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1.
Insect Biochem Mol Biol ; 126: 103451, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32841718

RESUMO

C-type lectins (CTLs) recognize various glycoconjugates through carbohydrate recognition domains (CRDs) and they play important roles in immune responses. In this study, comparative genomic analysis of CTLs were performed in 7 holometabolous species. CTL-S1 to S8 and CTL-X1 to X4 orthologous groups existed in the 7 species, while CTL-X5 group with dual-CRD, CTL-S11 group with triple-CRD, CTL-S9 group with a long C-terminus and Lepidopteran specific CTL-S10 group were not conserved. SliCTL-S12 to S14 cluster was only present in Spodoptera litura, and CTL-S genes were expanded on chromosomes 2 L and 2 R in Drosophila melanogaster. Most IMLs were clustered into three groups and the numbers of IMLs vary among species due to gene duplications. D. melanogaster specific CTLs and Lepidopteran IMLs within each of the three groups evolved more rapidly with higher dN/dS ratios. Two CRDs in IMLs clustered into two clades, with conserved Cys4-Cys5 and Cys1-Cys2 bonds in the first and second CRDs, respectively. The CTL-S and CTL-X family members in S. litura were mainly expressed in the fat body of 5th but not 6th instar larvae, and responded differently to S. litura nucleopolyhedrovirus (SpltNPV) and Nomuraea rileyi infection. The transcription levels of SliCTLs that expressed in fat body but not highly expressed in hemocytes were decreased at the middle and late stages of SpltNPV infection, and the mRNA levels of SliCTLs highly or specifically expressed in hemocytes were mainly decreased by SpltlNPV, N. rileyi and Bacillus thuringiensis infection. These results provide valuable information for further exploration of CTL functions in host-pathogen interaction.

2.
J Agric Food Chem ; 68(16): 4607-4615, 2020 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-32227950

RESUMO

Cry toxins produced by Bacillus thuringiensis are well known for their high insecticidal activities against Lepidoptera, Diptera, and Coleoptera; however, their activities against Aphididae are very low. Recently, it has been reported that a Cry41-related toxin exhibited moderate activity against the aphid Myzus persicae, and thus, it is highly desirable to uncover its unique mechanism. In this paper, we report that Cathepsin B, calcium-transporting ATPase, and symbiotic bacterial-associated protein ATP-dependent-6-phosphofructokinase were pulled down from the homogenate of M. persicae as unique proteins that possibly bound to Cry41-related toxin. Cathepsin B has been reported to cleave and inactivate antiapoptotic proteins and plays a role in caspase-initiated apoptotic cascades. In this study, Cathepsin B was expressed in Escherichia coli and purified, and in vitro interaction between recombinant Cathepsin B and Cry41-related toxin was demonstrated. Interestingly, we found that addition of Cry41-related toxin obviously enhanced Cathepsin B activity. We propose a model for the mechanism of Cry41-related toxin as follows: Cry41-related toxin enters the aphid cells and enhances Cathepsin B activity, resulting in acceleration of apoptosis of aphid cells.

3.
Dev Comp Immunol ; 107: 103661, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32097696

RESUMO

The diamondback moth, Plutella xylostella, is the first insect to develop resistance to Bacillus thuringiensis (Bt) in the field. To date, little is known about the molecular mechanism of the interaction between Bt and midgut immunity in P. xylostella. Here, we report immune responses in the P. xylostella midgut to Bt strain Bt8010 using a combined approach of transcriptomics and quantitative proteomics. Many genes in the Toll, IMD, JNK and JAK-STAT pathways and antimicrobial peptide genes were activated at 18 h post-infection. In the prophenoloxidase (PPO) cascade, four serpin genes were activated, and the PPO1 gene was suppressed by Bt8010. Inhibition of the two PPO proteins was observed at 18 h post-infection. Feeding Bt8010-infected larvae recombinant PPOs enhanced their survival. These results revealed that the Toll, IMD, JNK and JAK-STAT pathways were triggered and participated in the immune defence of the midgut against Bt8010, while the PPO cascade was inhibited and played an important role in this process.

4.
Arch Insect Biochem Physiol ; 103(1): e21626, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31562754

RESUMO

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Insetos/farmacologia , Spodoptera/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Bacillus subtilis/efeitos dos fármacos , Beauveria/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Insetos/química , Serratia marcescens/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos
5.
Pestic Biochem Physiol ; 162: 96-104, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31836060

RESUMO

Galectins are a family of ß-galactoside binding proteins, and insect galectins play a role in immune responses and may also affect Cry toxin activity. In this study, we aimed to further understand the function and molecular mechanism of Aedes aegypti galectin-6 in modulation of Cry11Aa toxicity. A. aegypti galectin-6 was cloned, and the recombinant galectin-6 was expressed and purified. Bioassays indicated that galectin-6 could reduce the toxicity of Cry11Aa, protecting A. aegypti larvae. To determine interactions among galectin-6, Cry11Aa and putative toxin receptors, Octet Red System, western blotting, far-western blotting and ELISA assays were performed. Octet Red System showed that galectin-6 bound to BBMVs of A. aegypti larvae with lower affinity than that of Cry11Aa. Western blotting and far-western blotting analyses demonstrated that galectin-6 bound to A. aegypti ALP1 and APN2 as well as to BBMVs, consistent with the results of ELISA and protein docking simulations. However, galectin-6 did not bind to Cadherin in far-western blotting or ELISA assay, though the protein docking simulations suggested their binding potential. These findings support the conclusion that galectin-6 may block Cry11Aa from binding to ALP1 and APN2 due to structural similarity, which might decrease the mosquitocidal toxicity of Cry11Aa.


Assuntos
Aedes , Bacillus thuringiensis , Animais , Proteínas de Bactérias , Endotoxinas , Galectinas , Proteínas Hemolisinas , Proteínas de Insetos , Larva
6.
Dev Comp Immunol ; 102: 103468, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31430488

RESUMO

Insects, which lack the adaptive immune system, have developed sophisticated innate immune system consisting of humoral and cellular immune responses to defend against invading microorganisms. Non-self recognition of microbes is the front line of the innate immune system. Repertoires of pattern recognition receptors (PRRs) recognize the conserved pathogen-associated molecular patterns (PAMPs) present in microbes, such as lipopolysaccharide (LPS), peptidoglycan (PGN), lipoteichoic acid (LTA) and ß-1, 3-glucans, and induce innate immune responses. In this review, we summarize current knowledge of the structure, classification and roles of PRRs in innate immunity of the model organism Drosophila melanogaster, focusing mainly on the peptidoglycan recognition proteins (PGRPs), Gram-negative bacteria-binding proteins (GNBPs), scavenger receptors (SRs), thioester-containing proteins (TEPs), and lectins.

7.
Adv Exp Med Biol ; 1209: 79-108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31728866

RESUMO

Pattern recognition receptors (PRRs) are sensors of exogenous and endogenous "danger" signals from pathogen-associated molecular patterns (PAMPs), and damage associated molecular patterns (DAMPs), while autophagy can respond to these signals to control homeostasis. Almost all PRRs can induce autophagy directly or indirectly. Toll-like receptors (TLRs), Nod-like receptors (NLRs), retinoic acid-inducible gene-I-like receptors (RLRs), and cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway can induce autophagy directly through Beclin-1 or LC3-dependent pathway, while the interactions with the receptor for advanced glycation end products (RAGE)/high mobility group box 1 (HMGB1), CD91/Calreticulin, and TLRs/HSPs are achieved by protein, Ca2+, and mitochondrial homeostasis. Autophagy presents antigens to PRRs and helps to clean the pathogens. In addition, the induced autophagy can form a negative feedback regulation of PRRs-mediated inflammation in cell/disease-specific manner to maintain homeostasis and prevent excessive inflammation. Understanding the interaction between PRRs and autophagy in a specific disease will promote drug development for immunotherapy. Here, we focus on the interactions between PRRs and autophagy and how they affect the inflammatory response.


Assuntos
Autofagia , Inflamação , Receptores de Reconhecimento de Padrão , Autofagia/imunologia , Humanos , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais
8.
Insect Sci ; 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31617302

RESUMO

Wolbachia are Gram-negative endosymbionts that are known to cause embryonic lethality when infected male insects mate with uninfected females or with females carrying a different strain of Wolbachia, a situation characterized as cytoplasmic incompatibility (CI). However, the mechanism of CI is not yet fully understood, although recent studies on Drosophila melanogaster have achieved great progress. Here, we found that Wolbachia infection caused changes in the expressions of several immunity-related genes, including significant upregulation of kenny (key), in the testes of D. melanogaster. Overexpression of key in fly testes led to a significant decrease in egg hatch rates when these flies mate with wild-type females. Wolbachia-infected females could rescue this embryonic lethality. Furthermore, in key overexpressing testes terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling signal was significantly stronger than in the control testes, and the level of reactive oxygen species was significantly increased. Overexpression of key also resulted in alterations of some other immunity-related gene expressions, including the downregulation of Zn72D. Knockdown of Zn72D in fly testes also led to a significant decrease in egg hatch rates. These results suggest that Wolbachia might induce the defect in male host fertility by immunity-related pathways and thus cause an oxidative damage and cell death in male testes.

9.
J Agric Food Chem ; 67(32): 8896-8904, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31339308

RESUMO

The mosquito Aedes aegypti is associated with the spread of many viral diseases in humans, including Dengue virus (DENVs), Yellow fever virus (YFV), Zika virus (ZIKV), and Chikungunya virus (CHIKV). Bacillus thuringiensis (Bt) is widely used as a biopesticide, which produces Cry toxins for mosquito control. The Cry toxins bind mainly to important receptors, including alkaline phosphatase (ALP) and aminopeptidase-N (APN). This work investigated the function of a C-type lectin, CTLGA9, in A. aegypti in response to Cry toxins. Our results showed by far-western blot and ELISA methods that the CTLTGA9 protein interacted with brush border membrane vesicles (BBMVs) of A. aegypti larvae and with ALP1, APN, and Cry11Aa proteins. Furthermore, molecular docking showed overlapping binding sites in ALP1 and APN for binding to Cry11Aa and CTLGA9. The toxicity assays further demonstrated that CTLGA9 inhibited the larvicidal activity of Cry toxins. According to the results of molecular docking, CTLGA9 may compete with Cry11Aa for binding to ALP1 and APN receptors and thus decreases the mosquitocidal toxicity of Cry11Aa. Our results provide further insights into better understanding the mechanism of Cry toxins and help improve the Cry toxicity for mosquito control.


Assuntos
Aedes/efeitos dos fármacos , Aedes/metabolismo , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/metabolismo , Aedes/química , Aedes/genética , Animais , Proteínas de Bactérias/química , Sítios de Ligação , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos
10.
Parasit Vectors ; 12(1): 319, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31238963

RESUMO

BACKGROUND: Bacillus thuringiensis israelensis (Bti) is a widely used mosquitocidal microbial pesticide due to its high toxicity. ATP-binding proteins (ABP) are prevalently detected in insects and are related to reaction against Bti toxins. However, the function of ABP in mosquito biocontrol is little known, especially in Aedes aegypti. Therefore, this study aimed to clarify the function of ABP in Ae. aegypti against Bti toxin. RESULTS: Aedes aegypti ABP (GenBank: XM_001661856.2) was cloned, expressed and purified in this study. Far-western blotting and ELISA were also carried out to confirm the interaction between ABP and Cry11Aa. A bioassay of Cry11Aa was performed both in the presence and absence of ABP, which showed that the mortality of Ae. aegypti is increased with an increase in ABP. CONCLUSIONS: Our results suggest that ABP in Ae. aegypti can modulate the toxicity of Cry11Aa toxin to mosquitoes by binding to Bti toxin. This could not only enrich the mechanism of Bt toxin, but also provide more data for the biocontrol of this transmission vector.


Assuntos
Aedes/genética , Bacillus thuringiensis/patogenicidade , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Aedes/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Bioensaio , Clonagem Molecular , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Mosquitos Vetores/microbiologia , Controle Biológico de Vetores , Ligação Proteica
11.
Fish Shellfish Immunol ; 92: 276-287, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31181341

RESUMO

In invertebrates, both fibrinogen-related proteins (FREPs) and C-type lectins are acknowledged to act as pattern recognition receptors (PRRs) to participate particularly in an innate immunity. Hereby, a unique C-type lectin designated as FmLFd was isolated from the hemocytes of Fenneropenaeus merguiensis. FmLFd contained one open reading frame which encoding a peptide of 312 amino acid residues and a signal peptide of 18 amino acids. The primary sequence of FmLFd was composed of a fibrinogen-like domain (Fd) with a Ca2+-binding site and possessing specificity to bind N-acetyl glucosamine (GlcNAc). The FmLFd transcripts were detected mainly in hemocytes of healthy shrimp. The expression of FmLFd was significantly up-regulated upon challenge shrimp with Vibrio parahaemolyticus and Vibrio harveyi which more potent than by white spot syndrome virus (WSSV). The knocking down shrimp with FmLFd double-stranded RNA caused dramatical gene down-regulation. The gene silencing with co-injection of pathogens resulted in reduction of the shrimp survival rate. Recombinant protein of FmLFd (rFmLFd) could agglutinate and bind directly to both Gram-negative and Gram-positive bacteria in a Ca2+-dependent manner and showed the sugar specificity to GlcNAc and bacterial saccharides; peptidoglycan (PGN), lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Recombinant protein of Fd domain (rFd) displayed the lower activity and specificity only to PGN. The binding between recombinant proteins of FmLFd and its domain confirming by ELISA demonstrated that both rFmLFd and rFd could bind to PGN, LPS and LTA with the highest affinity respected to PGN including a less extent of rFd. Besides, rFmLFd but not rFd could bind to WSSV proteins with the highest binding affinity to capsid VP15 and decreasing in order to envelope VP28 and tegument VP39A, respectively. It was presumed that entire molecule of FmLFd exhibited the antimicrobial ability by inhibiting the growth of pathogenic V. parahaemolyticus and this action was not affected by GlcNAc. Otherwise, FmLFd, a lectin containing fibrinogen-like domain, was firstly reported to be capable of promoting encapsulation by hemocytes. Altogether, we concluded that FmLFd belonged to a FREP family indentified by the existence of a conserved fibrinogen-like domain with possessing an ability to bind GlcNAc. It was a new C-type lectin existed in F. merguiensis and might presumably act as a kind of PRRs to participate in the shrimp immune defense towards bacterial and viral pathogens.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Penaeidae/genética , Penaeidae/imunologia , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Perfilação da Expressão Gênica , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Lectinas Tipo C/química , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Ácidos Teicoicos/farmacologia
12.
J Biol Chem ; 294(26): 10172-10181, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31088910

RESUMO

The Toll signaling pathway in Drosophila melanogaster regulates several immune-related functions, including the expression of antimicrobial peptide (AMP) genes. The canonical Toll receptor (Toll-1) is activated by the cytokine Spätzle (Spz-1), but Drosophila encodes eight other Toll genes and five other Spz genes whose interactions with one another and associated functions are less well-understood. Here, we conducted in vitro assays in the Drosophila S2 cell line with the Toll/interleukin-1 receptor (TIR) homology domains of each Toll family member to determine whether they can activate a known target of Toll-1, the promoter of the antifungal peptide gene drosomycin. All TIR family members activated the drosomycin promoter, with Toll-1 and Toll-7 TIRs producing the highest activation. We found that the Toll-1 and Toll-7 ectodomains bind Spz-1, -2, and -5, and also vesicular stomatitis virus (VSV) virions, and that Spz-1, -2, -5, and VSV all activated the promoters of drosomycin and several other AMP genes in S2 cells expressing full-length Toll-1 or Toll-7. In vivo experiments indicated that Toll-1 and Toll-7 mutants could be systemically infected with two bacterial species (Enterococcus faecalis and Pseudomonas aeruginosa), the opportunistic fungal pathogen Candida albicans, and VSV with different survival times in adult females and males compared with WT fly survival. Our results suggest that all Toll family members can activate several AMP genes. Our results further indicate that Toll-1 and Toll-7 bind multiple Spz proteins and also VSV, but they differentially affect adult survival after systemic infection, potentially because of sex-specific differences in Toll-1 and Toll-7 expression.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções Bacterianas/microbiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Receptores Toll-Like/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/genética , Infecções Bacterianas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/microbiologia , Feminino , Masculino , Regiões Promotoras Genéticas , Transdução de Sinais , Receptores Toll-Like/genética
13.
Mol Immunol ; 107: 142-164, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30739833

RESUMO

Immune sensing of exogenous molecules from microbes (e.g., pathogen-associated molecular patterns) and nonmicrobial molecules (e.g., asbestos, alum, and silica), as well as endogenous damage-associated molecular patterns (e.g., ATP, uric acid crystals, and amyloid A) activates innate immunity by inducing immune-related genes, including proinflammatory cytokines, which further facilitate the development of adaptive immunity. The roles of transcriptional responses downstream of immune sensing have been widely characterized in informing adaptive immunity; however, few studies focus on the effect of post-translational responses on the modulation of adaptive immune responses. Inflammasomes activated by the previously described endo- and exogenous stimuli autocatalytically induce intracellular pro-caspase-1, which cleaves the inactive precursors of interleukin-1ß (IL-1ß) and IL-18 into bioactive proinflammatory cytokines. IL-1ß and IL-18 not only contribute to the host defense against infections by activating phagocytes, such as monocytes, macrophages, dendritic cells, and neutrophils, but also induce T-helper 17 (Th17)- and Th1-mediated adaptive immune responses. In synergy with IL-6 and IL-23, IL-1ß activates IL-1 receptor (IL-1R) signaling to drive the differentiation of IL-17-producing Th17 cells, which not only play critical roles in host protective immunity to infections of bacteria, fungi, and certain viruses but also participate in the pathology of inflammatory disorders and tumorigenesis. Consequently, targeting inflammasomes and IL-1/IL-1R signaling may effectively improve the treatment of Th17-associated disorders, such as autoinflammatory diseases and cancers, thereby providing novel insights into drug development.


Assuntos
Imunidade Adaptativa/imunologia , Diferenciação Celular/imunologia , Inflamassomos/imunologia , Transdução de Sinais/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Citocinas/imunologia , Humanos , Fagócitos/citologia , Fagócitos/imunologia , Células Th17/citologia
14.
Dev Comp Immunol ; 95: 10-18, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30731096

RESUMO

Drosophila melanogaster possesses a sophisticated and effective immune system composed of humoral and cellular immune responses, and production of antimicrobial peptides (AMPs) is an important defense mechanism. Expression of AMPs is regulated by the Toll and IMD (immune deficiency) pathways. Production of AMPs can be systemic in the fat body or a local event in the midgut and epithelium. So far, most studies focus on systemic septic infection in adult flies and little is known about AMP gene activation after ingestion of killed bacteria. In this study, we investigated activation of AMP genes in the wild-type w1118, MyD88 and Imd mutant flies after ingestion of heat-killed Escherichia coli and Staphylococcus aureus. We showed that ingestion of E. coli activated most AMP genes, including drosomycin and diptericin, in the first to third instar larvae and pupae, while ingestion of S. aureus induced only some AMP genes in some larval stages or in pupae. In adult flies, ingestion of killed bacteria activated AMP genes differently in males and females. Interestingly, ingestion of killed E. coli and S. aureus in females conferred resistance to septic infection by both live pathogenic Enterococcus faecalis and Pseudomonas aeruginosa, and ingestion of E. coli in males conferred resistance to P. aeruginosa infection. Our results indicated that E. coli and S. aureus can activate both the Toll and IMD pathways, and systemic and local immune responses work together to provide Drosophila more effective protection against infection.


Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Bactérias/imunologia , Drosophila melanogaster/imunologia , Ingestão de Alimentos/imunologia , Sepse/imunologia , Animais , Bactérias/patogenicidade , Resistência à Doença/imunologia , Proteínas de Drosophila/imunologia , Proteínas de Drosophila/metabolismo , Feminino , Masculino , Sepse/microbiologia , Fatores Sexuais , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo
15.
Dev Comp Immunol ; 95: 50-58, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30735676

RESUMO

An important innate immune response in Drosophila melanogaster is the production of antimicrobial peptides (AMPs). Expression of AMP genes is mediated by the Toll and immune deficiency (IMD) pathways via NF-κB transcription factors Dorsal, DIF and Relish. Dorsal and DIF act downstream of the Toll pathway, whereas Relish acts in the IMD pathway. Dorsal and DIF are held inactive in the cytoplasm by the IκB protein Cactus, while Relish contains an IκB-like inhibitory domain at the C-terminus. NF-κB factors normally form homodimers and heterodimers to regulate gene expression, but formation of heterodimers between Relish and DIF or Dorsal and the specificity and activity of the three NF-κB homodimers and heterodimers are not well understood. In this study, we compared the activity of Rel homology domains (RHDs) of Dorsal, DIF and Relish in activation of Drosophila AMP gene promoters, demonstrated that Relish-RHD (Rel-RHD) interacted with both Dorsal-RHD and DIF-RHD, Relish-N interacted with DIF and Dorsal, and overexpression of individual RHD and co-expression of any two RHDs activated the activity of AMP gene promoters to various levels, suggesting formation of homodimers and heterodimers among Dorsal, DIF and Relish. Rel-RHD homodimers were stronger activators than heterodimers of Rel-RHD with either DIF-RHD or Dorsal-RHD, while DIF-RHD-Dorsal-RHD heterodimers were stronger activators than either DIF-RHD or Dorsal-RHD homodimers in activation of AMP gene promoters. We also identified the nucleotides at the 6th and 8th positions of the 3' half-sites of the κB motifs that are important for the specificity and activity of NF-κB transcription factors.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Drosophila melanogaster/imunologia , NF-kappa B/metabolismo , Transdução de Sinais/genética , Motivos de Aminoácidos/genética , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/imunologia , Imunidade Inata , NF-kappa B/genética , NF-kappa B/isolamento & purificação , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/genética , Domínios Proteicos/genética , Multimerização Proteica/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo
16.
Insect Biochem Mol Biol ; 106: 47-54, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30468769

RESUMO

Wolbachia are endosymbiotic bacteria present in a wide range of invertebrates. Although their dramatic effects on host reproductive biology have been well studied, little is known about the effects of Wolbachia on the learning and memory capacity (LMC) of hosts, despite their distribution in the host nervous system, including brain. In this study, we found that Wolbachia infection significantly enhanced LMC in both Drosophila melanogaster and D. simulans. Expression of LMC-related genes was significantly increased in the head of D. melanogaster infected with the wMel strain, and among these genes, crebA was up-regulated the most. Knockdown of crebA in Wolbachia-infected flies significantly decreased LMC, while overexpression of crebA in Wolbachia-free flies significantly enhanced the LMC of flies. More importantly, a microRNA (miRNA), dme-miR-92b, was identified to be complementary to the 3'UTR of crebA. Wolbachia infection was correlated with reduced expression of dme-miR-92b in D. melanogaster, and dme-miR-92b negatively regulated crebA through binding to its 3'UTR region. Overexpression of dme-miR-92b in Wolbachia-infected flies by microinjection of agomirs caused a significant decrease in crebA expression and LMC, while inhibition of dme-miR-92b in Wolbachia-free flies by microinjection of antagomirs resulted in a significant increase in crebA expression and LMC. These results suggest that Wolbachia may improve LMC in Drosophila by altering host gene expression through a miRNA-target pathway. Our findings help better understand the host-endosymbiont interactions and, in particular, the impact of Wolbachia on cognitive processes in invertebrate hosts.


Assuntos
Drosophila melanogaster/fisiologia , Drosophila simulans/fisiologia , Regulação da Expressão Gênica , MicroRNAs/genética , Wolbachia/fisiologia , Animais , Drosophila melanogaster/microbiologia , Drosophila simulans/microbiologia , Aprendizagem , Memória , MicroRNAs/metabolismo
17.
J Agric Food Chem ; 66(51): 13435-13443, 2018 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-30556692

RESUMO

The key step for the toxicity of Bacillus thuringiensis subsp. israelensis (Bti) is the interaction between toxins and putative receptors; thus, many studies focus on identification of new toxin receptors and engineering of toxins with higher affinity/specificity for receptors. In the larvae of Aedes aegypti, galectin-14 was one of the genes upregulated by Bti treatment. RNAi knockdown expression of galectin-14 and feeding recombinant galectin-14-thioredoxin fusion protein significantly affected survival of Ae. aegypti larvae treated with Bti toxins. Recombinant galectin-14 protein bound to brush border membrane vesicles (BBMVs) of Ae. aegypti larvae, ALP1 and APN2, and galectin-14 and Cry11Aa bound to BBMVs with a similarly high affinity. Competitive binding results showed that galectin-14 competed with Cry11Aa for binding to BBMVs and ALP1 to prevent effective binding of toxin to receptors. These novel findings demonstrated that midgut proteins other than receptors play an important role in modulating the toxicity of Cry toxins.


Assuntos
Aedes/metabolismo , Fosfatase Alcalina/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Galectinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/metabolismo , Aedes/química , Aedes/efeitos dos fármacos , Aedes/genética , Fosfatase Alcalina/química , Fosfatase Alcalina/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/toxicidade , Endotoxinas/química , Endotoxinas/toxicidade , Galectinas/química , Galectinas/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidade , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/química , Larva/genética , Larva/metabolismo , Ligação Proteica
18.
Insect Biochem Mol Biol ; 103: 12-21, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30321587

RESUMO

Antimicrobial peptides (AMPs) are important effector molecules of insect humoral immunity, and expression of AMPs is mainly regulated by the Toll and immune deficiency (IMD) pathways. FoxO, a key downstream regulator of the insulin-like signaling (ILS) pathway, has been recently reported to be involved in the regulation of AMPs in Drosophila melanogaster. In the present study, we investigated AMP gene expression and the regulation pathway controlled by the starvation in the silkworm Bombyx mori. We discovered that antibacterial activity in the hemolymph of B. mori larvae was increased by starvation, and expression of AMP genes (BmCecB6, BmAtta1, BmLeb3 and BmDefB) as well as the ILS target genes (FoxO, InR and Brummer) were strongly activated in the fat body by starvation. Moreover, phosphorylation of Akt kinase was reduced in the Bm-12 cells after starvation, suggesting that the ILS pathway was inhibited by starvation. We then showed that more FoxO protein was present in the cytoplasm than in the nucleus of Bm-12 cells under normal conditions, but more FoxO was detected in the nucleus after cells were starved for 8 h, indicating that FoxO was activated by starvation. In summary, our results indicated that starvation can activate AMP gene expression in B. mori via the ILS/FoxO signaling pathway.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Bombyx/genética , Bombyx/metabolismo , Proteínas de Insetos/genética , Receptor de Insulina/genética , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bombyx/crescimento & desenvolvimento , Corpo Adiposo/metabolismo , Privação de Alimentos/fisiologia , Regulação da Expressão Gênica , Hemolinfa/imunologia , Imunidade Inata/genética , Proteínas de Insetos/imunologia , Proteínas de Insetos/metabolismo , Insulina/metabolismo , Larva/genética , Larva/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
19.
Dev Comp Immunol ; 88: 94-103, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30009928

RESUMO

Lipopolysaccharide (LPS) is a common component of the outermost cell wall in Gram-negative bacteria. In mammals, LPS serves as an endotoxin that can be recognized by a receptor complex of TLR4 (Toll-like receptor 4) and MD-2 (myeloid differentiation-2) and subsequently induce a strong immune response to signal the release of tumor necrosis factor (TNF). In Drosophila melanogaster, no receptors for LPS have been identified, and LPS cannot activate immune responses. Here, we report a protein, BmEsr16, which contains an ML (MD-2-related lipid-recognition) domain, may function as an LPS receptor in the silkworm Bombyx mori. We showed that antibacterial activity in the hemolymph of B. mori larvae was induced by Escherichia coli, peptidoglycan (PGN) and LPS and that the expression of antimicrobial peptide genes was also induced by LPS. Furthermore, both the expression of BmEsr16 mRNA in the fat body and the expression of BmEsr16 protein in the hemolymph were induced by LPS. Recombinant BmEsr16 bound to LPS and lipid A, as well as to PGN, lipoteichoic acid, but not to laminarin or mannan. More importantly, LPS-induced immune responses in the hemolymph of B. mori larvae were blocked when the endogenous BmEsr16 protein was neutralized by polyclonal antibody specific to BmEsr16. Our results suggest that BmEsr16 may function as a key accessory protein for LPS signaling in B. mori.


Assuntos
Bombyx/imunologia , Imunidade Inata , Proteínas de Insetos/imunologia , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/imunologia , Animais , Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Hemolinfa/imunologia , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/imunologia , Receptores de Lipopolissacarídeos/química , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Simulação de Acoplamento Molecular , Peptidoglicano/química , Peptidoglicano/imunologia , Domínios Proteicos/imunologia , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Transdução de Sinais/imunologia
20.
Dev Comp Immunol ; 87: 137-146, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29935286

RESUMO

Antimicrobial peptides (AMPs) are produced by the stimulated humoral immune system. Most mature AMPs contain less than 50 amino acid residues. Some of them are generated from proproteins upon microbial challenges. Here, we report the antimicrobial activities of a proline-rich proprotein, named SlLebocin1 (SlLeb1), from the tobacco cutworm Spodoptera litura. SlLebocin1 cDNA contains a 477-bp open reading frame (ORF). It is mainly expressed in hemocytes and the midgut in naïve larvae. The transcript level was significantly induced in hemocytes but repressed in the midgut and fat body by bacterial challenges. The proprotein contains 158 amino acids with 3 RXXR motifs that are characteristic of some Lepidopteral lebocin proproteins. Four peptides corresponding to the predicted processed fragments were synthesized chemically, and their antimicrobial activities against two Gram-negative and two Gram-positive bacterial strains were analyzed. The peptides showed differential antimicrobial activities. For Escherichia coli and Bacillus subtilis, only the C-terminal fragment (124-158) showed strong inhibitory effects. For Staphylococcus aureus, all peptides showed partial inhibitions. None of them inhibited Serratia marcescens. Bacterial morphologies were examined by the scanning electron microscopy and confocal laser scanning microscopy. The antimicrobial peptides either disrupted cellular membrane or inhibited cell division and caused elongated/enlarged morphologies. The results may provide ideas for designing novel antibiotics.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Insetos/genética , Domínios Proteicos Ricos em Prolina/genética , Precursores de Proteínas/genética , Spodoptera/genética , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/classificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Bases , Sistema Digestório/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/ultraestrutura , Perfilação da Expressão Gênica , Hemócitos/metabolismo , Proteínas de Insetos/classificação , Proteínas de Insetos/farmacologia , Larva/genética , Microscopia Eletrônica de Varredura , Filogenia , Precursores de Proteínas/classificação , Precursores de Proteínas/farmacologia , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/ultraestrutura
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