex II in the ETC, in which SdhC is a subunit anchored in mitochondrial membrane. However, the physiological roles of SdhC remain enigmatic in filamentous fungi. In filamentous insect-pathogenic fungus B. bassiana, SdhC is required for maintaining mitochondrial functionality, which is critical for fungal stress response, development, and pathogenicity. These findings improve our understanding of physiological mechanisms of ETC components involved in pathogenicity of the entomopathogenic fungi.

Inductive Production of the Iron-Chelating 2-Pyridones Benefits the Producing Fungus To Compete for Diverse Niches.

Diverse 2-pyridone alkaloids have been identified with an array of biological and pharmaceutical activities, including the development of drugs. However, the biosynthetic regulation and chemical ecology of 2-pyridones remain largely elusive. Here, we report the inductive activation of the silent polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) (tenS) gene cluster for the biosynthesis of the tenellin-type 2-pyridones in the insect-pathogenic fungus Beauveria bassiana when cocultured with its natural competitor fungus Metarhizium robertsii. A pathway-specific transcription factor, tenR, was identified, and the overexpression of tenR well expanded the biosynthetic mechanism of 15-hydroxytenellin (15-HT) and its derivatives. In particular, a tandemly linked glycosyltransferase-methyltransferase gene pair located outside the tenS gene cluster was verified to mediate the rare and site-specific methylglucosylation of 15-HT at its N-OH residue. It was evident that both tenellin and 15-HT can chelate iron, which could benefit B. bassiana to outcompete M. robertsii in cocultures and to adapt to iron-replete and -depleted conditions. Relative to the wild-type strain, the deletion of tenS had no obvious negative effect on fungal virulence, but the overexpression of tenR could substantially increase fungal pathogenicity toward insect hosts. The results of this study well advance the understanding of the biosynthetic machinery and chemical ecology of 2-pyridones. IMPORTANCE Different 2-pyridones have been identified, with multiple biological activities but unclear chemical ecology. We found that the silent tenS gene cluster was activated in the insect pathogen Beauveria bassiana when the fungus was cocultured with its natural competitor Metarhizium robertsii. It was established that the gene cluster is regulated by a pathway-specific regulator, tenR, and the overexpression of this transcription factor expanded the biosynthetic machinery of the tenellin 2-pyridones. It was also found that the paired genes located outside the tenS cluster contribute to the site-specific methylglucosylation of the main compound 15-hydroxytenellin. Both tenellin and 15-hydroxytenellin can chelate and sequester iron to benefit the producing fungus to compete for different niches. This study well advances the biosynthetic mechanism and chemical ecology of 2-pyridones.

SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections.

Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to ß-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.

Iron homeostasis in the absence of ferricrocin and its consequences in fungal development and insect virulence in Beauveria bassiana.

The putative ferricrocin synthetase gene ferS in the fungal entomopathogen Beauveria bassiana BCC 2660 was identified and characterized. The 14,445-bp ferS encodes a multimodular nonribosomal siderophore synthetase tightly clustered with Fusarium graminearum ferricrocin synthetase. Functional analysis of this gene was performed by disruption with the bar cassette. ΔferS mutants were verified by Southern and PCR analyses. HPLC and TLC analyses of crude extracts indicated that biosynthesis of ferricrocin was abolished in ΔferS. Insect bioassays surprisingly indicated that ΔferS killed the Spodoptera exigua larvae faster (LT50 59 h) than wild type (66 h). Growth and developmental assays of the mutant and wild type demonstrated that ΔferS had a significant increase in germination under iron depletion and radial growth and a decrease in conidiation. Mitotracker staining showed that the mitochondrial activity was enriched in ΔferS under both iron excess and iron depletion. Comparative transcriptomes between wild type and ΔferS indicated that the mutant was increased in the expression of eight cytochrome P450 genes and those in iron homeostasis, ferroptosis, oxidative stress response, ergosterol biosynthesis, and TCA cycle, compared to wild type. Our data suggested that ΔferS sensed the iron excess and the oxidative stress and, in turn, was up-regulated in the antioxidant-related genes and those in ergosterol biosynthesis and TCA cycle. These increased biological pathways help ΔferS grow and germinate faster than the wild type and caused higher insect mortality than the wild type in the early phase of infection.

The Tudor Domain-Containing Protein BbTdp1 Contributes to Fungal Cell Development, the Cell Cycle, Virulence, and Transcriptional Regulation in the Insect Pathogenic Fungus Beauveria bassiana.

Beauveria bassiana is an insect pathogenic fungus that serves as a model system for exploring the mechanisms of fungal development and host-pathogen interactions. Clinical and experimental studies have indicated that SND1 is closely correlated with the progression and invasiveness of common cancers as a potential oncogene, but this gene has rarely been studied in fungi. Here, we characterized the contributions of an SND1 ortholog (Tdp1) by constructing a BbTdp1 deletion strain and a complemented strain of B. bassiana. Compared with the wild-type (WT) strain, the ΔBbTdp1 mutant lost conidiation capacity (∼87.7%) and blastospore (∼96.3%) yields, increased sensitivity to chemical stress (4.4 to 54.3%) and heat shock (∼44.2%), and decreased virulence following topical application (∼24.7%) and hemocoel injection (∼40.0%). Flow cytometry readings showed smaller sizes of both conidia and blastospores for ΔBbTdp1 mutants. Transcriptomic data revealed 4,094 differentially expressed genes (|log2 ratio| > 2 and a q value of <0.05) between ΔBbTdp1 mutants and the WT strain, which accounted for 41.6% of the total genes, indicating that extreme fluctuation in the global gene expression pattern had occurred. Moreover, deletion of BbTdp1 led to an abnormal cell cycle with a longer S phase and shorter G2/M and G0/G1 phases of blastospores, and enzyme-linked immunosorbent assay confirmed that the level of phosphorylated cyclin-dependent kinase 1 (Cdk1) in the ΔBbTdp1 strain was ∼31.5% lower than in the WT strain. In summary, our study is the first to report that BbTdp1 plays a vital role in regulating conidia and blastospore yields, fungal morphological changes, and pathogenicity in entomopathogenic fungi. IMPORTANCE In this study, we used Beauveria bassiana as a biological model to report the role of BbTdp1 in entomopathogenic fungi. Our findings indicated that BbTdp1 contributed significantly to cell development, the cell cycle, and virulence in B. bassiana. In addition, deletion of BbTdp1 led to drastic fluctuations in the transcriptional profile. BbTdp1 can be developed as a novel target for B. bassiana development and pathogenicity, which also provides a framework for the study of Tdp1 in other fungi.

Characterization of a fungal competition factor: Production of a conidial cell-wall associated antifungal peptide.

Competition is one of the fundamental driving forces of natural selection. Beauveria bassiana is a soil and plant phylloplane/root fungus capable of parasitizing insect hosts. Soil and plant environments are often enriched with other fungi against which B. bassiana competes for survival. Here, we report an antifungal peptide (BbAFP1), specifically expressed and localized to the conidial cell wall and is released into the surrounding microenvironment inhibiting growth of competing fungi. B. bassiana strains expressing BbAFP1, including overexpression strains, inhibited growth of Alternaria brassicae in co-cultured experiments, whereas targeted gene deletion of BbAFP1 significantly decreased (25%) this inhibitory effect. Recombinant BbAFP1 showed chitin and glucan binding abilities, and growth inhibition of a wide range of phytopathogenic fungi by disrupting membrane integrity and eliciting reactive oxygen species (ROS) production. A phenylalanine residue (F50) contributes to chitin binding and antifungal activity, but was not required for the latter. Expression of BbAFP1 in tomato resulted in transgenic plants with enhanced resistance to plant fungal pathogens. These results highlight the importance of fungal competition in shaping primitive competition strategies, with antimicrobial compounds that can be embedded in the spore cell wall to be released into the environment during the critical initial phases of germination for successful growth in its environmental niche. Furthermore, these peptides can be exploited to increase plant resistance to fungal pathogens.

Neuroprotective Effect of CR-777, a Glutathione Derivative of Withaferin A, Obtained through the Bioconversion of Withania somnifera (L.) Dunal Extract by the Fungus Beauveria bassiana.

The bioconversion of Withania somnifera extract by the fungus Beauveria bassiana leads to cysteine and glutathione derivatives of withaferin A at the C-6 position. The compounds were purified and fully characterized by 1D-NMR, 2D-NMR, and HRMS analysis. The glutathione derivative CR-777 was evaluated as a neuroprotective agent from damage caused by different neurotoxins mimicking molecular symptoms in Parkinson´s disease (PD), including 1-methyl-4-phenylpyridinium (MPP+), 6-hydroxydopamine (6-OHDA), and α-synuclein (α-Syn). CR-777, at nanomolar concentrations, protected dopaminergic and cortical neurons. In 6-OHDA-treated neurons, CR-777 increased cell survival and neurite network and decreased the expression of α-Syn. Using specific inhibitors of cell toxicity signaling pathways and specific staining experiments, the observed role of CR-777 seemed to involve the PI3K/mTOR pathway. CR-777 could be considered as a protective agent against a large panel of neuronal stressors and was engaged in further therapeutic development steps.

Identification of calmodulin binding proteins in the entomopathogenic fungus Beauveria bassiana.

Calmodulin (CaM) is a primary Ca2+ receptor and plays a pivotal role in a variety of cellular responses in eukaryotes. Even though a large number of CaM-binding proteins are well known in yeast, plants, and animals, little is known regarding CaM-targeted proteins in filamentous fungi. To identify CaM-binding proteins in filamentous fungi, we used a proteomics method coupled with co-immunoprecipitation (CoIP) and MALDI-TOF/TOF mass spectrometry (MS) in Beauveria bassiana. Through this method, we identified ten CaM-binding proteins in B. bassiana. One of the CaM-targeted proteins was the heat shock protein 70 (BbHSP70) in B. bassiana. Our biochemical study showed that ATP inhibits the molecular interaction between BbHSP70 and CaM, suggesting a regulatory mechanism between CaM and ATP for regulating BbHSP70.

In vivo gene expression profiling of the entomopathogenic fungus Beauveria bassiana elucidates its infection stratagems in Anopheles mosquito.

The use of entomopathogenic fungi to control mosquitoes is a promising tool for reducing vector-borne disease transmission. To better understand infection stratagems of insect pathogenic fungi, we analyzed the global gene expression profiling of Beauveria bassiana at 36, 60, 84 and 108 h after topical infection of Anopheles stephensi adult mosquitoes using RNA sequencing (RNA-Seq). A total of 5,354 differentially expressed genes (DEGs) are identified over the course of fungal infection. When the fungus grows on the mosquito cuticle, up-regulated DEGs include adhesion-related genes involved in cuticle attachment, Pth11-like GPCRs hypothesized to be involved in host recognition, and extracellular enzymes involved in the degradation and penetration of the mosquito cuticle. Once in the mosquito hemocoel, the fungus evades mosquito immune system probably through up-regulating expression of ß-1,3-glucan degrading enzymes and chitin synthesis enzymes for remodeling of cell walls. Moreover, six previous unknown SSCP (small secreted cysteine-rich proteins) are significantly up-regulated, which may serve as "effectors" to suppress host defense responses. B. bassiana also induces large amounts of antioxidant genes to mitigate host-generated exogenous oxidative stress. At late stage of infection, B. bassiana activates a broad spectrum of genes including nutrient degrading enzymes, some transporters and metabolism pathway components, to exploit mosquito tissues and hemolymph as a nutrient source for hyphal growth. These findings establish an important framework of knowledge for further comprehensive elucidation of fungal pathogenesis and molecular mechanism of Beauveria-mosquito interactions.

Differential Roles for Six P-Type Calcium ATPases in Sustaining Intracellular Ca2+ Homeostasis, Asexual Cycle and Environmental Fitness of Beauveria bassiana.

A global insight into the roles of multiple P-type calcium ATPase (CA) pumps in sustaining the life of a filamentous fungal pathogen is lacking. Here we elucidated the functions of five CA pumps (Eca1, Spf1 and PmcA/B/C) following previous characterization of Pmr1 in Beauveria bassiana, a fungal insect pathogen. The fungal CA pumps interacted at transcriptional level, at which singular deletions of five CA genes depressed eca1 expression by 76-98% and deletion of spf1 resulted in drastic upregulation of four CA genes by 36-50-fold. Intracellular Ca2+ concentration increased differentially in most deletion mutants exposed to the stresses of Ca2+, EDTA chelator, and/or endoplasmic reticulum and calcineurin inhibitors, accompanied with their changed sensitivities to not only the mentioned agents but also Fe2+, Cu2+ and Zn2+. Liquid culture acidification was delayed in the Δspf1, Δpmr1 and ΔpmcA mutants, coinciding well with altered levels of their extracellular lactic and oxalic acids. Moreover, all deletion mutants showed differential defects in conidial germination, vegetative growth, conidiation capacity, antioxidant activity, cell wall integrity, conidial UV-B resistance and/or virulence. Our results provide the first global insight into differential roles for six CA pumps in sustaining intracellular Ca2+ level, asexual cycle and environmental fitness of B. bassiana.

Isolation and characterization of 2-pyridone alkaloids and alloxazines from Beauveria bassiana.

Two novel compounds bearing heterocyclic nitrogen, 2-pyridone alkaloid (1) and alloxazine derivative (2), along with the known pretenellin B (3), pyridovericin (4) and lumichrome (5) were isolated from a culture of the entomopathogenic fungal strain Beauveria bassiana. The chemical structures of 2-pyridone alkaloid and alloxazine derivative were established on the basis of the interpretation of spectroscopic data. The isolated compounds were evaluated in a panel of five cancer cell lines and pyridovericin exhibited cytotoxicity (IC50, µM) against cancer cell lines: HL-60 (25.9 ± 0.3), HCT8 (34.6 ± 3.6), MDA-MB435 (34.8 ± 3.8) and SF295 (31.1 ± 0.6). Considering that other pyridone compounds display good cytotoxic activity, it would be suggested to obtain new semi synthetic derivatives of pyridovericin, for the development of new cytotoxic chemical entities.

Comparative performance evaluation of multi-metal resistant fungal strains for simultaneous removal of multiple hazardous metals.

In the present study, five fungal strains viz., Aspergillus terreus AML02, Paecilomyces fumosoroseus 4099, Beauveria bassiana 4580, Aspergillus terreus PD-17, Aspergillus fumigatus PD-18, were screened for simultaneous multimetal removal. Highest metal tolerance index for each individual metal viz., Cd, Cr, Cu, Ni, Pb and Zn (500mg/L) was recorded for A. fumigatus for the metals (Cd, 0.72; Cu, 0.72; Pb, 1.02; Zn, 0.94) followed by B. bassiana for the metals (Cd, 0.56; Cu, 0.14; Ni, 0.29; Zn, 0.85). Next, the strains were exposed to multiple metal mixture (Cd, Cr, Cu, Ni, Pb and Zn) of various concentrations (6, 12, 18, 30mg/L). Compared to other strains, B. bassiana and A. fumigatus had higher cube root growth (k) constants indicating their better adaptability to multi metal stress. After 72h, multimetal accumulation potential of B. bassiana (26.94±0.07mg/L) and A. fumigatus (27.59±0.09mg/L) were higher than the other strains at initial multimetal concentration of 30mg/L. However, considering the post treatment concentrations of individual metals in multimetal mixture (at all the tested concentrations), A. fumigatus demonstrated exceptional performance and could bring down the concentrations of Cd, Cu, Ni, Pb and Zn below the threshold level for irrigation prescribed by Food and Agriculture Organization (FAO).

Regulative roles of glutathione reductase and four glutaredoxins in glutathione redox, antioxidant activity, and iron homeostasis of Beauveria bassiana.

Multiple glutaredoxins (Grx) and glutathione reductase (Glr) are vital for the thiol-disulfide redox system in budding yeast but generally unexplored in filamentous fungi. Here we characterized the Beauveria bassiana redox system comprising dithiol Grx1, monothiol Grx2-4, Grx-like Grx5, and Glr orthologue. Each grx or glr deletion was compensated by increased transcripts of some other grx genes in normal cultures. Particularly, grx3 compensated the absence of grx1, grx2, grx5, or glr under oxidative stress while its absence was compensated only by undeletable grx4 under normal conditions but by most of other undeleted grx and glr genes in response to menadione. Consequently, the redox state was disturbed in Δglr more than in Δgrx3 but not in Δgrx1/2/5. Superoxide dismutases were more active in normal Δgrx1-3 cultures but less in Δgrx5 or Δglr response to menadione. Total catalase activity increased differentially in all the mutant cultures stressed with or without H2O2 while total peroxidase activity decreased more in the normal or H2O2-stressed culture of Δglr than of Δgrx3. Among the mutants, Δgrx3 showed slightly increased sensitivity to menadione or H2O2; Δglr exhibited greater sensitivity to thiol-oxidizing diamide than thiol-reducing 1-chloro-2,4-dinitrobenzene as well as increased sensitivity to the two oxidants. Intriguingly, all the mutants grew slower in a Fe(3+)-inclusive medium perhaps due to elevated transcripts of two Fe(3+) transporter genes. More or fewer phenotypes linked with biocontrol potential were altered in four deletion mutants excluding Δgrx5. All the changes were restored by targeted gene complementation. Overall, Grx3 played more critical role than other Grx homologues in the Glr-dependent redox system of the fungal entomopathogen.

A Distinctive Pattern of Beauveria bassiana-biotransformed Ginsenoside Products Triggers Mitochondria/FasL-mediated Apoptosis in Colon Cancer Cells.

Ginseng is one of the most commonly used adaptogens. Transformation into the minor ginsenosides produces compounds with more effective action. Beauveria bassiana, a teleomorph of Cordyceps bassiana, is a highly efficient producer of mammalian steroids and produces large amounts of sugar-utilizing enzymes. However, the fermentation of steroid glycosides in ginseng with B. bassiana has never been studied. Thus, we evaluated the bioconversion of the major ginsenosides in white ginseng by B. bassiana. Interestingly, B. bassiana increased the total amount of protopanaxadiols and hydrolyzed Rb1 into minor ginsenosides, exhibiting high levels of Rd and Rg3, as well as moderate levels of Rb2 and Rc analyzed by high-performance liquid chromatography coupled with evaporative light-scattering detection. The ß-glucosidase activity was highly increased, which led to the selective elimination of sugar moiety at the 20-C position of Rb1 to Rd, followed by Rg3. Rb2 and Rc accumulated because of the minimal activities of α-L-arabinopyranosidase and α-L-arabinofuranosidase, respectively. The fermentation product exerted dose-dependent cytotoxicity in HCT-15 cells, which are resistant to ginseng. The product, but not white ginseng, exhibited apoptotic effects via the Fas ligand and caspase 8/9. This study demonstrates for the first time that the B. bassiana-fermented metabolites have potent apoptotic activity in colon cancer cells, linking to a therapeutic use.

A novel Ras GTPase (Ras3) regulates conidiation, multi-stress tolerance and virulence by acting upstream of Hog1 signaling pathway in Beauveria bassiana.

Two Ras ATPases (Ras1 and Ras2) are well known to regulate antagonistically or cooperatively various cellular events in many fungi. Here we show the significance of a novel Ras homolog (Ras3) for Beauveria bassiana. Ras3 possesses five domains and two GTP/GDP switches typical for Ras family and was proven to localize to plasma membrane despite the position change of a membrane-targeting cysteine in C-terminal CAAX motif. Deletion of ras3 altered temporal transcription pattern of ras1 instead of ras2. Compared with wild-type, Δras3 grew significantly faster in a rich medium but slower in some minimal media, and produced far fewer conidia with impaired quality, which was evident with slower germination, attenuated virulence, reduced thermotolerance and decreased UV-B resistance. Moreover, Δras3 was much more sensitive to the oxidative stress of menadione than of H2O2 and to the stress of high osmolarity than of cell wall perturbation during growth. The high sensitivity of Δras3 to menadione was concurrent with reductions in both gene transcripts and total activity of superoxide dismutases. Intriguingly, the high osmosensitivity was concurrent with not only reduced transcripts of a critical transcription factor (Msn2) and most signaling proteins in the high-osmolarity-glycerol pathway of Δras3 but nearly undetectable phosphorylation signal of Hog1 hallmarking the pathway. All the changes were restored by ras3 complementation. Taken together, Ras3 is involved in the Hog1 pathway required for osmoregulation and hence can positively regulate conidiation, germination, multi-stress tolerance and virulence linked to the biological control potential of the filamentous insect pathogen.

Variations in oxygen concentration cause differential antioxidant response and expression of related genes in Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana is widely used in pest biocontrol strategies. We evaluated both the antioxidant response mediated by compatible solutes, trehalose or mannitol, and the expression of related genes using oxygen pulses at three oxygen concentrations in solid state culture (SSC): normal atmosphere (21% O2), low oxygen (16% O2) and enriched oxygen (26% O2). Trehalose concentration decreased 75% after atmospheric modifications in the cultures, whereas mannitol synthesis was three-fold higher under the 16% O2 pulses relative to normal atmosphere (100 and 30 µg mannitol mg(-1) biomass, respectively). Confirming this result, expression of the mpd gene, coding for mannitol-1-P dehydrogenase (MPD), increased up to 1.4 times after O2 pulses. The expression of the bbrgs1 gene, encoding a regulatory G protein related to conidiation, was analysed to explain previously reported differences in conidial production. Surprisingly, expression of bbrgs1 decreased after atmospheric modification. Finally, principal component analysis (PCA) indicated that 83.39% of the variability in the data could be explained by two components. This analysis corroborated the positive correlation between mannitol concentration and mpd gene expression, as well as the negative correlation between conidial production and bbrgs1 gene expression. This study contributes to understanding of antioxidant and molecular response of B. bassiana induced under oxidant conditions.