Role Of A Fungal Pathogen,beauveria Bassiana,-Secreted Laccase 2 In Evasion Of Insect Immune Defense

Entomopathogenic fungi are a kind of micriobial pathogens that infect host insects by direct penetration of the cuticle.The infection process initiates with spore adhesion to insect cuticle and germination.The germ tubes often differentiate to form a specialized infection structure,termed appressorium,which further stretched with an infection hypha and penetrate the cuticle through action of mechanical pressure and robust degradative enzymes.Once intrude the insect hemocoel,penetrating hyphae elaborate yeast-like cells termed in vivo blastospores or the haemolymph-derived cells,hyphal bodies that evade the insect immune system and proliferate in the haemolymph.Beauveria bassiana is an entomogenous fungus,worldwide used as a biological control agent against variety of agricultural,forestry and sanitary insect pests,which is also a model fungal species for investigation of interaction between the fungal pathogens and host/environment.It is demonstrated that a highly ordered outermost brush-like structure is uniquely present in the cell surface of the B.bassiana hyphal bodies,which displays distinct surface characteristics as compared to vitro spores,e.g.,conidia and blastospores,suggesting involvement of the surface structure in evading the insect hemolymph immune response.Our previous study revealed that the outermost brush-like structure on hyphal bodies is mainly composed of cell surface proteins.Followed comparative proteomics identified surface/secreted proteins of hyphal bodies and in-vitro spores,including conidia and blastospores.Six unique proteins have been identified in the protein profile of hyphal bodies.Further characterization of these proteins would provide new insight into the tactics that fungal pathogen deploys to evade host immune response,and pave theoretical basis for improvement of fungal agent via genetic engineering.Laccases are widely distributed in bacteria,fungi,plants and invertebrates(including insects),which participate in a variety of physiology and biological processes.A secretory laccase 2(BBA?08183),termed Op S5,has demonstrated to participate in biosynthesis of oosporein,in B.bassiana.In our previous study,the laccase 2 was specifically present in cell surface/secreted protein profile of hyphal bodies,suggesting it's involvement in cell surface structure formation and addition role in interaction between the fungal pathogen and host insect besides oosporein biosynthesis.In this study,role of the laccase 2(named as BbLac2)in evasion of insect immune response were characterized via analysis of gene expression patterns,investigation of protein distribution in fungal cells and secretion characteristics,and enzyme function analysis,as well as examination of effects of gene disruption and overexpression on pathogensis.The main findings of BbLac2 are as follows:1.BbLac2 is induced by insect derived nutrient,oxidative stress and phenolic compoundsTo reveal the expression patterns of BbLac2,RT-qPCR analysis and GFP fluorescence inspection in fungal cells of PBbLac2::GFP strain,in which GFP was driven by BbLac2 promoter,were performed.The results indicated that BbLac2 was exclusively expressed in haemolymph-derived cells,hyphal bodies,and dramatically induced by insect nutrition(haemolymph and cuticle of silkworm),oxidative stresses(menadione,tert-Butyl hydroperoxide and H₂O₂)and insect endogenous phenolic compounds(L-thyrosine,3,4-Dihydroxy-L-phenylalanine(L-DOPA)and 5,6-dihydroxyindole).To investigate extra role of BbLac2 besides oosporein biosynthesis,a strain was constructed with GFP controlled by the Op S1(encoding oosporein polyketide synthase)promoter,termed POp S1::GFP strain.Expression patterns of BbLac2 and Op S1 were analyzed by examination of GFP signal(as compared with PBbLac2::GFP strain)and using RT-qPCR analysis during infection after inoculation of conidia via intrahemocoel injection.Op S1 expression(GFP signal)was detected in the fungal cells 72 h post-inoculation(12 h post-insect death),and gradually decreased 84 h post-inoculation(24 h post-insect death).Whereas,BbLac2 expression(GFP signal)was probed when the hyphal bodies formed 36 h post-inoculation,and gradually increased during 48-72 h,and then dramatically decreased 84 h post-inoculation.These data suggested additional role of BbLac2 in the interaction between the pathogen and host insect besides oosporein synthesis,might be involved in insect nutrient utilization,oxidative stress response and detoxification of phenolic substances in the infection processes.2.BbLac2 is either distributes on surface of hyphal bodies or is secretedTo reveal the distribution of BbLac2 in fungal cells,GFP fluorescence was detected in fungal cells expressing BbLac2::GFP fusion gene.The GFP signal were only distributed on the cell wall or surface of the hyphal bodies.To reveal whether BbLac2 could be secreted or not,GFP was fused to BbLac2 ORF with/without signal peptide sequences and controlled by a B.bassiana constitutive promoter PBbgpd(named PB3)generated PB3::BbLac2::GFP/PB3::BbLac2-sp::GFP strain.The GFP signal were probed on surface of all PB3::BbLac2::GFP cells.Whereas,the fluorescence was only detected within PB3::BbLac2-sp::GFP cells.The fusion proteins in PB3::BbLac2::GFP culture broth were detected using Western blotting.These data suggested that BbLac2 was secreted as an extracellular substrates.3.BbLac2 involved in oxidative stress response and is independent of oosporein synthesisTo reveal role of BbLac2 in the fungal pathogenicity,gene disruption(?BbLac2),reverse complementation(Comp)and overexpression(BbLac2^OE)strains were generated.The results showed that disruption of BbLac2 increased the fungal sensitivity to oxidative stress.Whereas,overexpressing of BbLac2 displayed slightly increase in tolerance to oxidative stress as compared to the wild type parent.However,no obvious difference in sensitivity to oxidative stress was examined between the?Op S3 strain,a mutant that is not capable to synthesize oosporein and wild type strain,suggesting that involvement of BbLac2(Op S5)in oxidative stress response is independent of oosporein synthesis.4.BbLac2 is involved in surface features of hyphal bodiesSince BbLac2 was a cell wall or cell surface protein in hyphal bodies,we examined the effect of BbLac2 disruption or overexpression on cell surface features.Cell surface carbohydrate epitopes were detected using three fluorescent-labeled lectins,concanavalin A(Con A),which binds to?-glucose,mannose and?-?-acetylglucosamine(Glc NAc),wheatgerm agglutinin(WGA),which recognizes?-Glc NAc and sialic acid residues,and Galanthus nivalis(GNL),which recognizes mannose residues,and fluorescent-labeled?-1,3 glucan antibody that specific recognizes?-1,3 glucan.No obvious difference in surface carbohydrate epitopes was examined by the three lectins between?BbLac2 and wild type hyphal bodies.However,significant increase of binding activity to?-1,3 glucan antibody was measured in?BbLac2 hyphal bodies as compared to wild type cells.In contrast,BbLac2^OE hyphal bodies displayed increased affinity to the three lectins,but distinctly decreased bond towards?-1,3 glucan antibody.These data demonstrated that BbLac2 acted as a cell surface protein that masked pathogen associated molecular patterns(PAMPs)included?-1,3 glucan.5.BbLac2 is a fungal virulence factor and involved in evasion of immune responseTo evaluate role of BbLac2 in pathogenicity,third-instar larvae of the greater wax moth,Galleria mellonella was used for bioassay through both topical infection and intrahaemocoel injection.In both assays,disruption of the gene resulted in a significant decrease in fungal virulence,with weakened lethal rates and higher LT₅₀ values(the mean lethal time)as compared to the wild type strain.However,increased virulence was examined in the overexpression strain(BbLac2^OE)with reduced LT₅₀ values.After host death,all of BbLac2^OE,Comp and wild type,as well as?Op S3 cells,penetrated outwards from the insect cadaver for growth and sporulation.However,fewer?BbLac2cells were penetrated outwards and grew on the insect cadavers,indicating impaired cuticle penetration ability.These data suggested that BbLac2 is required for full virulence of B.bassiana.It was noticed that melanization reaction was significantly increased in?BbLac2-infected insects after 48 h of intrahaemoceol injection with conidia as compared to wild type-infected insect,implying activation of insect immune response.Whereas,no obvious melanization reaction was observed in BbLac2^OE-treated insects.Microscopical observation indicated that BbLac2^OE conidia germinated and the germ tubes quickly escaped from haemocyte encapsulation 36-48 h following the injection of fungal spores,more easily than wild type cells.However,the?BbLac2 spores or germ tubes were heavely attacked and melanized by insect haemocytes as compared to wild type cells.The qPCR detection indicated that BbLac2^OE strain proliferated more quickly than the wild type strain in host haemocoel.Whereas,growth of?BbLac2 in insect decrease compared to wild type strain.In contrast,count of haemocytes in BbLac2^OE-inoculated insect was significantly lower than that of WT-treatment,but increased in?BbLac2-infected insect.These data suggest role of BbLac2 in the evasion of insect immune response.6.BbLac2 is involved in detoxification of reactive oxygen species and interferes phenoloxidase cascadeTo reveal possible mechanism of BbLac2 involved in evasion of immune response,the host insect generated reactive oxygen species(ROS)and phenoloxidase(PO)activities were measured during host infection.A fluorescence oxidant-sensitive probe,5-(and-6)-chloromethyl-2?,7?-dichloro-dihydrofluorescein diacetate,acetyl ester(CM-H2DCFDA)was used to probe ROS levels of haemocytes around the phagocytosed conidia.Exceptional weaked fluorescence signals were detected in BbLac2^OE-infected samples as compared to wild type-infected samples.Whereas,no obvious difference in fluorescence signals was observed between?BbLac2 and wild type-infected samples.The similar trends were verified by quantitative determination of H₂O₂(representing ROS)in the samples,suggesting role of BbLac2 in scavenging insect immune response-generated ROS.To further understand the underlying mechanism of BbLac2 in scavenging ROS,the protein was expressed in Pichia pastoris,which displayed typical laccase activities against laccase substrates.High ROS scavenging activities were examined at p H values from 7.0 to 13,and at 20^oC to 50^oC.The ability to scavenge H₂O₂enhanced with increased BbLac2 protein concentrations at p H 8.0 and 40^oC.PO activity in G.mellonella haemolymph was assayed over time after challenge with fungal cells.The enzyme activities in all of fungal conidia-inoculated insect samples distinctly increased during 0-10 h after inoculation,and then decreased.However,?BbLac2-inoculated samples displayed significantly higher activities than wild type-treatment.Whereas,the activities in BbLac2^OE-infected insects were distinctly lower than those of wild type 6 h post-inoculation,indicating easy to evade immune recognition.To reveal the underlying mechanism of BbLac2 in interference with PO cascade,L-DOPA,a typical phenolic substrate of PO,was used to assay BbLac2 activity.Relatively high activities of BbLac2 against L-DOPA were detected at p H 6.0 to 9.0.The melanin formation was also observed by addition of the yeast-expressed BbLac2 on the L-DOPA-contained Czapek agar.These data indicated that BbLac2 has PO activity against the phenolic substrates,which result in interference with PO synthesis and its activities.To further reveal the effect of BbLac2 mutation or overexpression on immune system of the host insect,expression of 3 Toll-pathway genes,Sp(?)tzle,Dorsal and BGBP1(?-1,3 glucan binding protein gene)and 14 antimicrobial peptide(AMP)genes were examined at 12 h after infection of fungi.Significantly upregulation of 3Toll-pathway genes and 11 AMP genes were examined in the?BbLac2-inoculated insects compared to WT-treated.However,3 Toll-pathway members and 13 AMP genes were distinctly downregulated in the BbLac2^OE-inoculated larvae.These data confirmed the role of BbLac2 in evasion of immune response.Overall,our results demonstrated that insect fungal pathogens specifically secrete a laccase 2 during infection that play a multifunctional role in evasion of insect immune defenses.BbLac2 was uniquely expressed by fungal pathogens after invaded into insect haemocoel and secreted into hemolymph.Beside oosporein synthesis,laccase 2 acts as a secreted protein that either detoxifies immunity response-generated ROS or represses PO activity.Moreover,the enzyme masks PAMPs(such as?-1,3 glucan)as a cell surface protein that enables pathogen to evade immune recognition.