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1.
Proc Natl Acad Sci U S A ; 120(12): e2205140120, 2023 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-36917667

RESUMO

The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. Here, we demonstrate that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Receptores Toll-Like/metabolismo , Transdução de Sinais , Peptídeos/metabolismo , Fungos/metabolismo , Bactérias Gram-Positivas/metabolismo
2.
BMC Microbiol ; 23(1): 96, 2023 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-37016280

RESUMO

Destruxin A (DA) is a cyclo-hexadepsipeptidic insecticidal mycotoxin isolated from the entomopathogenic fungi, Metarhizium spp. However, its mode of action is unknown. In this study, we isolated 149 candidate DA-binding proteins by drug affinity response target stability, and determined the interactions of 80 canditates with DA in vitro by surface plasmon resonance. The affinity coefficients (KD) ranged from 24 to 469 µM. Binding proteins were functionally diverse and included cytoskeletal components and cell motility, protein transcription and translation pathways, ubiquitin dependent protein metabolic processes, nucleus pore entry and exit, and endoplasmic reticulum vesicle transport and etc. Electron microscopy revealed that DA damaged the cytoskeleton and multiple organelles, disrupted cell adhesion and motility, and led to cell death. DA appeared to have a multi-targeted approach to cellular structures and multiple life processes, leading to cell death. The results of this study could provide molecular evidence for the analysis of the insecticidal toxicology of DA and further improve the study of the pathogenic insect mechanism of Metarhizium.


Assuntos
Depsipeptídeos , Inseticidas , Metarhizium , Animais , Proteínas de Transporte , Depsipeptídeos/farmacologia , Depsipeptídeos/química , Depsipeptídeos/metabolismo , Insetos/metabolismo , Inseticidas/farmacologia , Proteínas de Insetos/metabolismo
3.
Pestic Biochem Physiol ; 197: 105654, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38072529

RESUMO

Destruxin A, a non-ribosomal peptide toxin produced by Metarhizium, exhibits potent insecticidal activity by targeting various tissues, organs, and cells of insects. Our previous research has revealed that DA possesses the ability to bind to multiple proteins. In this study, we aimed to identify the most sensitive binding proteins of DA and investigate the physiological processes in which DA regulated. Through RNAi technology, we screened 22 binding proteins of DA in silkworm hemolymph. Among them, the juvenile hormone binding protein (JHBP), a hormone transport protein crucial for growth and development regulation, exhibited the highest sensitivity to DA. Subsequent experiments demonstrated that DA could inhibit the body weight gain of silkworm larvae, accelerate the pupation occurrence, and modulate the content of free juvenile hormone (JH) in the hemolymph. We also observed that DA could induce conformational changes in both the JHBP and the JHBP-JH binding complex. Notably, at low dosage, DA influenced the binding of JHBP to JH, while at high dosage, it irreversibly affected the binding of JHBP to JH. Molecular docking and point-mutant experiments suggested that DA might affect the N-arm of JHBP, which is responsible for JH binding. Additionally, we discovered that JHBP is widely distributed in various tissues of the silkworm, including the epidermis, gut, fat body, Malpighian tubule, gonad, muscle, trachea, and hemocyte. This study provides novel insights into the insecticidal mechanism of DA and enhances our understanding of the pathogenic process of Metarhizium.


Assuntos
Bombyx , Mariposas , Animais , Hormônios Juvenis/farmacologia , Hormônios Juvenis/metabolismo , Simulação de Acoplamento Molecular , Proteínas de Transporte/química , Mariposas/metabolismo , Bombyx/metabolismo , Proteínas de Insetos/metabolismo
4.
Molecules ; 27(22)2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36431809

RESUMO

Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, has insecticidal activity, but its molecular mechanism of action is still not clear. Three proteins with modification-related functions, calreticulin (BmCRT), dipeptidyl peptidase Ⅲ (BmDPP3), and protein disulfide isomerase A5 (BmPDIA5), were selected to verify the interactions with DA in this study. The kinetic data of the interactions were measured by surface plasmon resonance (SPR) and bio-layer interferometry (BLI) in vitro. The KD values of DA with BmCRT, BmDPP3, and BmPDIA5 ranged from 10-4 to 10-5 mol/L, which suggested that the three proteins all had fairly strong interactions with DA. Then, it was found that DA in a dose-dependent manner affected the interactions of the three proteins with their partners in insect two-hybrid tests in SF-9 cells. Furthermore, the results of enzyme activities by ELISA indicated that DA could inhibit the activity of BmDPP3 but had no significant effect on BmPDIA5. In addition, DA induced the upregulation of BmDPP3 and the downregulation of BmCRT. The results prove that BmCRT, BmDPP3, and BmPDIA5 are all binding proteins of DA. This study might provide new insights to elucidate the molecular mechanism of DA.


Assuntos
Bombyx , Depsipeptídeos , Animais , Ressonância de Plasmônio de Superfície , Regulação para Baixo
5.
BMC Genomics ; 20(1): 1036, 2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31888481

RESUMO

BACKGROUND: Alternaria brassicae, a necrotrophic pathogen, causes Alternaria Leaf Spot, one of the economically important diseases of Brassica crops. Many other Alternaria spp. such as A. brassicicola and A. alternata are known to cause secondary infections in the A. brassicae-infected Brassicas. The genome architecture, pathogenicity factors, and determinants of host-specificity of A. brassicae are unknown. In this study, we annotated and characterised the recently announced genome assembly of A. brassicae and compared it with other Alternaria spp. to gain insights into its pathogenic lifestyle. RESULTS: We also sequenced the genomes of two A. alternata isolates that were co-infecting B. juncea using Nanopore MinION sequencing for additional comparative analyses within the Alternaria genus. Genome alignments within the Alternaria spp. revealed high levels of synteny between most chromosomes with some intrachromosomal rearrangements. We show for the first time that the genome of A. brassicae, a large-spored Alternaria species, contains a dispensable chromosome. We identified 460 A. brassicae-specific genes, which included many secreted proteins and effectors. Furthermore, we have identified the gene clusters responsible for the production of Destruxin-B, a known pathogenicity factor of A. brassicae. CONCLUSION: The study provides a perspective into the unique and shared repertoire of genes within the Alternaria genus and identifies genes that could be contributing to the pathogenic lifestyle of A. brassicae.

6.
J Physiol ; 595(2): 523-539, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27373966

RESUMO

KEY POINTS: The digestive tract of larval and adult Drosophila is an excellent analogue of the mammalian gut. Enterocytes of the posterior midgut are separated by septa, with no paracellular path, and therefore perform both immune and transport functions. Using microperfusion electrophysiology, we show that larvae emerging from the embryo into sterile medium have symmetrical apical and basal membrane conductances while larvae emerging into non-sterile medium have apical membranes fivefold more conductive than basal membranes. The channels inserted into the apical membranes could originate in microbiata or host and mediate recognition of microbes. Entomopathogenic cyclic peptide toxins deplete intracellular ions reversibly, forming transient ion channels that do not conduct water, unlike an ionophore like nystatin that depletes ions irreversibly. We show the feasibility of studying the interaction of a single microbial species, or tractable combinatorials of microbial species, with only enterocytes in the primary epithelial barrier. ABSTRACT: Microbiota colonizing exposed epithelial surfaces are vital for sustenance of metazoan life, but communication between microbiota, epithelial cells and the host immune system is only beginning to be understood. We address this issue in the posterior midgut epithelium of Drosophila larvae where nutrient transport and immune functions are exclusively transcellular. We showed that larvae emerging into a sterile post-embryonic environment have symmetrical apical and basal membranes. In contrast, larvae emerging into non-sterile media, the source of microbiota, have markedly asymmetrical membranes, with apical membrane conductance more than fivefold higher than the basal membrane. As an example of pathogen action, we showed that the entomopathogenic fungal toxin destruxin A (Dx) depleted intracellular ions. Reversibility of action of Dx was verified by bilayer reconstitution in forming transient non-specific channels that conduct ions but not water. Dx was also less effective from the apical side as compared to the basal side of the epithelium. We also showed that intercellular septa are not conductive in non-sterile cells, even though most cells are isopotential. Luminal microbiota therefore impart asymmetry to the epithelium, by activation of apical membrane conductance, enhancing inter-enterocyte communication, separated by insulating septa, via the gut lumen. These results also open the possibility of studying the basis of bidirectional molecular conversation specifically between enterocytes and microbiota that enables discrimination between commensals and pathogens, establishment of the former, and elimination of the latter.


Assuntos
Eletrólitos/metabolismo , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiologia , Animais , Depsipeptídeos/farmacologia , Drosophila melanogaster , Mucosa Intestinal/efeitos dos fármacos , Larva , Macrolídeos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Micotoxinas/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores
7.
BMC Genomics ; 17: 367, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27189621

RESUMO

BACKGROUND: Aschersonia badia [(Ab) Teleomorph: Hypocrella siamensis] is an entomopathogenic fungus that specifically infects scale insects and whiteflies. We present the whole genome sequence of Ab and its comparison with two clavicipitaceous fungi Metarhizium robertsii (MR: generalist entomopathogen) and M. acridum (MAC: acridid-specific entomopathogen) that exhibit variable host preferences. Here, through comparative analysis of pathogen-host interacting genes, carbohydrate active enzymes, secondary metabolite biosynthesis genes, and sexuality genes, we explore the proteins with possible virulence functions in clavicipitaceous fungi. Comprehensive overview of GH18 family chitinases has been provided to decipher the role of chitinases in claviceptaceous fungi that are either host specific or generalists. RESULTS: We report the 28.8 Mb draft genome of Ab and its comparative genome analysis with MR and MAC. The comparative analyses suggests expansion in pathogen-host interacting gene families and carbohydrate active enzyme families in MR, whilst their contraction in Ab and MAC genomes. The multi-modular NRPS gene (dtxS1) responsible for biosynthesis of the secondary metabolite destruxin in MR is not conserved in Ab, similar to the specialist pathogen MAC. An additional siderophore biosynthetic gene responsible for acquisition of iron was identified in MR. Further, the domain survey of chitinases suggest that the CBM50 (LysM) domains, which participate in chitin-binding functions, were not observed in MAC, but were present in Ab and MR. However, apparent differences in frequency of CBM50 domains associated with chitinases of Ab and MR was identified, where MR chitinases displayed a higher proportion of associated CBM50 domains than Ab chitinases. CONCLUSIONS: This study suggests differences in distribution of dtxS1 and chitinases in specialists (Ab and MAC) and generalists (MR) fungi. Our analysis also suggests the presence of a siderophore biosynthetic gene in the MR genome which perhaps aids in enhanced virulence potential and host range. The variation in association of CBMs, being higher in generalists (MR) and lower in specialists (Ab and MAC) fungi may further be responsible for the differences in host affiliation.


Assuntos
Ascomicetos/genética , Genoma Fúngico , Genômica , Insetos/microbiologia , Animais , Ascomicetos/classificação , Quitinases , Biologia Computacional/métodos , Elementos de DNA Transponíveis , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Anotação de Sequência Molecular , Mutação , Filogenia
8.
Curr Genet ; 62(4): 799-807, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26936154

RESUMO

Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate knockout mutants of two candidate non-ribosomal peptide synthetases (NRPS29 and NRPS30). Comparative studies of secondary metabolites in the two deletion mutants and wild type confirmed the absence of sansalvamide in the NRPS30 deletion mutant, implicating this synthetase in the biosynthetic pathway for sansalvamide. Sansalvamide is structurally related to the cyclic hexadepsipeptide destruxin, which both contain an α-hydroxyisocaproic acid (HICA) unit. A gene cluster responsible for destruxin production has previously been identified in Metarhizium robertsii together with a hypothetical biosynthetic pathway. Using comparative bioinformatic analyses of the catalytic domains in the destruxin and sansalvamide NRPSs, we were able to propose a model for sansalvamide biosynthesis. Orthologues of the gene clusters were also identified in species from several other genera including Acremonium chrysogenum and Trichoderma virens, which suggests that the ability to produce compounds related to destruxin and sansalvamide is widespread.


Assuntos
Depsipeptídeos/biossíntese , Depsipeptídeos/farmacologia , Fusarium/genética , Fusarium/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Antineoplásicos , Depsipeptídeos/química , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Metaboloma , Metabolômica , Modelos Biológicos , Família Multigênica , Filogenia , Metabolismo Secundário , Deleção de Sequência , Transcrição Gênica
9.
Appl Microbiol Biotechnol ; 100(21): 9217-9228, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27521024

RESUMO

The insect pathogenic fungus Metarhizium anisopliae is an important insect biological control agent commercialized for use worldwide. Fungal infection is percutaneous, and rapid germination and growth has been linked to virulence. Using a simple in vitro growth screen to isolate mutants with increased virulence, M. anisopliae SM04 conidia were exposed to UV radiation for 20, 40, and 60 min, and mutants were subsequently screened for more rapid growth on standard potato dextrose agar. From a screen of >6,000 colonies, mutants were selected based on larger colony diameters as compared to the wild-type parent. Insect bioassays using the diamondback moth, Plutella xylostella, revealed one mutant, designated as MaUV-40.1 as displaying both more rapid growth and increased virulence. The mean lethal time to kill (LT50 using 106 conidia/ml) was 57.6 and 115.4 h for the MaUV-40.1 mutant and wild-type strains, respectively. Total conidial production, UV and thermal tolerances of the MaUV-40.1 strain were increased, but a reduced secretome was seen for the mutant compared to wild type. Analyses of culture supernatants indicated significant shifts in secondary metabolite production in the mutant. The insecticidal activity of EthOAc extracts derived from MaUV-40.1 mutant cell-free culture supernatants were ~20 times more potent that wild-type extracts. These data indicate that mutagenesis coupled to a growth screen can be a simple approach to isolate strains with greater stress resistance and virulence and that cell-free extracts may hold promise as an alternative to the living organism for insect control.


Assuntos
Lepidópteros/microbiologia , Lepidópteros/fisiologia , Metarhizium/crescimento & desenvolvimento , Metarhizium/efeitos da radiação , Viabilidade Microbiana , Mutação , Raios Ultravioleta , Animais , Bioensaio , Meios de Cultura/química , Programas de Rastreamento , Análise de Sobrevida , Virulência
10.
Molecules ; 22(1)2016 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-28036076

RESUMO

Destruxin A (DA), a cyclodepsipeptidic mycotoxin of entomopathogenic fungus, Metarhizium anisopliae, has anti-immunity activity against insects, but the mechanism of immune regulation is not clear yet. In our previous experiment, the significant expression changes of Bm_nscaf2838_045, Bm_nscaf2674_066, and Bm_nscaf2767_133 genes in a silkworm's hemocytes were found, which suggested that these genes might be involved in insect's innate immunity. In the current experiment, the silkworm cell line Bm12 was used to survey the expression levels of these genes after the cells were treated with DA and the transcription factors BmRel, BmRelish1 and BmRelish2 were silenced by specific siRNA. The results indicated that, after the cells were treated by DA, the gene expression level of BmRelish2 was significantly downregulated, but BmRel and BmRelish1 were not changed. The results also showed that the gene expression levels of Bm_nscaf2838_045 and Bm_nscaf2674_066 had similar phenomena, i.e., downregulation with individual BmRelish1 gene silence or DA treatment, upregulation with combination of BmRelish1 gene silence and DA treatment, upregulation with individual BmRelish2 gene silence, and downregulation with combination of BmRelish2 gene silence plus DA treatment, but no changes in the BmRel gene silence combined with DA treatment. For the Bm_nscaf2767_133 gene, the downregulated expressions were found in individual BmRelish2 gene silence or DA treatment, upregulation in the combination treatment of BmRelish2 gene silence plus DA, and the individual treatment of BmRel or BmRelish1 silence. It is suggested that expressions of the Bm_nscaf2838_045 and Bm_nscaf2674_066 genes are closely related to the Imd signal pathway, but Bm_nscaf2767_133 genes might involve in both Toll and Imd pathways. Furthermore, the BmRelish1 gene acts as an activator and the BmRelish2 gene acts as a repressor for both Bm_nscaf2838_045 and Bm_nscaf2674_066 gene expressions. It also implies that DA may participate in the splicing process of BmRelish where BmRelish2 was promoted. Our research will provide new insights on the understanding of the activity mechanisms of destruxins.


Assuntos
Bombyx/genética , Depsipeptídeos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Proteínas de Insetos/genética , Micotoxinas/farmacologia , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacos , Animais , Bombyx/imunologia , Linhagem Celular , Imunidade Inata/genética , Proteínas de Insetos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Fatores de Transcrição/metabolismo
11.
Arch Insect Biochem Physiol ; 86(1): 33-45, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24719308

RESUMO

Destruxin A (DA), a cyclodepsipeptidic secondary metabolite of the entomopathogenic fungus, Metarhizium anisopliae, is an important anti-immunity agent against insect hemocytes. To understand the mechanism of the molecular responses to DA, fifth-instar larvae of the silkworm, Bombyx mori, were injected with 2 µg of DA. The proteomics of hemocytes were then investigated using two-dimensional electrophoresis and mass spectrometry, and validated qPCR. As a result, a total of 47 differently expressed protein spots were detected and 22 proteins in 26 spots were identified. There are eight immunity-related proteins, including three downregulated proteins (antitrypsin isoform 3, p50 protein, and calreticulin precursor) and five upregulated proteins (C-type lectin 10 precursor, serine proteinase-like protein, paralytic peptide, PPO-1, and PPO-2). Four resistance- and/or stress-related proteins (arginine kinase, carboxylesterase clade H, member 1, aminoacylase, and thiol peroxiredoxin) were upregulated. Ten proteins with other or unknown functions were also recorded. Five selected proteins were verified with qPCR. These results provide new insights into the molecular mechanism of host immune response to DA challenge.


Assuntos
Bombyx/efeitos dos fármacos , Bombyx/imunologia , Depsipeptídeos/toxicidade , Hemócitos/efeitos dos fármacos , Hemócitos/imunologia , Micotoxinas/toxicidade , Animais , Proteínas de Insetos , Larva , Proteoma
12.
Drug Metab Pharmacokinet ; 58: 101028, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-39265438

RESUMO

Cyclic peptides have attracted attention as new modalities for drug development owing to their unique pharmacokinetic and pharmacodynamic properties. Destruxin E, a 19-membered cyclodepsipeptide, is a promising candidate drug for cancer therapy. The purpose of the present study was to clarify the molecular mechanisms underlying membrane transport, metabolism, and the binding for target molecules of destruxin E in human cervical carcinoma HeLa cells used as a model of cancer cells. The influx transport and the intracellular metabolism of destruxin E were non-saturable and saturable, respectively, at up to 10 µM. The intracellular amounts of destruxin E and destruxin E-diol after incubation of destruxin E with the cells significantly decreased at 4 °C compared to those at 37 °C. Destruxin E-diol, but not destruxin E, undergoes efflux transport out of cells via P-gp/MDR1/ABCB1 and BCRP/ABCG2. The epoxide hydrolase EPHX2 functions as a potent metabolizing enzyme that can convert the epoxide of destruxin E to the destruxin E-diol. Treatment with an EPHX2 inhibitor increased the intracellular destruxin E levels and enhanced the inhibitory activity of vacuolar type-H+ ATPase. These results suggest that epoxide hydrolase could be a regulatory factor for intracellular destruxin E levels and its pharmacological activity.

13.
Front Microbiol ; 14: 1210647, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37333627

RESUMO

Introduction: Destruxin A (DA) is a mycotoxin isolated from the entomopathogenic fungus Metarhizium anisopliae which has demonstrated inhibitory activity against various insect species. However, the mechanism of inhibition on target sites in insects remains unknown. Methods: In this research, the dose-response relationship between DA and morphological changes in body tissues and organs of domestic silkworm, Bombyx mori, were investigated by histopathological methods to identify the target sites that responded to DA. Results and Discussion: The results showed that responses of individual tissues and organs varied with DA dosage and treatment time. At low doses (i.e., 0.01µg/g), the hemocytes were the most sensitive to DA with morphological changes apparent at 6 h after treatment. However, the muscle cells, fat body, and Malpighian tubules were unaltered. At higher doses (i.e., > 0.1µg/g), morphological changes were observed in muscle cells, fat body, and Malpighian tubules at 24 h after treatment. The results indicated that DA can be an immunosuppressant by damaging host cells like hemocytes, and at higher doses may potentially impact other physiological processes, including muscle function, metabolism, and excretion. The information presented in the current study will facilitate development of mycopesticides and novel immunosuppressants.

14.
PeerJ ; 11: e15726, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37583910

RESUMO

Species of the genus Metarhizium are characterized by a multitrophic lifestyle of being arthropod parasites, rhizosphere colonizers, endophytes, and saprophytes. The process of adaptation to various organisms and substrates may lead to specific physiological alterations that can be elucidated by passaging through different hosts. Changes in virulence and cultivation properties of entomopathogenic fungi subcultured on different media or passaged through a live insect host are well known. Nevertheless, comparative in-depth physiological studies on fungi after passaging through insect or plant organisms are scarce. Here, virulence, plant colonization, hydrolytic enzymatic activities, toxin production, and antimicrobial action were compared between stable (nondegenerative) parent strain Metarhizium robertsii MB-1 and its reisolates obtained after eight passages through Galleria mellonella larvae or Solanum lycopersicum or after subculturing on the Sabouraud medium. The passaging through the insect caused similar physiological alterations relative to the plant-based passaging: elevation of destruxin A, B, and E production, a decrease in protease and lipase activities, and lowering of virulence toward G. mellonella and Leptinotarsa decemlineata as compared to the parent strain. The reisolates passaged through the insect or plant showed a slight trend toward increased tomato colonization and enhanced antagonistic action on tomato-associated bacterium Bacillus pumilus as compared to the parental strain. Meanwhile, the subculturing of MB-1 on the Sabouraud medium showed stability of the studied parameters, with minimal alterations relative to the parental strain. We propose that the fungal virulence factors are reprioritized during adaptation of M. robertsii to insects, plants, and media.


Assuntos
Metarhizium , Mariposas , Animais , Virulência , Insetos/microbiologia , Mariposas/microbiologia , Plantas
15.
Pest Manag Sci ; 78(5): 1915-1924, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35080798

RESUMO

BACKGROUND: Destruxin A (DA) is a mycotoxin secreted by entomogenous fungi, such as Metarhizium anisopliae, which has broad-spectrum insecticidal activity. Insect innate immunity provides resistance against the invasion of entomopathogenic fungi. Previous studies have shown that DA could inhibit the immune response, however, the suppressive mechanism of DA on prophenoloxidase system is still unknown. RESULTS: Based on the transcriptome of Aphis citricola, we screened the scavenger receptor class B(AcSR-B)and identified that it significantly responds to DA. Spatio-temporal expression analysis showed that AcSR-B is highly expressed in adult stage and is mainly distributed in the abdominal region. We further revealed that both M. anisopliae and Escherichia coli could suppress the expression of AcSR-B at 24 h, and that the expressed recombinant protein rAcSR-B possessed agglutination activity to M. anisopliae and E. coli. DA could suppress the protein expression of AcSR-B. In addition, RNA interference of AcSR-B caused death of A. citricola in a dose-dependent manner, and RNA interference of AcSR-B increased mortality in A. citricola under the same lethal concentration of DA. The inhibiting effect of AcSR-B silencing was similar with the DA treatment upon phenol oxidase (PO) activity of A. citricola hemolymph. DA could not decrease PO activity further after AcSR-B silencing. CONCLUSION: Destruxin A inhibits melanization by suppressing AcSR-B in A. citricola. Our findings are helpful in understanding the underlying molecular mechanism of the DA suppressing immune system, and uncover a potential molecular target for double-stranded RNA (dsRNA) insecticides.


Assuntos
Afídeos , Depsipeptídeos , Inseticidas , Animais , Afídeos/metabolismo , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Receptores Depuradores
16.
J Fungi (Basel) ; 7(6)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201102

RESUMO

Destruxin A (DA), a mycotoxin isolated from the entomopathogenic fungus Metarhizium anisopliae, has good insecticidal and immune-inhibitory activity, but the action mechanism has not yet been elucidated. In order to identify the DA-binding proteins, we conducted drug affinity responsive target stability (DARTS) experiments, which indicated that the silkworm's (Bombyx mori) transmembrane protein 214 (BmTEME214) and protein transport protein SEC23A isoform X2 (BmSEC23) are the potential DA-binding proteins. The current research was focused on validation of the interaction between DA and these two proteins via bio-layer interferometry (BLI) in vitro, insect two-hybrid (I2H) in Sf9 cells, and RNAi in the insect. The results of the BLI tests showed that DA has strong affinity to bind BmTEME214 and BmSEC23 proteins with a KD value of 0.286 and 0.291 µM, respectively. In the I2H experiments, DA inhibited (at 0.02 µg/mL) and activated (at 0.002-0.0002 µg/mL) the protein interactions of BmSEC23-BmSEC13, but it only inhibited the BmTMEM214-BmSEC13L interaction. Furthermore, in the RNAi tests, an apparent increase in the silkworm's mortality was recorded in the joint treatment of DA with dsBmSEC23 or dsBmTMEM214 when compared with the single treatment of DA (1.5 µg/g body), 40 µg/g body dsBmSEC23, or dsBmTMEM214. This research confirmed that BmSEC23 and BmTMEM214 are the DA-binding proteins and provided new insights to understand the action mechanism of DA.

17.
J Fungi (Basel) ; 7(8)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34436132

RESUMO

Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, exhibits insecticidal activities in a wide range of pests and is known as an innate immunity inhibitor. However, its mechanism of action requires further investigation. In this research, the interactions of DA with the six aminoacyl tRNA synthetases (ARSs) of Bombyx mori, BmAlaRS, BmCysRS, BmMetRS, BmValRS, BmIleRS, and BmGluProRS, were analyzed. The six ARSs were expressed and purified. The BLI (biolayer interferometry) results indicated that DA binds these ARSs with the affinity indices (KD) of 10-4 to 10-5 M. The molecular docking suggested a similar interaction mode of DA with ARSs, whereby DA settled into a pocket through hydrogen bonds with Asn, Arg, His, Lys, and Tyr of ARSs. Furthermore, DA treatments decreased the contents of soluble protein and free amino acids in Bm12 cells, which suggested that DA impedes protein synthesis. Lastly, the ARSs in Bm12 cells were all downregulated by DA stress. This study sheds light on exploring and answering the molecular target of DA against target insects.

18.
Toxins (Basel) ; 11(2)2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30682818

RESUMO

Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin secreted by the entomopathogenic fungus Metarhizium anisopliae, was reported to have an insecticidal effect and anti-immunity activity. However, its molecular mechanism of action remains unclear. Previously, we isolated several potential DA-affinity (binding) proteins in the Bombyx mori Bm12 cell line. By docking score using MOE2015, we selected three proteins-BmTudor-sn, BmPiwi, and BmAGO2-for further validation. First, using Bio-Layer Interferometry in vitro, we found that BmTudor-sn had an affinity interaction with DA at 125, 250, and 500 µM, while BmPiwi and BmAGO2 had no interaction signal with DA. Second, we employed standard immunoblotting to verify that BmTudor-sn is susceptible to DA, but BmPiwi and BmAGO2 are not. Third, to verify these findings in vivo, we used a target engagement strategy based on shifts in protein thermal stability following ligand binding termed the cellular thermal shift assay and found no thermal stability shift in BmPiwi and BmAGO2, whereas a shift was found for BmTudor-sn. In addition, in BmTudor-sn knockdown Bm12 cells, we observed that cell viability increased under DA treatment. Furthermore, insect two-hybrid system results indicated that the key site involved in DA binding to BmTudor-sn was Leu704. In conclusion, in vivo and in vitro experimental evidence indicated that BmTudor-sn is a binding protein of DA in silkworm Bm12 cells at the 100 µM level, and the key site of this interaction is Leu704. Our results provide new perspectives to aid in elucidating the molecular mechanism of action of DA in insects and developing new biopesticide.


Assuntos
Proteínas de Transporte/metabolismo , Depsipeptídeos/toxicidade , Endonucleases/metabolismo , Proteínas de Insetos/metabolismo , Animais , Bombyx , Proteínas de Transporte/química , Linhagem Celular , Depsipeptídeos/química , Endonucleases/química , Proteínas de Insetos/química , Simulação de Acoplamento Molecular , Domínio Tudor
19.
Toxins (Basel) ; 11(6)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216655

RESUMO

Destruxin A (DA), a major secondary metabolite of Metarhizium anisopliae, has anti-immunity to insects. However, the detailed mechanism and its interactions with target proteins are elusive. Previously, three immunophilins, peptidyl-prolyl cis-trans isomerase (BmPPI), FK506 binding-protein 45 (BmFKBP45) and BmFKBP59 homologue, were isolated from the silkworm, Bombyx mori Bm12 cell line following treatment with DA, which suggested that these proteins were possible DA-binding proteins. To validate the interaction between DA and the three immunophilins, we performed bio-layer interferometry (BLI) assay, and the results showed that DA has interaction with BmPPI, whose affinity constant value is 1.98 × 10-3 M and which has no affinity with FKBP45 and FKBP59 homologue in vitro. Furthermore, we investigated the affinity between DA and human PPI protein (HsPPIA) and the affinity constant (KD) value is 2.22 × 10-3 M. Additionally, we compared the effects of silkworm and human PPI proteins produced by DA and immunosuppressants, cyclosporine A (CsA), and tacrolimus (FK506), by employing I2H (insect two-hybrid) in the SF-9 cell line. The results indicated that in silkworm, the effects created by DA and CsA were stronger than FK506. Furthermore, the effects created by DA in silkworm were stronger than those in humans. This study will offer new thinking to elucidate the molecular mechanism of DA in the immunity system of insects.


Assuntos
Depsipeptídeos/toxicidade , Imunofilinas/metabolismo , Proteínas de Insetos/metabolismo , Micotoxinas/toxicidade , Animais , Bombyx , Imunofilinas/genética , Proteínas de Insetos/genética , Células Sf9 , Técnicas do Sistema de Duplo-Híbrido
20.
Front Immunol ; 9: 185, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29472927

RESUMO

Plutella xylostella, a global key pest, is one of the major lepidopteran pests of cruciferous vegetables owing to its strong ability of resistance development to a wide range of insecticides. Destruxin A, a mycotoxin of the entomopathogenic fungus, Metarhizium anisopliae, has broad-spectrum insecticidal effects and has been used as an alternative control strategy to reduce harmful effects of insecticides. However, microRNA (miRNA)-regulated reactions against destruxin A have not been elucidated yet. Therefore, here, to identify immunity-related miRNAs, we constructed four small RNA libraries from destruxin A-injected larvae of P. xylostella at three different time courses (2, 4, and 6 h) with a control, and sequenced by Illumina. Our results showed that totally 187 known and 44 novel miRNAs were identified in four libraries by bioinformatic analysis. Interestingly, among differentially expressed known miRNAs, some conserved miRNAs, such as miR-263, miR-279, miR-306, miR-2a, and miR-308, predicted to be involved in regulating immunity-related genes, were also identified. Worthy to mention, miR-306 and miR-279 were also listed as common abundantly expressed miRNA in all treatments. The Kyoto Encyclopedia of Genes and Genomes pathway analysis also indicated that differentially expressed miRNAs were involved in several immunity-related signaling pathways, including toll signaling pathway, IMD signaling pathway, JAK-STAT signaling pathway, and cell adhesion molecules signaling pathway. To the best of our knowledge, this is the first comprehensive report of destruxin A-responsive immunity-related miRNAs in P. xylostella. Our findings will improve in understanding the role of destruxin A-responsive miRNAs in the host immune system and would be useful to develop biological control strategies for controlling P. xylostella.


Assuntos
Depsipeptídeos/farmacologia , MicroRNAs/imunologia , Mariposas/imunologia , Animais , Perfilação da Expressão Gênica , Biblioteca Gênica , Genoma de Inseto , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Larva/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Transdução de Sinais , Transcriptoma
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