Fungal Virulence Research Articles

ApWD40a, a Member of the WD40-Repeat Protein Family, Is Crucial for Fungal Development, Toxin Synthesis, and Pathogenicity in the Ginseng Alternaria Leaf Blight Fungus Alternaria panax

Alternaria panax, the primary pathogen that causes ginseng Alternaria leaf blight disease, can lead to a 20–30% reduction in ginseng yield. WD40 repeat-containing proteins are evolutionarily conserved proteins with diverse functions between different organisms. In this study, we characterized the roles of a WD40 repeat-containing protein in A. panax. The deletion of ApWD40a impaired the mycelial growth, reduced the sporulation, and significantly decreased the efficiency in utilizing various carbon sources. The ΔApwd40a mutant showed increased sensitivity to osmotic stress and metal ion stress induced by sorbitol, NaCl, and KCl, but decreased the sensitivity to a cell wall stress factor (SDS) and oxidative stress factors (paraquat and H2O2). Pathogenicity assays performed on detached ginseng leaves and roots revealed that the disruption of ApWD40a significantly decreased the fungal virulence through attenuating melanin and mycotoxin production by A. panax. A comparative transcriptome analysis revealed that ApWD40a was involved in many metabolic and biosynthetic processes, including amino acid metabolism, carbon metabolism, sulfate metabolic pathways, and secondary metabolite pathways. In particular, a significantly upregulated gene that encoded a sulfate permease 2 protein in ΔApwd40a, named ApSulP2, was deleted in the wild-type strain of A. panax. The deletion of ApSulP2 resulted in reduced biomass under sulfate-free conditions, demonstrating that the sulfate transport was impaired. Taken together, our findings highlight that ApWD40a played crucial roles in different biological processes and the pathogenicity of A. panax through modulating the expressions of genes involved in various primary and secondary metabolic processes.

Novel genomic features in entomopathogenic fungus Beauveria bassiana ILB308: accessory genomic regions and putative virulence genes involved in the infection process of soybean pest Piezodorus guildinii.

Biological control methods involving entomopathogenic fungi like Beauveria bassiana have been shown to be a valuable approach in integrated pest management as an environmentally friendly alternative to control pests and pathogens. Identifying genetic determinants of pathogenicity in B. bassiana is instrumental for enhancing its virulence against insects like the resistant soybean pest Piezodorus guildinii. This study focused on comparative genomics of different B. bassiana strains and gene expression analyses to identify virulence genes in the hypervirulent strain ILB308, especially in response to infection of P. guildinii and growth on hydrocarbon HC15, a known virulence enhancer. Strain ILB308 showed the highest number of virulence-related features, such as candidate virulence proteins, effectors, small secreted proteins and biosynthetic gene clusters. ILB308 also had a high percentage of unique DNA sequences, including six accessory scaffolds. Gene expression analysis at 4 days post inoculation revealed upregulation of known virulence factors, including Tudor domain proteins, LysM motif-containing proteins, subtilisin-like proteases and novel genes encoding secreted effectors and heat-labile enterotoxins. Growth on HC15 led to the upregulation of genes associated with oxidoreductase activity related to cuticular alkane degradation and fermentation metabolism/antioxidant responses in the hemolymph. The low number of known B. bassiana virulence genes points to novel or unknown mechanisms acting on the interaction between P. guildinii and strain ILB308. The presence of accessory genomic regions and unique virulence genes in ILB308 may contribute to its higher virulence. These genes could be considered as potential targets for enhancing fungal virulence through genetic manipulation. © 2025 Society of Chemical Industry.

Ex Vivo Fungal Nail Penetration Study: Effects of Causative Organisms, Nail Polish and Age.

Few exvivo studies have investigated the virulence factors of fungi causing onychomycosis. The effect of nail polish in predisposing or protecting against onychomycosis remains debatable. This exvivo study aimed to identify the nail invasion ability of dermatophytes, non-dermatophytes and yeast, with and without nail polishing, in the nails of young and elderly individuals. Six fungal species were tested: dermatophytes (Trichophyton rubrum, T. mentagrophytes, Microsporum canis), non-dermatophytes (Neoscytalidium dimidiatum, Fusarium spp.) and Candida albicans. Nail plates from eight volunteers (four aged ≥ 70 years; four aged < 70 years) were divided into polished and non-polished groups, incubated with each fungus and evaluated at 2, 4 and 8 weeks. Positive results were determined the presence of fungal hyphae or pseudohyphae penetrating the nail plate, with the enlargement of invasive fungal elements confirmed by histology. At 2 weeks, N. dimidiatum exhibited the highest nail invasion rate (15/16, 93.75%), whereas C. albicans showed the lowest (1/16, 6.25%). Fungal penetration into nail plates increased with longer incubation durations. At 8 weeks, C. albicans did not invade any polished nail plates; however, the difference in invasion rates between polished and unpolished nail plates was not statistically significant. Additionally, age did not significantly affect the invasion of most fungi in this exvivo study. This exvivo study supported the concept that fungal virulence is the main determining factor for nail invasion. N. dimidiatum caused the most and fastest nail plate penetration. Nail polishing may slow the penetration of low-virulence organisms.

Insights into phosphatidic acid phosphatase and its potential role as a therapeutic target.

Phosphatidic acid phosphatase, a conserved eukaryotic enzyme that catalyzes the Mg2+-dependent dephosphorylation of phosphatidic acid to produce diacylglycerol, has emerged as a vital regulator of lipid homeostasis. By controlling the balance of phosphatidic acid and diacylglycerol, the enzyme governs the use of the lipids for synthesis of the storage lipid triacylglycerol and the membrane phospholipids needed for cell growth. The mutational, biochemical, and cellular analyses of yeast phosphatidic acid phosphatase have provided insights into the structural determinants of enzyme function with the understanding of its regulation by phosphorylation and dephosphorylation. The key role that the enzyme plays in triacylglycerol synthesis indicates it may be a potential drug target to ameliorate obesity in humans. The enzyme activity, which is critical to the growth and virulence of pathogenic fungi, is a proposed target for therapeutic development to ameliorate fungal infections.

Insect hypovirulence-associated mycovirus confers entomopathogenic fungi with enhanced resistance against phytopathogens

ABSTRACT Mycoviruses can alter the biological characteristics of host fungi, including change virulence or pathogenicity of phytopathogens and entomopathogenic fungi (EPF). However, most studies on the mycoviruses found in EPF have focused on the effects of the viruses on the virulence of host fungi towards insect pests, with relatively few reports on the effects to the host fungi with regard to plant disease resistance in hosts. The present study investigated the effects of the mycovirus Beauveria bassiana chrysovirus 2 (BbCV2) virus infection on host biological characteristics, evaluated antagonistic activity of BbCV2 against two phytopathogenic fungi (Sclerotinia sclerotiorum and Botrytis cinerea), and transcriptome analysis was used to reveal the interactions between viruses and hosts. Our results showed that BbCV2 virus infection increased B. bassiana‘s growth rate, spore production, and biomass, it also enhanced the capacity of host fungi and their metabolic products to inhibit phytopathogenic fungi. BbCV2 virus infection reduced the contents of the two pathogens in tomato plants significantly, and transcriptome analysis revealed that the genes related to competition for ecological niches and nutrition, mycoparasitism and secondary metabolites in B. bassiana were significantly up-regulated after viral infection. These findings indicated that the mycovirus infection is an important factor to enhance the ability of B. bassiana against plant disease after endophytic colonization. We suggest that mycovirus infection causes a positive effect on B. bassiana against phytopathogens, which should be considered as a potential strategy to promote the plant disease resistance of EPF.

The magnitude of Ascochyta blight of chickpea and its relationship with prevailing environmental conditions in the Thal region of Punjab, Pakistan

This study assessed Ascochyta blight disease in chickpea across five districts in the ‘Thal’ region of Punjab, Pakistan: Bhakkar, Jhang, Layyah, Muzaffargarh, and Mianwali. The disease prevalence, incidence, and severity were recorded over two consecutive years, 2020-2021 and 2021-2022, during the chickpea-growing season. Disease prevalence ranged from 76% in Layyah to 40% in Muzaffargarh. Layyah also exhibited the highest disease incidence (84.2%) and severity (59.41%), while Muzaffargarh recorded the lowest disease incidence (20.55%) and severity (8.55%). All thirty-seven fungal isolates obtained from diseased samples were morphologically identified as Ascochyta rabiei. Twenty-five isolates, including five representatives from each district, were characterized based on their pathogenicity. The isolate ARL1 from Layyah exhibited the highest pathogenicity, with disease rating scores of 8.5 in detached leaf assays and 7.3 in attached leaf assays. Pathogen virulence showed a positive relationship with the disease intensity observed in the respective districts. Disease incidence and severity were further correlated with various prevailing environmental factors in these districts. Disease incidence was positively correlated with relative humidity and wind speed, while disease severity showed a positive correlation with relative humidity and rainfall but a negative correlation with maximum temperature. Other epidemiological factors did not exhibit significant relationships with the disease. This study concludes that the ‘Thal’ region is highly threatened by Ascochyta blight disease due to relatively higher relative humidity, rainfall, wind speed, and fungal virulence. Therefore, proper and timely management practices are essential to mitigate the impact of Ascochyta blight disease and address these influencing factors effectively.

A positively charged carbon dot complex improves the bioactivity of Isaria fumosorosea against Plutella xylostella (Linnaeus)

BackgroundNanomaterials compounded with entomopathogenic fungi have been proven to be effective in insect pest management. This study reports the synthesis and bioactivity of Isaria fumosorosea-based nanoparticles by compounding the fungus with differentially (positively or negatively) charged carbon dots. Initially, negatively charged carbon dots (N-CDs) and carbon dots (P-CDs) were synthesized via a one-step hydrothermal method. The N-CDs and P-CDs were then individually compounded with I. fumosorosea to develop N-CD@I. fumosorosea and P-CD@I. fumosorosea nanocomposites.ResultsCharacterization of the nanoparticles revealed that positively or negatively charged carbon dots were attached to I. fumosorosea by electrostatic bonding. Finally, the virulence of both types of nanoparticles was observed in Plutella xylostella. The bioassay results indicated that the highest P. xylostella mortality (92.7 ± 2.04%) was associated with the P-CD@I. fumosorosea treatment. The results of nontargeted metabolomic analysis revealed that different treatments affected the metabolism of P. xylostella by interfering with the riboflavin metabolism pathway by downregulating the expression of two main metabolites of the riboflavin metabolism pathway (2-5-diamino-6-pyrimidin-4-one and 7-hydroxy-6-methyl-8-ribiotin), although the extent of the expression downregulation varied among the different treatments.ConclusionOverall, this work provides insight into the mechanism by which positively charged nanomaterials improve the virulence of entomopathogenic fungi. This work provides a new direction for the design and application of nanomaterials for insect pest management.Graphical

A virulent milRNA of Fusarium oxysporum f. sp. cubense impairs plant resistance by targeting banana AP2 transcription factor coding gene MaPTI6L

Abstract Fungi produce microRNA-like RNAs (milRNAs) with functional importance in various biological processes. Our previous research identified a new milRNA Foc-milR87 from Fusarium oxysporum f. sp. cubense, which contributes to fungal virulence by targeting the pathogen glycosyl hydrolase encoding gene. However, the potential roles of fungal milRNAs in interactions with hosts are not well understood. This study demonstrated that Foc-milR87 specifically suppressed the expression of MaPTI6L, a pathogenesis-related gene that encodes a transcriptional activator in the banana (Musa acuminata Cavendish group cv. ‘Baxi Jiao’) genome, by targeting the 3'untranslated region (UTR) of MaPTI6L. Transient overexpression of MaPTI6L activated plant defense responses that depend on its nuclear localization, yet co-expression with Foc-milR87 attenuated these responses. MaPTI6L enhanced plant resistance by promoting transcription of the salicylic acid (SA) signaling pathway marker gene MaEDS1. Sequence analysis of the MaPTI6L gene in 19 banana varieties, particularly those resistant to Fusarium wilt, uncovered single nucleotide polymorphisms (SNPs) at Foc-milR87 target sites. Experimental validation showed that these SNPs significantly reduce the microRNA's ability to suppress target gene expression. Our findings reveal that Foc-milR87 plays an important role in impairing plant resistance by targeting MaPTI6L mRNA and reducing MaEDS1 transcription during the early infection stage, suggesting the 3'UTR of MaPTI6L as a promising target for genome editing in generation of disease-resistant banana cultivars.

Role of the Anaphase-Promoting Complex Activator Cdh1 in the Virulence of Cryptococcus neoformans.

Cryptococcus neoformans is a globally distributed human fungal pathogen that can cause cryptococcal meningitis with high morbidity and mortality. In this study, we identified an anaphase-promoting complex (APC) activator, Cdh1, and examined its impact on the virulence of C. neoformans. Our subcellular localization analysis revealed that Cdh1 is situated in the nucleus of C. neoformans. Disrupting or overexpressing the CDH1 gene caused abnormal capsule formation in C. neoformans. The cdh1Δ mutant displayed slight sensitivity when grown at 37 °C, indicating that Cdh1 plays a role in maintaining the growth of C. neoformans at 37 °C. A fungal virulence assay showed that Cdh1 is closely associated with the virulence of C. neoformans, and both the cdh1Δ mutant and CDH1OE overexpression strains significantly diminished the virulence of C. neoformans. The Cryptococcus-macrophage interaction assay revealed that both the cdh1∆ mutant and the CDH1OE strains had significantly lower proliferation ability inside macrophages. Furthermore, the infection of the cdh1Δ mutant significantly activated neutrophil recruitment, as well as Th2 and Th17 immune responses, in lung tissue. In summary, our findings indicate that Cdh1 is crucial for producing virulence factors and fungal virulence in C. neoformans. The findings of this study can offer valuable insights and form the basis for further study of the regulatory mechanisms governing the pathogenicity of C. neoformans, potentially leading to the development of novel therapeutic strategies.

Two secretory T2 RNases from a fungal pathogen target distinct insect cell transmembrane proteins to cause cytotoxicity.

Fungal pathogens produce secretory ribonuclease (RNase) T2 proteins during infection, which contribute to fungal virulence via their enzyme functions in degradation of host cell RNA. However, the details of those proteins entering the host cells are unclear. Our previous study demonstrated that the two secretory RNase T2 members, BbRNT2 and BbTrv, produced by the insect fungal pathogen Beauveria bassiana, caused cytotoxic damage to insect cells and contributed to fungal virulence. Here, the Spodoptera frugiperda ovarian epithelial cells (sf9 cells) were used as models to investigate the interactions of the two fungus-produced RNase T2 proteins with the insect cells. Two transmembrane proteins, an ABC transporter (SfABCG) and an Innexin 7-like protein (Sfinx), were identified from the sf9 cells as interacting with BbRNT2 and BbTrv, respectively, through protein immunoprecipitation, yeast-two hybrid tests and protein pull-down assays. Although a slight decrease in the sf9 cell viability was examined by transfection of RNA interference of SfABCG or Sfinx, the transfected cells displayed a dramatically decreased sensitivity to BbRNT2 or BbTrv, suggesting the requirement of the twotransmembrane proteins for BbRNT2 and BbTrv to enter the insect cells. These results reveal a mechanism of the cytotoxic molecules, T2 RNases, produced by the fungal pathogen, entering the insect cells via interaction with specific insect cell transmembrane proteins and causing cytotoxic damage.

Inhibitory effects of berberine on fungal growth, biofilm formation, virulence, and drug resistance as an antifungal drug and adjuvant with prospects for future applications.

Berberine (BBR), an isoquinoline alkaloid found in medicinal plants such as Coptidis rhizoma, Berberis sp., and Hydrastis canadensis, is a distinctive compound known for its dual ability to exhibit broad-spectrum antifungal activity while offering beneficial effects to the host. These attributes make it a highly valuable candidate for antifungal therapy and as an antibiotic adjuvant. This review provides a comprehensive evaluation of BBR's antifungal properties, focusing on its in vitro and in vivo activity, underlying mechanisms, and its influence on fungal pathogenicity, including virulence, biofilm formation, and resistance. Additionally, the antifungal potential of BBR extracts, derivatives, and nanoformulations is examined in detail. BBR demonstrates fungicidal effects through multiple mechanisms. It targets critical fungal components such as mitochondria, cell membranes, and cell walls, while also inhibiting enzymatic activity and transcription processes. Furthermore, it suppresses the expression of virulence factors, effectively diminishing fungal pathogenicity. Beyond its direct antifungal activity, BBR exerts beneficial effects on the host by modulating gut microbiota, thereby bolstering host defenses against fungal infections and reducing potential adverse effects. BBR's interaction with conventional antifungal drugs presents a unique complexity, particularly in the context of resistance mechanisms. When used in combination therapies, conventional antifungal drugs enhance the intracellular accumulation of BBR, thereby amplifying its antifungal potency as the primary active agent. These synergistic effects position BBR as a promising candidate for combination strategies, especially in addressing drug-resistant fungal infections and persistent biofilms. As antifungal resistance and biofilm-associated infections continue to rise, the multifaceted properties of BBR and its advanced formulations highlight their significant therapeutic potential. However, the scarcity of robust in vivo and clinical studies limits a full understanding of its efficacy and safety profile. To bridge this gap, future investigations should prioritize well-designed in vivo and clinical trials to thoroughly evaluate the therapeutic effectiveness and safety of BBR in diverse clinical settings. This approach could pave the way for its broader application in combating fungal infections.

A cysteine-rich domain of the Cuf1 transcription factor is required for high copper stress sensing and fungal virulence.

Copper is an essential micronutrient required for survival in all kingdoms of life as it is used as a catalytic cofactor for many essential processes in the cell. In turn, this reactivity of copper ions makes elevated levels of free copper toxic for the cell. This dual nature of copper-essential for life but toxic at elevated levels- is used by our innate immune system in a process called nutritional immunity to combat and kill invading pathogens. In this work we explore how the fungal human pathogen Cryptococcus neoformans senses high copper stress, a copper microenvironment encountered within the host lung. We identified a specific cysteine-rich region within the copper responsive transcription factor Cuf1 to be essential for high copper stress sensing. Mutation of this region led to an impaired high copper stress adaptation, which did not affect fitness of the yeast but did impact immune recognition and inflammation inside the host lung.

Boosting the efficacy of fungal biocontrol: miRNA339-5p-mediated mosquito immunity regulation.

Aedes mosquitoes are vectors for numerous viral diseases, including dengue, zika, chikungunya, and yellow fever. Therefore, underscoring the urgent need for eco-friendly alternatives to combat insecticide resistance and the scarcity of effective vaccines. Entomopathogenic fungi present a sustainable alternative to chemical insecticides; however, their widespread application is limited by their relatively low virulence. Here, we investigated the immunological interactions between Metarhizium anisopliae and Aedes albopictus, demonstrating that fungal infection significantly up-regulated immune-related genes in the Toll and melanization pathways, thereby enhancing antifungal and antibacterial defenses at 48 h post-infection (hpi). Small RNA sequencing identified miR339-5p as a crucial modulator, targeting the immune genes Gram-Negative Binding Protein 1 (GNBP1) and CLIP-domain Serine Protease B15 (CLIPB15), which are critical for Toll and phenoloxidase (PO) pathway activation. The administration of a synthetic miR339-5p mimic increased fungal virulence, resulting in a higher mortality rate among adult mosquitoes and a significant increase in the mortality rate of mosquito larvae within 24 hpi. GNBP1 was found to regulate both Toll and PO pathways, while CLIPB15 specifically modulated the PO system by cleaving prophenoloxidase (PPO). This research highlights the potential of leveraging Ae. albopictus-encoded miR339-5p through advanced genetic engineering techniques to bolster the efficacy of existing fungal-based mosquito control strategies, providing a promising approach in the fight against mosquito-borne diseases. © 2024 Society of Chemical Industry.

Protein PARylation: a novel regulator of fungal virulence.

Protein PARylation is a reversible post-translational modification; however, its role in fungal virulence has remained elusive. Recently, Gao et al. demonstrated that PARylation of two 14-3-3 regulatory proteins by poly(ADP-ribose) polymerase is essential for the virulence of rice blast fungus, highlighting the critical regulatory function of PARylation in fungal pathogenicity.

M6A demethylase CpALKBH regulates CpZap1 mRNA stability to modulate the development and virulence of chestnut blight fungus.

N6-methyladenosine (m6A) is the most abundant eukaryotic mRNA modification and is involved in various biological processes. Methyltransferases and demethylases regulate the m6A modification, but the regulatory role of m6A demethylases in fungi remains poorly understood. Here, we demonstrated that CpALKBH functions as a demethylase in Cryphonectria parasitica. The deletion of CpALKBH leads to a significant increase in m6A levels and a reduction in fungal growth, sporulation, and virulence. We identified CpZap1 as a downstream target of CpALKBH, with CpALKBH regulating CpZap1 mRNA stability in an m6A-dependent manner. Additionally, our findings indicate that methylation at position 1935A of CpZap1 is regulated by both the CpALKBH demethylase and the CpMTA1 methyltransferase. Given its critical role in fungal development and virulence, overexpression of CpZap1 can rescue abnormal phenotypes of ∆CpALKBH mutant. Overall, these findings contribute to improving our understanding of the role of m6A demethylase in fungi.

Importance of benzoyltransferase GcnE and lysine benzoylation of alcohol dehydrogenase AdhB in pathogenesis and aflatoxin production in Aspergillus flavus.

Lysine benzoylation (Kbz) is a newly identified post-translational modification associated with active transcription and metabolism in eukaryotes. However, whether Kbz exists in pathogenic fungi and its function remains unknown. Here, we demonstrated for the first time that Kbz is present in Aspergillus flavus and identified 60 benzoylated sites on 46 benzoylated proteins by global benzoylome analysis. Our data demonstrated that alcohol dehydrogenase B (AdhB) is regulated by benzoylation on lysine 321 (K321), and mutations of Kbz site in AdhB significantly reduced the alcohol dehydrogenase activity in vivo and in vitro. Both adhB deletion mutant and benzoylated site mutants (K321R and K321A) exhibited similar phenotype, including decreased conidiation and seed colonization, increased sclerotia formation and aflatoxin production, and more sensitive to cell wall damage stress. We also found that GcnE has benzoyltransferase activity in vitro and in vivo, and its repression leads to decreased Kbz level and enzymatic activity of AdhB. The catalytic site E139 is important for the benzoyltransferase function of GcnE. Our study uncovers a previously unknown mechanism by which benzoylation regulates AdhB activity to affect the development, secondary metabolism, pathogenicity, and stress response of A. flavus. Meanwhile, it points out the important role of Kbz in the pathogenicity of pathogenic fungi.IMPORTANCEAspergillus flavus is a ubiquitous opportunistic pathogen of plants and animals, which produces carcinogenic and toxic secondary metabolite aflatoxin. A. flavus and aflatoxin contamination have emerged as a global food safety concern. Currently, post-translational modification plays crucial modulatory roles in the fungal development and virulence, but the role of benzoylation in fungal pathogenicity remains undetermined, which limits the development of prevention and control technique. Here, we first identified 46 benzoylated proteins in A. flavus, and found that benzoyltransferase GcnE exerted effects on pathogenicity and aflatoxin production by regulating the benzoylation of AdhB. This finding not only provided valuable information for prevention and control of A. flavus contamination, but also offered basic knowledge for investigation of the regulation mechanism of secondary metabolism in other fungi.

Research progress on the function and regulatory pathways of amino acid permeases in fungi.

Nitrogen sources are pivotal for the formation of fungal mycelia and the biosynthesis of metabolites, playing a crucial role in the growth and development of fungi. Amino acids are integral to protein construction, constitute an essential nitrogen source for fungi. Fungi actively uptake amino acids from their surroundings, a process that necessitates the involvement of amino acid permeases (AAPs) located on the plasma membrane. By sensing the intracellular demand for amino acids and their extracellular availability, fungi activate or suppress relevant pathways to precisely regulate the genes encoding these transporters. This review aims to illustrate the function of fungal AAPs on uptake of amino acids and the effect of AAPs on fungal growth, development and virulence. Additionally, the complex mechanisms to regulate expression of aaps are elucidated in mainly Saccharomyces cerevisiae, including the Ssy1-Ptr3-Ssy5 (SPS) pathway, the Nitrogen Catabolite Repression (NCR) pathway, and the General Amino Acid Control (GAAC) pathway. However, the physiological roles of AAPs and their regulatory mechanisms in other species, particularly pathogenic fungi, merit further exploration. Gaining insights into these aspects could reveal how AAPs facilitate fungal adaptation and survival under diverse stress conditions, shedding light on their potential impact on fungal biology and pathogenicity.

CRISPR-Cas9-mediated enhancement of Beauveria bassiana virulence with overproduction of oosporein

Biocontrol agents play a pivotal role in managing pests and contribute to sustainable agriculture. Recent advancements in genetic engineering can facilitate the development of entomopathogenic fungi with desired traits to enhance biocontrol efficacy. In this study, a CRISPR-Cas9 ribonucleoprotein system was utilized to genetically improve the virulence of Beauveria bassiana, a broad-spectrum insect pathogen used in biocontrol of arthropod pests worldwide. CRISPR-Cas9-based disruption of the transcription factor-encoding gene Bbsmr1 led to derepression of the oosporein biosynthetic gene cluster resulting in overproduction of the red-pigmented dibenzoquinone oosporein involved in host immune evasion, thus increasing fungal virulence. Mutants defective for Bbsmr1 displayed a remarkable enhanced insecticidal activity by reducing lethal times and concentrations, while concomitantly presenting negligible or minor pleiotropic effects. In addition, these mutants displayed faster germination on the insect cuticle which correlated with higher density of free-floating blastospores in the hemolymph and accelerated mortality of the host. These findings emphasize the utility of genetic engineering in developing enhanced fungal biocontrol agents with customized phenotypic traits, and provide an efficient and versatile genetic transformation tool for application in other beneficial entomopathogenic fungi.

Docking and Simulation Studies on Novel Analogues of 3,4,5-Trihydroxy Benzoic Acid as HSP90Alpha Inhibitors

HSP90 assists as a crucial molecular chaperone that responds to environmental stressors and helps in the survival of cells in microorganisms. This protein is integral to the stress response, aiding in the stabilization of various proteins essential for microbial survival. Consequently, the ability of a number of tissues to adjust to endogenous stress depends critically on appropriate chaperone activity. Modulators of chaperone activity, however, have emerged as a novel and developing area of drug discovery due to the association between changed chaperone function and the development of numerous illnesses. Inhibition of HSP90alpha can disrupt proper protein folding, thus impairing growth and virulence in fungi. In this work, we selected novel leads of gallic acid derivatives with the help of OSIRIS Property Explorer and DruLiTo software. Selected leads were subjected to ADME-T studies for further screening. Docking and molecular simulation studies on selected compounds were performed using Schrodinger v21 and GROMACS software to predict the bioactivity of novel leads of 3,4,5 trihydroxy benzoic acid for suppression of the HSP90alpha enzyme. Compounds 4N, 18N, 15N, and 14N showed good docking scores of -6.5, -6.4, -5.91, and -5.98, respectively, which was comparable to standard ciprofloxacin. Compound 4N and compound 14N demonstrated notable binding interactions and were selected for further investigation through molecular dynamics studies with HSP90alpha (PDB ID: 1YC1). RMSD, H BOND, and RMSF analysis confirmed the stable binding of compounds 4N and 14 N with the HSP90 enzyme. The RMSF plot showed less than 0.35 nm fluctuation for the HSP90alpha enzyme in complex with different ligands. It can be concluded that ligand binding can cause stability to the conformation of the protein. Compounds 4N and 14N are considered to be the best theoretical lead, which can further be studied experimentally as HSP90 alpha inhibitors for antimicrobial activity.

Exploring secretory signal sequences useful in excreting recombinant proteins in Beauveria bassiana as biocontrol fungus.

Entomopathogenic fungi excrete a group of proteins to assimilate nutrients and defeat the host immune defense. Functional secretory signal sequences are needed for efficient secretion of the virulence-related proteins in recombinant strain. In this study, secretome analysis was used to explore the secreted proteins of Beauveria bassiana. Enrichment analysis indicated that B. bassiana secretome was mainly associated with metabolism of glucoside, polysaccharide, extracellular ester compound, and so on. In addition, proteins associated with biogenesis of cellular components were also enriched, including those involved in biogenesis of cell wall and vacuole. Then, four secretory signal sequences were functionally verified with green fluorescent protein as reporter. Finally, a signal sequence was used to excrete three insect venom protein serpins in B. bassiana, in which over-expression of serpin 8 gene resulted in a significant increase in fungal virulence. This study highlights that functional secretory signal sequences are potential molecular elements useful in excretion of virulence-related proteins in insect pathogenic fungi.