Insect fungal pathogens secrete a cell wall-associated glucanase that acts to help avoid recognition by the host immune system.

Fungal insect pathogens have evolved diverse mechanisms to evade host immune recognition and defense responses. However, identification of fungal factors involved in host immune evasion during cuticular penetration and subsequent hemocoel colonization remains limited. Here, we report that the entomopathogenic fungus Beauveria bassiana expresses an endo-ß-1,3-glucanase (BbEng1) that functions in helping cells evade insect immune recognition/ responses. BbEng1 was specifically expressed during infection, in response to host cuticle and hemolymph, and in the presence of osmotic or oxidative stress. BbEng1 was localized to the fungal cell surface/ cell wall, where it acts to remodel the cell wall pathogen associated molecular patterns (PAMPs) that can trigger host defenses, thus facilitating fungal cell evasion of host immune defenses. BbEng1 was secreted where it could bind to fungal cells. Cell wall ß-1,3-glucan levels were unchanged in ΔBbEng1 cells derived from in vitro growth media, but was elevated in hyphal bodies, whereas glucan levels were reduced in most cell types derived from the BbEng1 overexpressing strain (BbEng1OE). The BbEng1OE strain proliferated more rapidly in the host hemocoel and displayed higher virulence as compared to the wild type parent. Overexpression of their respective Eng1 homologs or of BbEng1 in the insect fungal pathogens, Metarhizium robertsii and M. acridum also resulted in increased virulence. Our data support a mechanism by which BbEng1 helps the fungal pathogen to evade host immune surveillance by decreasing cell wall glucan PAMPs, promoting successful fungal mycosis.

Multifunctional role of a fungal pathogen-secreted laccase 2 in evasion of insect immune defense.

Laccases are widely present in bacteria, fungi, plants and invertebrates and involved in a variety of physiological functions. Here, we report that Beauveria bassiana, an economic important entomopathogenic fungus, secretes a laccase 2 (BbLac2) during infection that detoxifies insect immune response-generated reactive oxygen species (ROS) and interferes with host immune phenoloxidase (PO) activation. BbLac2 is expressed in fungal cells during proliferation in the insect haemocoel and can be found to distribute on the surface of haemolymph-derived in vivo fungal hyphal bodies or be secreted. Targeted gene-knockout of BbLac2 increased fungal sensitivity to oxidative stress, decreased virulence to insect, and increased host PO activity. Strains overexpressing BbLac2 showed increased virulence, with reduced host PO activity and lowered ROS levels in infected insects. In vitro assays revealed that BbLac2 could eliminate ROS and oxidize PO substrates (phenols), verifying the enzymatic functioning of the protein in detoxification of cytotoxic ROS and interference with the PO cascade. Moreover, BbLac2 acted as a cell surface protein that masked pathogen associated molecular patterns (PAMPs), enabling the pathogen to evade immune recognition. Our data suggest a multifunctional role for fungal pathogen-secreted laccase 2 in evasion of insect immune defenses.

The fungal mitochondrial membrane protein, BbOhmm, antagonistically controls hypoxia tolerance.

Adaptation to low-oxygen (LO) environment in host tissues is crucial for microbial pathogens, particularly fungi, to successfully infect target hosts. However, the underlying mechanisms responsible for hypoxia tolerance in most pathogens are poorly understood. A mitochondrial protein, BbOhmm, is demonstrated to limit oxidative stress resistance and virulence in the insect fungal pathogen, Beauveria bassiana. Here, we found that BbOhmm negatively affected hypoxic adaptation in the insect haemocoel while regulating respiration-related events, heme synthesis and mitochondrial iron homeostasis. A homologue of the mammalian sterol regulatory element-binding proteins (SREBPs), BbSre1, was shown to be involved in BbOhmm-mediated LO adaptation. Inactivation of BbSre1 resulted in a significant increase in sensitivity to hypoxic and oxidative stress. Similar to ΔBbOhmm, ΔBbSre1 or the ΔBbOhmmΔBbSre1 double mutant accumulated high levels of heme and mitochondrial iron, regulating the similar pathways during hypoxic stress. BbSre1 transcriptional activity and nuclear import were repressed in ΔBbOhmm cells and affected by intracellular reactive oxygen species (ROS) and oxygen levels. These findings have led to a new model in which BbOhmm affects ROS homeostasis in combination with available oxygen to control the transcriptional activity of BbSre1, which in turn mediates LO adaptation by regulating mitochondrial iron homeostasis, heme synthesis and respiration-implicated genes.

MADS-box transcription factor Mcm1 controls cell cycle, fungal development, cell integrity and virulence in the filamentous insect pathogenic fungus Beauveria bassiana.

MADS-box transcription factor Mcm1 plays crucial roles in regulating mating processes and pathogenesis in some fungi. However, its roles are varied in fungal species, and its function remains unclear in entomopathogenic fungi. Here, Mcm1 orthologue, Bbmcm1, was characterized in a filamentous entomopathogenic fungus, Beauveria bassiana. Disruption of Bbmcm1 resulted in a distinct reduction in growth with abnormal conidiogenesis, and a significant decrease in conidial viability with abnormal germination. ΔBbmcm1 displayed impaired cell integrity, with distorted cell wall structure and altered cell wall component. Abnormal cell cycle was detected in ΔBbmcm1 with longer G2 /M phase but shorter G1 /G0 and S phases in unicellular blastospores, and sparser septa in multicellular hyphae, which might be responsible for defects in development and differentiation due to the regulation of cell cycle-involved genes, as well as the corresponding cellular events-associated genes. Dramatically decreased virulence was examined in ΔBbmcm1, with impaired ability to escape haemocyte encapsulation, which was consistent with markedly reduced cuticle-degrading enzyme production by repressing their coding genes, and downregulated fungal effector protein-coding genes, suggesting a novel role of Mcm1 in interaction with host insect. These data indicate that Mcm1 is a crucial regulator of development, cell integrity, cell cycle and virulence in insect fungal pathogens.

An aldo-keto reductase, Bbakr1, is involved in stress response and detoxification of heavy metal chromium but not required for virulence in the insect fungal pathogen, Beauveria bassiana.

The aldo-keto reductases (AKRs) belong to the NADP-dependent oxidoreductase superfamily, which play important roles in various physiological functions in prokaryotic and eukaryotic organisms. However, many AKR superfamily members remain uncharacterized. Here, a downstream target gene of the HOG1 MAPK pathways coding for an aldo-keto reductase, named Bbakr1, was characterized in the insect fungal pathogen, Beauveria bassiana. Bbakr1 expression increased in response to osmotic and salt stressors, and oxidative and heavy metal (chromium) stress. Deletion of Bbakr1 caused a reduction in conidiation, as well as delayed conidial germination. ΔBbakr1 displayed increased sensitivity to osmotic/high-salt stress with decreased compatible solute accumulation. In addition, the mutant was more sensitive to high concentrations of the heavy metal, chromium, and to oxidative stress than the wild type cells, with impaired ability to detoxify active aldehyde that might accumulate due to lipid peroxidation. However, over-expressing Bbakr1 in either the wild type strain or a ΔBbhog1 background did not cause any obvious changes in phenotypes as compared to their controls. Little effect on virulence was seen for either the ΔBbakr1 or overexpression strains in insect bioassays via cuticle infection or intrahemocoel injection assays, suggesting that Bbakr1 is not required for virulence.

Comparative transcriptome and gene co-expression network analysis reveal genes and signaling pathways adaptively responsive to varied adverse stresses in the insect fungal pathogen, Beauveria bassiana.

Sensing, responding, and adapting to the surrounding environment are crucial for all living organisms to survive, proliferate, and differentiate in their biological niches. Beauveria bassiana is an economically important insect-pathogenic fungus which is widely used as a biocontrol agent to control a variety of insect pests. The fungal pathogen unavoidably encounters a variety of adverse environmental stresses and defense response from the host insects during application of the fungal agents. However, few are known about the transcription response of the fungus to respond or adapt varied adverse stresses. Here, we comparatively analyzed the transcriptome of B. bassiana in globe genome under the varied stationary-phase stresses including osmotic agent (0.8 M NaCl), high temperature (32 °C), cell wall-perturbing agent (Congo red), and oxidative agents (H2O2 or menadione). Total of 12,412 reads were obtained, and mapped to the 6767 genes of the B. bassiana. All of these stresses caused transcription responses involved in basal metabolism, cell wall construction, stress response or cell rescue/detoxification, signaling transduction and gene transcription regulation, and likely other cellular processes. An array of genes displayed similar transcription patterns in response to at least two of the five stresses, suggesting a shared transcription response to varied adverse stresses. Gene co-expression network analysis revealed that mTOR signaling pathway, but not HOG1 MAP kinase pathway, played a central role in regulation the varied adverse stress responses, which was verified by RNAi-mediated knockdown of TOR1. Our findings provided an insight of transcription response and gene co-expression network of B. bassiana in adaptation to varied environments.

Correlation of cell surface proteins of distinct Beauveria bassiana cell types and adaption to varied environment and interaction with the host insect.

The insect fungal pathogen Beauveria bassiana produces a number of distinct cell types that include aerial conidia, blastospores and haemolymph-derived cells, termed hyphal bodies, to adapt varied environment niches and within the host insect. These cells display distinct biochemical properties and surface structures, and a highly ordered outermost brush-like structure uniquely present on hyphal bodies, but not on any in vitro cells. Here, we found that the outermost structure on the hyphal bodies mainly consisted of proteins associated to structural wall components in that most of it could be removed by dithiothreitol (DTT) or proteinase K. DTT-treatment also caused delayed germination, decreased tolerance to ultraviolet irradiation and virulence of conidia or blastospores, with decreased adherence and alternated carbohydrate epitopes, suggesting involvement in fungal development, stress responses and virulence. To characterize these cell surface molecules, proteins were released from the living cells using DTT, and identified and quantitated using label-free quantitative mass spectrometry. Thereafter, a series of bioinformatics programs were used to predict cell surface-associated proteins (CSAPs), and 96, 166 and 54 CSAPs were predicted from the identified protein pools of conidia, blastospores and hyphal bodies, respectively, which were involved in utilization of carbohydrate, nitrogen, and lipid, detoxification, pathogen-host interaction, and likely other cellular processes. Thirteen, sixty-nine and six CSAPs were exclusive in conidia, blastospores and hyphal bodies, respectively, which were verified by eGFP-tagged proteins at their N-terminus. Our data provide a crucial cue to understand mechanism of B. bassiana to adapt to varied environment and interaction with insect host.

A fungal pathogen secretes a cell wall-associated ß-N-acetylhexosaminidase that is co-expressed with chitinases to contribute to infection of insects.

BACKGROUND: ß-N-acetylhexosaminidases (HEXs) are widely distributed in fungi and involved in cell wall chitin metabolism and utilization of chitin-containing substrates. However, details of the fungal pathogens-derived HEXs in the interaction with their hosts remain limited. RESULTS: An insect nutrients-induced ß-N-acetylhexosaminidase, BbHex1, was identified from the entomopathogenic fungus Beauveria bassiana, which was involved in cell wall modification and degradation of insect cuticle. BbHex1 was localized to cell wall and secreted, and displayed enzyme activity to degrade the chitinase-hydrolyzed product (GlcNAc)2. Disruption of BbHex1 resulted in a significant decrease in the level of cell wall chitin in the presence of insect nutrients and during infection of insects, with impaired ability to penetrate insect cuticle, accompanying downregulated cell wall metabolism-involved and cuticle-degrading chitinase genes. However, the opposite phenotypes were examined in the gene overexpression strain. Distinctly altered cell wall structures caused by BbHex1 mutation and overexpression led to the easy activation and evasion (respectively) of insect immune response during fungal infection. As a result, BbHex1 contributed to fungal virulence. Bioinformatics analysis revealed that promoters of some co-expressed chitinase genes with the BbHex1 promoter shared conserved transcription factors Skn7, Msn2 and Ste12, and CreA-binding motifs, implying co-regulation of those genes with BbHex1. CONCLUSION: These data support a mechanism that the fungal pathogen specifically expresses BbHex1, which is co-expressed with chitinases to modify cell wall for evasion of insect immune recognition and to degrade insect cuticle, and contributes to the fungal virulence against insects. © 2024 Society of Chemical Industry.

Fus3/Kss1-MAP kinase and Ste12-like control distinct biocontrol-traits besides regulation of insect cuticle penetration via phosphorylation cascade in a filamentous fungal pathogen.

BACKGROUND: Homolog of the yeast Fus3/Kss1 mitogen-activated protein kinase (MAPK) pathway and its target transcription factor, Ste12-like, are involved in penetration of host cuticle/pathogenicity in many ascomycete pathogens. However, details of their interaction during fungal infection, as well as their controlled other virulence-associated traits, are unclear. RESULTS: Ste12-like (BbSte12) and Fus3/Kss1 MAPK homolog (Bbmpk1) interacted in nucleus, and phosphorylation of BbSte12 by Bbmpk1 was essential for penetration of insect cuticle in an insect fungal pathogen, Beauveria bassiana. However, some distinct biocontrol-traits were found to be mediated by Ste12 and Bbmpk1. In contrast to ΔBbmpk1 colony that grew more rapid than wild-type strain, inactivation of BbSte12 resulted in the opposite phenotype, which was consistent with their different proliferation rates in insect hemocoel after direct injection of conidia bypass the cuticle. Reduced conidial yield with decreased hydrophobicity was examined in both mutants, however they displayed distinct conidiogenesis, accompanying with differently altered cell cycle, distinct hyphal branching and septum formation. Moreover, ΔBbmpk1 showed increased tolerance to oxidative agent, whereas the opposite phenotype was seen for ΔBbSte12 strain. RNA sequencing analysis revealed that Bbmpk1 controlled 356 genes depending on BbSte12 during cuticle penetration, but 1077 and 584 genes were independently controlled by Bbmpk1 and BbSte12. CONCLUSION: BbSte12 and Bbmpk1 separately participate in additional pathways for control of conidiation, growth and hyphal differentiation, as well as oxidative stress response besides regulating cuticle penetration via phosphorylation cascade. © 2023 Society of Chemical Industry.

A secretory phospholipase A2 of a fungal pathogen contributes to lipid droplet homeostasis, assimilation of insect-derived lipids, and repression of host immune responses.

Secretory phospholipase A2s (sPLA2s) are found in a wide range of organisms from bacteria to higher plants and animals and are involved in varied and cellular processes. However, roles of these enzymes in microbial pathogens remain unclear. Here, an sPLA2 (BbPLA2) was characterized in the filamentous insect pathogenic fungus, Beauveria bassiana. BbPLA2 was exclusively expressed in insect hemolymph-derived cells (hyphal bodies), and its expression was induced by insect-derived nutrients and lipids, and nutrient starvation. High levels of secretion of BbPLA2 were observed as well as its distribution in hyphal body lipid drops (LDs). Overexpression of BbPLA2 increased the ability of B. bassiana to utilize insect-derived nutrients and lipids, and promoted LD accumulation, indicating functions for BbPLA2 in mediating LD homeostasis and assimilation of insect-derived lipids. Strains overexpressing BbPLA2 showed moderately increased virulence, including more efficient penetration of the insect cuticle and evasion of host immune responses as compared to the wild type strain. In addition, B. bassiana-activated host immune genes were downregulated in the BbPLA2 overexpression strain, but upregulated by infections with a ΔBbPLA2 strain. These data demonstrate that BbPLA2 contributes to LD homeostasis, assimilation of insect-derived lipids, and repression of host immune responses.