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1.
Clin Immunol ; 266: 110309, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-39002795

RESUMO

Psoriasis is a common inflammatory systemic disease characterized by pro-inflammatory macrophages activation (M1 macrophage) infiltrated in the dermal layer. How M1 macrophage contributes to psoriasis remains unknown. In this study, we found that adenosine A2A receptor (A2AR) agonist CGS 21680 HCl alleviated the imiquimod (IMQ) and mouse IL-23 Protein (rmIL-23)-induced psoriasis inflammation through reducing infiltration of M1. Conversely, Adora2a deletion in mice exacerbated psoriasis-like phenotype. Mechanistically, A2AR activation inhibited M1 macrophage activation via the NF-κB-KRT16 pathway to reduce the secretion of CXCL10/11 and inhibit Th1/17 differentiation. Notably, the KRT16 expression was first found in M1 macrophage in our study, not only in keratinocytes (KCs). CXCL10/11 are first identified as primarily derived from macrophages and dendritic cells (DCs) rather than KCs in psoriasis using single cell RNA sequencing (scRNA-Seq). In total, the study emphasizes the importance of M1 as an innate immune cell in pathogenesis of psoriasis.

2.
World J Microbiol Biotechnol ; 40(2): 58, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38165488

RESUMO

Bacillus biocontrol agent(s) BCA(s) such as Bacillus cereus, Bacillus thuringiensis and Bacillus subtilis have been widely applied to control insects' pests of plants and pathogenic microbes, improve plant growth, and facilitate their resistance to environmental stresses. In the last decade, researchers have shown that, the application of Bacillus biocontrol agent(s) BCA(s) optimized agricultural production yield, and reduced disease risks in some crops. However, these bacteria encountered various abiotic stresses, among which ultraviolet (UV) radiation severely decrease their efficiency. Researchers have identified several strategies by which Bacillus biocontrol agents resist the negative effects of UV radiation, including transcriptional response, UV mutagenesis, biochemical and artificial means (addition of protective agents). These strategies are governed by distinct pathways, triggered by UV radiation. Herein, the impact of UV radiation on Bacillus biocontrol agent(s) BCA(s) and their mechanisms of resistance were discussed.


Assuntos
Bacillus thuringiensis , Bacillus , Raios Ultravioleta , Bacillus cereus , Bacillus subtilis
3.
Environ Sci Technol ; 57(9): 3980-3989, 2023 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-36808949

RESUMO

Nanopesticides are considered to be a promising alternative strategy for enhancing bioactivity and delaying the development of pathogen resistance to pesticides. Here, a new type of nanosilica fungicide was proposed and demonstrated to control late blight by inducing intracellular peroxidation damage to Phytophthora infestans, the pathogen associated with potato late blight. Results indicated that the structural features of different silica nanoparticles were largely responsible for their antimicrobial activities. Mesoporous silica nanoparticles (MSNs) exhibited the highest antimicrobial activity with a 98.02% inhibition rate of P. infestans, causing oxidative stress responses and cell structure damage in P. infestans. For the first time, MSNs were found to selectively induce spontaneous excess production of intracellular reactive oxygen species in pathogenic cells, including hydroxyl radicals (•OH), superoxide radicals (•O2-), and singlet oxygen (1O2), leading to peroxidation damage in P. infestans. The effectiveness of MSNs was further tested in the pot experiments as well as leaf and tuber infection, and successful control of potato late blight was achieved with high plant compatibility and safety. This work provides new insights into the antimicrobial mechanism of nanosilica and highlights the use of nanoparticles for controlling late blight with green and highly efficient nanofungicides.


Assuntos
Fungicidas Industriais , Phytophthora infestans , Solanum tuberosum , Phytophthora infestans/fisiologia , Fungicidas Industriais/farmacologia , Doenças das Plantas/prevenção & controle
4.
Pestic Biochem Physiol ; 193: 105429, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37248007

RESUMO

Bacillus thuringiensis (Bt) is a popular and environment-friendly biopesticide. However, similar to other microbial pesticides, Bt is limited by ultraviolet (UV) radiation during its application, which greatly reduces its toxicity and persistence. To further know the mechanism of Bt against UV radiation, metabolomic profiles between Bt LLP29 and its UV-resistant mutant LLP29-M19 were compared, analyzed, and annotated in this study, and then a total of 61 metabolites with different abundances were detected. With P < 0.05 as the standard, a total of 12 metabolic pathways were enriched, including the TCA cycle. According to the result of RT-qPCR, the expression levels of the TCA cycle key genes in Bt LL29-M19, such as icd1 citZ, citB, sdhA, sdhB, sdhC, fumA, and mdh, were found down-regulated for 85.58%, 37.02%, 70.87%, 85.97%, 76.33%, 83.15%, 87.28%, and 35.77% than those in Bt LLP29. It was consistent with the down-regulation trend of the TCA cycle key enzymes activity in Bt LLP29-M19. Consistently, the enzyme activities of ICDH, SDH, and PDH in LLP29-M19 were detected 86.28%, 43.93%, and 83.03% lower than those in Bt LLP29. It was revealed that the reduced TCA cycle was required for Bt UV radiation resistance, which was also demonstrated by the addition of inhibitors furfural and malonic acid, respectively. Based on the result of RT-qPCR, the gene transcription levels of the main reactive oxygen species (ROS) generation pathways were down-regulated, such as EMP, however, the activity of the main degrading enzymes was up-regulated, which showed the reduction of ROS generation rate was a way for the TCA cycle to regulate the anti-ultraviolet resistance of Bt. All of these provide solid evidence for reprogramming metabolomics to strengthen Bt UV radiation resistance.


Assuntos
Bacillus thuringiensis , Praguicidas , Bacillus thuringiensis/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Praguicidas/metabolismo , Endotoxinas/metabolismo , Proteínas de Bactérias/metabolismo
5.
Int J Mol Sci ; 24(6)2023 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-36982883

RESUMO

Bacillus thuringiensis (Bt) is a widely used microbial pesticide. However, its duration of effectiveness is greatly shortened due to the irradiation of ultraviolet rays, which seriously hinders the application of Bt preparations. Therefore, it is of great importance to study the resistance mechanism of Bt to UV at the molecular level to improve the UV-resistance of Bt strains. In order to know the functional genes in the UV resistance, the genome of UV-induced mutant Bt LLP29-M19 was re-sequenced and compared with the original strain Bt LLP29. It was shown that there were 1318 SNPs, 31 InDels, and 206 SV between the mutant strain and the original strain Bt LLP29 after UV irradiation, which were then analyzed for gene annotation. Additionally, a mutated gene named yqhH, a member of helicase superfamily II, was detected as an important candidate. Then, yqhH was expressed and purified successfully. Through the result of the enzymatic activity in vitro, yqhH was found to have ATP hydrolase and helicase activities. In order to further verify its function, the yqhH gene was knocked out and complemented by homologous recombinant gene knockout technology. The survival rate of the knockout mutant strain Bt LLP29-ΔyqhH was significantly lower than that of the original strain Bt LLP29 and the back-complemented strain Bt LLP29-ΔyqhH-R after treated with UV. Meanwhile, the total helicase activity was not significantly different on whether Bt carried yqhH or not. All of these greatly enrich important molecular mechanisms of Bt when it is in UV stress.


Assuntos
Bacillus thuringiensis , Praguicidas , Bacillus thuringiensis/genética , Raios Ultravioleta , Controle Biológico de Vetores , Análise de Sequência de DNA , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia
6.
Molecules ; 28(17)2023 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-37687108

RESUMO

On the basis of the three-component synthetic methodology developed by us, a total of twenty-six pyrazole compounds bearing aryl OCF3 were designed and synthesized. Their chemical structures were characterized by 1H and 13C nuclear magnetic resonance and high-resolution mass spectrometry. These compounds were evaluated systematically for antifungal activities in vitro against six plant pathogenic fungi by the mycelium growth rate method. Most of the compounds showed some activity against each of the fungi at 100 µg/mL. Compounds 1t and 1v exhibited higher activity against all the tested fungi, and 1v displayed the highest activity against F. graminearum with an EC50 value of 0.0530 µM, which was comparable with commercial pyraclostrobin. Structure-activity relationship analysis showed that, with respect to the R1 substituent, the straight chain or cycloalkyl ring moiety was a key structural moiety for the activity, and the R2 substituent on the pyrazole ring could have significant effects on the activity. Simple and readily available pyrazoles with potent antifungal activity were obtained, which are ready for further elaboration to serve as a pharmacophore in new potential antifungal agents.


Assuntos
Antifúngicos , Pirazóis , Antifúngicos/farmacologia , Pirazóis/farmacologia , Espectrometria de Massas , Micélio
7.
Cell Biol Int ; 46(10): 1682-1692, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35880837

RESUMO

Hepatocellular carcinoma (HCC) is a common liver cancer that accounts for 90% of cases. Doxorubicin exhibits a broad spectrum of antitumor activity and is one of the most active agents in HCC. WW domain-containing protein 2 (WWP2) is highly expressed in HCC tissues and activates protein kinase B (AKT) signaling pathway to enhance tumor metastasis. However, the role of WWP2 in the glycolysis and antitumor effects of doxorubicin and the epigenetic alterations of WWP2 in HCC remain to be elucidated. The levels of WWP2 and N6-methyladenosine methyltransferase-like 3 (METTL3) in clinical samples and cells were investigated. WWP2 were silenced or overexpressed to study the role of WWP2 in regulating cell proliferation, colony formation, and glycolysis. RNA immunoprecipitation was performed to test m6 A levels. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blot were used to measure mRNA and protein, respectively. WWP2 silencing inhibits cell proliferation, colony formation, and glycolysis, while WWP2 overexpression has the inverse effects via the AKT signaling pathway. Silencing WWP2 enhances doxorubicin's antitumor effect, while WWP2 overexpression suppresses doxorubicin's antitumor effect. Data also support that METTL3 mediates WWP2 m6A modification, and m6A reader, IGF2BP2, binds to the methylated WWP2 to promote the stability of WWP2, leading to upregulation of WWP2. METTL3 mediates WWP2 m6A modification, which can be recognized and bound by IGF2BP2 to increase the stability of WWP2, leading to WWP2 overexpression which inhibits the antitumor effects of doxorubicin through METTL3/WWP2/AKT/glycolysis axis.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Ubiquitina-Proteína Ligases , Carcinoma Hepatocelular/tratamento farmacológico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Humanos , Neoplasias Hepáticas/metabolismo , Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA , Ubiquitina-Proteína Ligases/metabolismo
8.
J Nerv Ment Dis ; 210(10): 754-759, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35849536

RESUMO

ABSTRACT: Virtual reality therapy (VRT) is a new psychotherapeutic approach integrating virtual reality technology and psychotherapy. This case series aimed to study effectiveness of VRT in treating psychological problems. We described four cases of first-line health care professionals with emerging clinically significant early psychological problems during the COVID-19 outbreak, and specifically received the VRT treatment. We compared the Patient Health Questionnaire 9 items (PHQ-9), Generalized Anxiety Disorder-7 (GAD-7), PHQ-15, and Athens Insomnia Scale to evaluate psychological symptoms and sleep quality before and after sessions. All four cases showed a reduction in scale comparison. General scores of the PHQ-9 reduced 65%, GAD-7 reduced 52.17%, PHQ-15 decreased 38.17%, and scores of the Athens Insomnia Scale reduced 67.44%. Meanwhile, a reduction in depression, anxiety, psychosomatic, and sleeping symptoms was also found, which decreased 76.92% in general. These results are highly significant statistically. This case series demonstrated the effectiveness of VRT on psychological problems as a promising approach to apply on various psychological distress and disorders.


Assuntos
COVID-19 , Distúrbios do Início e da Manutenção do Sono , Realidade Virtual , Ansiedade/psicologia , Depressão/psicologia , Pessoal de Saúde/psicologia , Humanos , Pandemias , Distúrbios do Início e da Manutenção do Sono/epidemiologia , Distúrbios do Início e da Manutenção do Sono/terapia
9.
Mar Drugs ; 20(8)2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35892947

RESUMO

The applications of alginate lyase are diverse, but efficient commercial enzymes are still unavailable. In this study, a novel alginate lyase with high activity was obtained from the marine bacteria Vibrio sp. Ni1. The ORF of the algB gene has 1824 bp, encoding 607 amino acids. Homology analysis shows that AlgB belongs to the PL7 family. There are two catalytic domains with the typical region of QIH found in AlgB. The purified recombinant enzyme of AlgB shows highest activity at 35 °C, pH 8.0, and 50 mmol/L Tris-HCl without any metal ions. Only K+ slightly enhances the activity, while Fe2+ and Cu2+ strongly inhibit the activity. The AlgB preferred polyM as substrate. The end products of enzymatic mixture are DP2 and DP3, without any metal ion to assist them. This enzyme has good industrial application prospects.


Assuntos
Polissacarídeo-Liases , Vibrio , Alginatos/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Concentração de Íons de Hidrogênio , Íons , Metais/farmacologia , Polissacarídeo-Liases/metabolismo , Especificidade por Substrato , Vibrio/metabolismo
10.
BMC Genomics ; 22(1): 271, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33858340

RESUMO

BACKGROUND: Infestation by tea green leafhoppers (Empoasca (Matsumurasca) onukii) can cause a series of biochemical changes in tea leaves. As a typical cell-rupture feeder, E. onukii secretes proteases while using its stylet to probe the tender shoots of tea plants (Camellia sinensis). This study identified and analyzed proteases expressed specifically in the salivary gland (SG) and gut of E. onukii through enzymatic activity assays complemented with an integrated analysis of transcriptomic and proteomic data. RESULTS: In total, 129 contigs representing seven types of putative proteases were identified. Transcript abundance of digestive proteases and enzymatic activity assays showed that cathepsin B-like protease, cathepsin L-like protease, and serine proteases (trypsin- and chymotrypsin-like protease) were highly abundant in the gut but moderately abundant in the SG. The abundance pattern of digestive proteases in the SG and gut of E. onukii differed from that of other hemipterans, including Nilaparvata lugens, Laodelphax striatellus, Acyrthosiphum pisum, Halyomorpha halys and Nephotettix cincticeps. Phylogenetic analysis showed that aminopeptidase N-like proteins and serine proteases abundant in the SG or gut of hemipterans formed two distinct clusters. CONCLUSIONS: Altogether, this study provides insightful information on the digestive system of E. onukii. Compared to five other hemipteran species, we observed different patterns of proteases abundant in the SG and gut of E. onukii. These results will be beneficial in understanding the interaction between tea plants and E. onukii.


Assuntos
Microbioma Gastrointestinal , Hemípteros , Animais , Hemípteros/genética , Peptídeo Hidrolases/genética , Filogenia , Proteômica , Glândulas Salivares , Transcriptoma
11.
Opt Express ; 28(12): 17307-17319, 2020 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-32679941

RESUMO

Polymer-templated nematic liquid crystal (LC) holographic gratings via visible-light recording are presented in the presence of reactive mesogens (RMs) and rose bengal (RB)/N-phenylglycine (NPG) photoinitiation systems. By optimizing the concentration of RMs in the polymer-templated LC gratings, the template after being washed out can be refilled with suitable fluidic components. And the dependence of the first-order diffraction efficiency (DE) on the concentration of RB and NPG molecules was discussed in detail. The polarization-dependency of diffraction properties was also investigated. It is revealed that the diffractive behaviors of polymer-templated LC gratings can be dynamically reconfigured by varying temperature or refilling organic solutions with different refractive index (RI). Furthermore, the potential for recording holograms using green light is explored. We expect that the reconfigurable polymer-templated LC gratings fabricated via visible-light interference would provide a facile approach to regulate the diffraction properties of holographic gratings apart from electric field, thus paving a way towards a class of novel anti-counterfeiting devices.

12.
Arch Microbiol ; 202(6): 1275-1284, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32185410

RESUMO

Insect and mite pests are damaging stressors that are threatening the cultivation of tea plants, which result in enormous crop loss. Over the years, the effectiveness of synthetic pesticides has allowed for its prominent application as a control strategy. However, the adverse effects of synthetic pesticides in terms of pesticide residue, environmental contamination and insect pest resistance have necessitated the need for alternative strategies. Meanwhile, microbial pesticides have been applied to tackle the damaging activities of the insect and mite pests of tea plants, and their performances were scientifically adjudged appreciable and environmental friendly. Herein, entomopathogenic microbes that were effective against tea geometrid (Ectropis obliqua Prout), tea green leafhopper (Empoasca onukii Matsuda), paraguay tea ampul (Gyropsylla spegazziniana), tea mosquito bug (Helopeltis theivora Waterhouse) and red spider mite (Oligonychus coffea Nietner) have been reviewed. The current findings revealed that microbial pesticides were effective and showed promising performances against these pests. Overall, this review has provided the basic and integrative information on the integrated pest management (IPM) tool(s) that can be utilized towards successful control of the aforementioned insect and mite pests.


Assuntos
Camellia sinensis/parasitologia , Insetos , Ácaros , Controle Biológico de Vetores/normas , Animais , Insetos/microbiologia , Insetos/virologia , Praguicidas/normas
13.
Arch Microbiol ; 202(7): 1693-1700, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32296870

RESUMO

Bacillus thuringiensis (Bt) is efficient, strongly specific, and avirulent to humans, making it one of the most popular biopesticides in the world. Bt LLP29 is a mosquitocidal strain that was first isolated from Magnolia denudata. To understand its molecular mechanism against mosquitoes, the genome of Bt LLP29 was sequenced and annotated in this study. The LLP29 genome was found to have a total length of 5.99 Mb, with an average G + C content of 35.21%. A total of 6107 coding sequences were also detected, together with 42 rRNAs and 124 tRNAs and 135 other RNAs. With the help of annotation databases, including GO, COG, KEGG, Nr and Swiss-Prot, most unigene functions were identified. At the same time, a collinear analysis was performed on the genome of LLP29. There were also some virulence genes detected, including cry, chitinase, zwittermicin and vip.


Assuntos
Bacillus thuringiensis/genética , Genoma Bacteriano/genética , Sequenciamento Completo do Genoma , Proteínas de Bactérias/genética , Anotação de Sequência Molecular , Análise de Sequência , Fatores de Virulência/genética
14.
Arch Microbiol ; 202(4): 755-763, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31807807

RESUMO

Thuricin 4AJ1, produced by Bacillus thuringiensis strain 4AJ1, showed inhibition activity against Bacillus cereus 0938 and ATCC 10987. It began to appear in the stationary phase and reached its maximum activity level of 209.958 U at 18 h against B. cereus 0938 and 285.689 U at 24 h against B. cereus ATCC 10987. Tricine-SDS-PAGE results showed that the partly purified thuricin 4AJ1 was about 6.5 kDa. The molecular weights of the known B. thuringiensis bacteriocins and the ones obtained by the two mainstream websites for predicting bacteriocins were inconsistent with the size of the thuricin 4AJ1, indicating that the bacteriocin obtained in this study may have a novel structure. Based on the biochemical properties, the thuricin 4AJ1 activities increased after treatment with proteinase K and lipase II, and were not affected by a-amylase, catalase, α-chymotrypsin VII and α-chymotrypsin II. It was heat tolerant, being active up to 90º C. In the pH 3-10 range, it maintained most of its activity. Finally, the sensitivity of the strain 4AJ1 to commonly used antibiotics was tested. In view of its stability and antibacterial activity, thuricin 4AJ1 may be applied as a food biopreservative.


Assuntos
Bacillus thuringiensis/metabolismo , Bacteriocinas/isolamento & purificação , Antibacterianos/farmacologia , Bacillus cereus/efeitos dos fármacos , Bacillus thuringiensis/química , Bacteriocinas/química , Bacteriocinas/farmacologia , Eletroforese em Gel de Poliacrilamida , Microbiologia de Alimentos , Peso Molecular
15.
Pestic Biochem Physiol ; 167: 104599, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32527443

RESUMO

As an ATP-dependent DNA helicase, RecG can repair DNA replication forks in many organisms. However, knowledge of recG in Bacillus thuringiensis (Bt) is limited. In our previous study, recG was found damaged in Bt LLP29-M19, which was more resistant to ultraviolet light (UV) after exposing Bt LLP29 to UV for 19 generations. To further understand the function of recG in the mechanism of Bt UV resistance, recG was knocked out and recovered with homologous recombination technology in Bt LLP29. Comparing the resistance of the different mutants to UVB, Bt ∆recG-LLP29 lacking recG was found more sensitive to UVB, hydroxyurea (HU) and H2O2 than LLP29 and the complementation strain. To compare the expression level of recG in the Bt strains under different UV treatments, Quantitative Real-time PCR (RT-qPCR) of recG was performed in the tested Bt strains, which showed that the expression level of recG in Bt ∆recG-LLP29 was substantially lower than that in the original strain and complementation strain. Interestingly, when exposed to UV for 20 min, RecG expression in both Bt LLP29 and Bt recG-R was the highest. The unwinding activity of recG in Bt LLP29 and the complementation strain were also found higher than that of the recG knockout strain, Bt ∆recG-LLP29. These results demonstrate that recG is involved with the resistance of Bt to UV. These findings not only enhance the understanding of the Bt UV resistance mechanism, but also provide an important theoretical basis for the application of Bt.


Assuntos
Bacillus thuringiensis , Praguicidas , Proteínas de Bactérias , Peróxido de Hidrogênio , Raios Ultravioleta
16.
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
17.
World J Microbiol Biotechnol ; 35(2): 23, 2019 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-30666554

RESUMO

A novel Bacillus thuringiensis (Bt) bacteriocin BtCspB, active against a food-borne pathogen Bacillus cereus, was identified and purified by a traditional four-step chromatographic process with low yield (44.5 µg/L) in our lab previously. The aim of this study was to dramatically increase its yield by heterologous expression of BtCspB. The BtCspB gene from Bt BRC-ZYR2 was successfully heterologously expressed in Escherichia coli BL21 (DE3). Affinity chromatography was used to obtain the pure BtCspB up to 20 mg/L. The purified BtCspB showed a MIC value of 12.5 µg/mL and a MBC value of 50.0 µg/mL against Bacillus cereus ATCC 10987. The bacteriocin activity of BtCspB against B. cereus ATCC 10987 was further directly detected in a gel-overlay assay. The anti-B. cereus activity, however, was lower than the bacteriocin purified by the traditional four-step chromatographic process probably because of structural modifications. Compared with the traditional method, the yield of the bacteriocin by heterologous expression increased by 449 times, and the purification step was dramatically simplified, which laying a foundation for the industrial production of this novel cold-shock protein-like bacteriocin BtCspB active against B. cereus.


Assuntos
Bacillus cereus/efeitos dos fármacos , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/farmacologia , Escherichia coli/crescimento & desenvolvimento , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Bacteriocinas/farmacologia , Escherichia coli/genética , Testes de Sensibilidade Microbiana
18.
Indian J Microbiol ; 59(1): 64-72, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30728632

RESUMO

Bacterial strains from karst landform soil were enriched via chemostat culture in the presence of sodium bicarbonate. Two chemolithotrophic strains were isolated and identified as Serratia marcescens Wy064 and Bacillus sp. Wy065. Both strains could grow using sodium bicarbonate as the sole carbon source. Furthermore, the supplement of the medium with three electron donors (Na2S, NaNO2, and Na2S2O3) improved the growth of both strains. The activities of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) could be detected in the crude enzyme of strain Wy064, implying that the strain Wy064 might employ Calvin cycle to fix CO2. S. marcescens genome mining revealed four potential CA genes designated CA1-CA4. The proteins encoded by genes CA1-3 were cloned and expressed in Escherichia coli. The purified recombinant enzymes of CA1 and CA3 exhibited CO2 hydration activities, whereas enzyme CA2 was expressed in inclusion bodies. A CO2 hydration assay demonstrated that the specific activity of CA3 was significantly higher than that of CA1. The maximum CO2 hydration activities for CA1 and CA3 were observed at pH 7.5 and 40 °C. The activities of CA1 and CA3 were significantly enhanced by several metal ions, especially Zn2+, which resulted in 21.1-fold and 26.1-fold increases of CO2 hydration activities, respectively. The apparent K m and V max for CO2 as substrate were 27 mM and 179 WAU/mg for CA1, and 14 mM and 247 WAU/mg for CA3, respectively. Structure modeling combined with sequence analysis indicated that CA1 and CA3 should belong to the Type II ß-CA.

19.
Phytopathology ; 108(1): 44-51, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28945518

RESUMO

The nematicidal activity of 92 Bacillus thuringiensis strains against the pine wood nematode Bursaphelenchus xylophilus, one of the world's top 10 plant-parasitic nematodes, was determined. The insecticidal crystal proteins (ICPs) from Bacillus thuringiensis BRC-XQ12 were the most toxic to Bursaphelenchus xylophilus, with a lethal concentration 50 (LC50) of 32.13 µg/ml. Because the ICPs expressed by Bacillus thuringiensis BRC-XQ12 were closest to Cry1Ea6 and B. thuringiensis BRC-XQ12 contained four kinds of cry1 subgenes (cry1Aa, cry1Cb, cry1Ea, and cry1Ia), Cry1Ea was most likely to be the key active component against the nematode. The 3,516-bp cry1Ea11 gene from BRC-XQ12, as designated by the B. thuringiensis δ-endotoxin nomenclature committee, was expressed in Escherichia coli. Purified Cry1Ea11 showed an LC50 of 32.53 and 23.23 µg/ml at 24 and 48 h, with corresponding virulence equations of Y = 32.15X + 1.38 (R2 = 0.9951) and Y = 34.29X + 3.16 (R2 = 0.9792), respectively. In order to detect the pathway of B. thuringiensis Cry1Ea11 into Bursaphelenchus xylophilus, the nematode was fed with NHS-rhodamine-labeled GST-Cry1Ea11. The results of confocal laser-scanning microscopy showed that the 159-kDa GST-Cry1Ea11 could be detected in the stylet and the esophageal lumen of the pine wood nematode, indicating that GST-Cry1Ea11 could enter into the nematode through the stylet. As far as we know, no Cry1 proteins have been shown to have activity against plant-parasitic nematodes before. These results demonstrate that Cry1Ea11 is a promising nematicidal protein for controlling pine wilt disease rendered by B. xylophilus, further dramatically broadening the spectrum of Bacillus thuringiensis ICPs.


Assuntos
Antinematódeos/farmacologia , Bacillus thuringiensis/química , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Nematoides/efeitos dos fármacos , Pinus/parasitologia , Doenças das Plantas/prevenção & controle , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/fisiologia , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Endotoxinas/genética , Endotoxinas/isolamento & purificação , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/isolamento & purificação , Nematoides/microbiologia , Nematoides/patogenicidade , Controle Biológico de Vetores , Doenças das Plantas/parasitologia , Virulência
20.
Genomics ; 109(1): 58-66, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27867104

RESUMO

Tea production has been significantly impacted by the false-eye leafhopper, Empoasca vitis (Göthe), around Asia. To identify the key genes which are responsible for nutrition absorption, xenobiotic metabolism and immune response, the transcriptome of either alimentary tracts or bodies minus alimentary tract of E. vitis was sequenced and analyzed. Over 31 million reads were obtained from Illumina sequencing. De novo sequence assembly resulted in 52,182 unigenes with a mean size of 848nt. The assembled unigenes were then annotated using various databases. Transcripts of at least 566 digestion-, 224 detoxification-, and 288 immune-related putative genes in E. vitis were identified. In addition, relative expression of highly abundant transcripts was verified through quantitative real-time PCR. Results from this investigation provide genomic information about E. vitis, which will be helpful in further study of E. vitis biology and in the development of novel strategies to control this devastating pest.


Assuntos
Digestão/genética , Hemípteros/genética , Sistema Imunitário , Inativação Metabólica/genética , Transcriptoma , Animais , Hemípteros/imunologia , Hemípteros/metabolismo , Hemípteros/fisiologia , Ninfa/genética
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