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1.
Appl Biochem Biotechnol ; 190(3): 1060-1073, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31667755

RESUMO

Microbial proteases are widely used as commercial enzymes, which have an active role in several industrial processes. The aim of this study was to investigate the production and properties of extracellular proteases from Barrientosiimonas sp. strain V9. The cultivation conditions for protease production were studied using different carbon and nitrogen sources. Maximum protease production was obtained in medium containing 25 g L-1 sucrose, 7 g L-1 KNO3, and initial pH 7.0 at 35 °C and 150 rpm during 72 h. Under these conditions, maximum proteolytic activity reached 1200 U mL-1. The enzyme extract showed optimum activity at 60 °C, pH 9.0, and was stable from 30 to 50 °C within a pH range from 4.0 to 10.0 and NaCl concentration up to 2.5 M. The enzyme was stable in the presence of EDTA, urea, Triton X-100 and laundry detergent (sodium lauryl sulfate as main component). The addition of 1% sodium dodecyl sulfate, Tween-80, or Tween-20 increased the activity by 183% and 119% respectively, while 2-mercaptoethanol reduced the activity to 71%. Casein zymogram analysis revealed three hydrolysis zones suggesting that Barrientosiimonas sp. V9 expresses proteases with molecular weights about 60, 45, and 35 kDa, which were inhibited in the presence of phenylmethylsulfonyl fluoride. Barrientosiimonas sp. V9 produces halotolerant serine proteases with great biotechnological potential.


Assuntos
Actinobacteria/enzimologia , Extremófilos/enzimologia , Peptídeo Hidrolases/metabolismo , Meios de Cultura , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/biossíntese , Proteólise , Temperatura
2.
Microb Cell Fact ; 18(1): 215, 2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31847856

RESUMO

BACKGROUND: Serratia marcescens, a Gram-negative nosocomial pathogen secretes a 50 kDa multi-domain zinc metalloprotease called serratiopeptidase. Broad substrate specificity of serratiopeptidase makes it suitable for detergent and food processing industries The protein shows potent anti-inflammatory, anti-edemic, analgesic, antibiofilm activity and sold as an individual or fixed-dose enteric-coated tablets combined with other drugs. Although controversial, serratiopeptidase as drug is used in the treatment of chronic sinusitis, carpal tunnel syndrome, sprains, torn ligaments, and postoperative inflammation. Since the native producer of serratiopeptidase is a pathogenic microorganism, the current production methods need to be replaced by alternative approaches. Heterologous expression of serratiopeptidase in E. coli was tried before but not found suitable due to the limited yield, and other expression related issues due to its inherent proteolytic activity such as cytotoxicity, cell death, no expression, minimal expression, or inactive protein accumulation. RESULTS: Recombinant expression of mature form serratiopeptidase in E. coli seems toxic and resulted in the failure of transformation and other expression related issues. Although E. coli C43(DE3) cells, express protein correctly, the yield was compromised severely. Optimization of protein expression process parameters such as nutrient composition, induction point, inducer concentration, post-induction duration, etc., caused significant enhancement in serratiopeptidase production (57.9 ± 0.73% of total cellular protein). Expressed protein formed insoluble, enzymatically inactive inclusion bodies, and gave 40-45 mg/l homogenous (> 98% purity) biologically active and conformationally similar serratiopeptidase to the commercial counterpart upon refolding and purification. CONCLUSION: Expression of mature serratiopeptidase in E. coli C43(DE3) cells eliminated the protein expression associated with toxicity issues. Further optimization of process parameters significantly enhanced the overexpression of protein resulting in the higher yield of pure and functionally active recombinant serratiopeptidase. The biological activity and conformational features of recombinant serratiopeptidase were very similar to the commercially available counterpart suggesting it-a potential biosimilar of therapeutic and industrial relevance.


Assuntos
Medicamentos Biossimilares/metabolismo , Escherichia coli/enzimologia , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
3.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(5): 714-719, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31762243

RESUMO

OBJECTIVE: To select and identify the bacterium which highly produces protease and ß-D-glucosidase from 72 strains of Shuidouchi from Sichuan, and to provide evidence for further research on its nutritional value and fermentation strain exploiting. METHODS: Casein degradation test and pNPG chemical test were applied respectively to detect the capacity to produce protease and ß-D-glucosidase of each strain. Characteristics of morphology, biochemistry, 16S rRNA and MALDI-TOF-MS were used to identify the fermentation strain, which genetic stability, curves of growth and enzyme producing were also obtained. RESULTS: The strain with the highest enzyme activity of ß-D-glucosidase (0.084 U/L) among the top 10 strains for producing protease was selected as the fermentation strain and was identified as Bacillus subtilis, which curves of growth and enzyme producing conformed as well. The result of genetic stability showed that capacity of enzyme producing was stable until the 10th generation. CONCLUSIONS: The fermentation strain which highly produced protease and ß-D-glucosidase was selected from 72 strains of shuidouchi from Sichuan and was identified as Bacillus subtilis.


Assuntos
Bacillus subtilis/enzimologia , Alimentos e Bebidas Fermentados/microbiologia , Glucosidases/biossíntese , Peptídeo Hidrolases/biossíntese , Alimentos de Soja/microbiologia , China , Fermentação , RNA Ribossômico 16S
4.
Vet Res ; 50(1): 43, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164171

RESUMO

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Genetic analyses suggest that this pathogen has a novel protein secretion system, known as the "type IX secretion system" (T9SS). We previously reported that deletion of the AS87_RS08465 gene significantly reduced the bacterial virulence of the R. anatipestifer strain Yb2, but the mechanism remained unclear. The AS87_RS08465 gene is predicted to encode the gliding motility protein GldM (GldM) protein, a key component of the T9SS complex. In this study, Western blotting analysis demonstrated that R. anatipestifer GldM was localized to the cytomembrane. Further study revealed that the adhesion and invasion capacities of the mutant strain RA2281 (designated Yb2ΔgldM) in Vero cells and the bacterial loads in the blood of infected ducks were significantly reduced. RNA-Seq and PCR analyses showed that six genes were upregulated and five genes were downregulated in the mutant strain Yb2ΔgldM and that these genes were mainly involved in the secretion of proteins. Yb2ΔgldM was also found to be defective in gliding motility and protein secretion. Liquid chromatography-tandem mass spectrometry analysis revealed that nine of the proteins had a conserved T9SS C-terminal domain and were differentially secreted by Yb2ΔgldM compared to Yb2. The complementation strain cYb2ΔgldM recovered the adhesion and invasion capacities in Vero cells and the bacterial loads in the blood of infected ducks as well as the bacterial gliding motility and most protein secretion in the mutant strain Yb2ΔgldM to the levels of the wild-type strain Yb2. Taken together, these results indicate that R. anatipestifer GldM is associated with T9SS and is important in bacterial virulence.


Assuntos
Aderência Bacteriana/genética , Expressão Gênica , Riemerella/genética , Riemerella/patogenicidade , Sistemas de Secreção Tipo IV/genética , Mutação , Peptídeo Hidrolases/biossíntese , Riemerella/enzimologia , Sistemas de Secreção Tipo IV/metabolismo , Virulência/genética , Fatores de Virulência/genética
5.
Vet Microbiol ; 231: 93-99, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955831

RESUMO

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our previous study, we demonstrated that the deletion of the AS87_08785 gene significantly reduced the virulence of R. anatipestifer strain Yb2, but the mechanism remained unclear. In this study, R. anatipestifer strains with mutated or complemented AS87_08785 genes were constructed and characterized. A sequence analysis indicated that the AS87_08785 gene encoded a putative GldK protein, which localized to the membrane fraction in a western blotting analysis. The mutant strain Yb2ΔgldK displayed defective gliding motility on agar plates, reduced protease activity, and a reduced capacity for protein secretion. RNA sequencing and quantitative PCR analyses indicated that the transcription of 13 genes was downregulated in mutant Yb2ΔgldK. Animal experiments showed that the bacterial loads in the blood of Yb2ΔgldK-infected ducks were significantly reduced relative to those in wild-type strain Yb2 infected ducks. Most of the defective biological properties of the mutant were restored in complementation strain cYb2ΔgldK. Our results demonstrated that R. anatipestifer gene AS87_08785 encoded a component of the type IX secretion system, GldK, which functioned in bacterial gliding motility, protein secretion, and bacterial virulence.


Assuntos
Infecções por Flavobacteriaceae/veterinária , Doenças das Aves Domésticas/microbiologia , Riemerella/genética , Sistemas de Secreção Tipo IV/genética , Animais , Aderência Bacteriana , Carga Bacteriana , Patos/microbiologia , Expressão Gênica , Mutação , Peptídeo Hidrolases/biossíntese , Reação em Cadeia da Polimerase , Riemerella/enzimologia , Análise de Sequência de RNA , Sistemas de Secreção Tipo IV/metabolismo , Virulência/genética , Fatores de Virulência/genética
6.
PLoS One ; 14(4): e0215328, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30973915

RESUMO

The nitrogen (N) cycle is closely related to the stability of marine ecosystems. Microbial communities have been directly linked to marine N-cycling processes. However, systematic research on the bacterial community composition and diversity involved in N cycles in different seas is lacking. In this study, microbial diversity in the Bohai Sea (BHS), Yellow Sea (YS) and South China Sea (SCS) was surveyed by targeting the hypervariable V4 regions of the 16S rRNA gene using next-generation sequencing (NGS) technology. A total of 2,505,721 clean reads and 15,307 operational taxonomic units (OTUs) were obtained from 86 sediment samples from the three studied China seas. LEfSe analysis demonstrated that the SCS had more abundant microbial taxa than the BHS and YS. Diversity indices demonstrated that Proteobacteria and Planctomycetes were the dominant phyla in all three China seas. Canonical correspondence analysis (CCA) indicated that pH (P = 0.034) was the principal determining factors, while the organic matter content, depth and temperature had a minor correlated with the variations in sedimentary microbial community distribution. Cluster and functional analyses of microbial communities showed that chemoheterotrophic and aerobic chemoheterotrophic microorganisms widely exist in these three seas. Further research found that the cultivable protease-producing bacteria were mainly affiliated with the phyla Proteobacteria, Firmicutes and Bacteroidetes. It was very clear that Pseudoalteromonadaceae possessed the highest relative abundance in the three sea areas. The predominant protease-producing genera were Pseudoalteromonas and Bacillus. These results shed light on the differences in bacterial community composition, especially protease-producing bacteria, in these three China seas.


Assuntos
Bactérias/classificação , Bactérias/genética , Sedimentos Geológicos/microbiologia , Microbiota , Organismos Aquáticos/classificação , Organismos Aquáticos/enzimologia , Organismos Aquáticos/genética , Bactérias/enzimologia , Bacteroidetes/classificação , Bacteroidetes/enzimologia , Bacteroidetes/genética , Biodiversidade , China , DNA Bacteriano/genética , Ecossistema , Firmicutes/classificação , Firmicutes/enzimologia , Firmicutes/genética , Microbiota/genética , Oceanos e Mares , Peptídeo Hidrolases/biossíntese , Proteobactérias/classificação , Proteobactérias/enzimologia , Proteobactérias/genética , RNA Ribossômico 16S/genética , Água do Mar/microbiologia
7.
Biotechnol Appl Biochem ; 66(3): 361-368, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30694578

RESUMO

Protease, cellulase, and α-amylase producing Bacillus subtilis strain was cultivated by solid-state fermentation technique using soybean meal as a substrate. The aim of the present study was to establish a highly efficient enzymes' extraction method as a first stage in downstream processing. The conventional extraction procedure was optimized by determining pH, stirring rate, solid/liquid ratio, and time of extraction on enzymes' recoveries from fermented soybean meal. Yields of leached enzymes were compared to the amounts of enzymes that are achieved with ultrasound-assisted extraction (UAE). UAE was established to be superior method for obtaining higher yields of proteases (up to 330 IU) and α-amylases (825 IU), under significantly shorter extraction time and gaining more concentrated product. However, the obtained model predicts that conventional process led to a product with a higher cellulolytic activity (≥7.5 IU).


Assuntos
Bacillus subtilis/enzimologia , Celulase/isolamento & purificação , Fermentação , Peptídeo Hidrolases/isolamento & purificação , Soja/metabolismo , Ondas Ultrassônicas , alfa-Amilases/isolamento & purificação , Celulase/biossíntese , Celulase/metabolismo , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/metabolismo , Soja/química , alfa-Amilases/biossíntese , alfa-Amilases/metabolismo
8.
Food Chem ; 271: 606-613, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30236722

RESUMO

Soy sauce materials of soybean meal and wheat bran were evaluated in solid-state (koji) fermentation (SSF) and submerged fermentation (SmF) by Aspergillus oryzae. Proteinase production in SSF (2331 ±â€¯39 U g-1) was about 4.9 times higher than that in SmF (477 ±â€¯13 U g-1), and glycoside hydrolase was approximately 2 times higher in SSF than that in SmF. In addition, protein expression of iTRAQ analysis deepens our understanding of the secreting mechanism. Abundant proteinases (dipeptidase, dipeptidyl aminopeptidase, puromycin-sensitive aminopeptidase, Xaa-pro aminopeptidase, neutral protease 2 and leucine aminopeptidase 2), along with the glycoside hydrolase (glycoamylase, glucosidase and ß-xylanase) were secreted at the late stage of SSF, but tripeptidyl peptidase sed 2 was proposed as an indispensable protease in SmF or the early stage of SSF. Several metabolites associated with the carbon flux and amino acid biosynthesis were proved to be regulated by the proteinase and glycoside hydrolase production.


Assuntos
Aspergillus oryzae/enzimologia , Fermentação , Microbiologia de Alimentos , Peptídeo Hidrolases/biossíntese , Carbono/metabolismo , Nitrogênio/metabolismo
9.
Appl Biochem Biotechnol ; 187(1): 282-297, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29936594

RESUMO

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the São Paulo Zoo Park (SPZPF), São Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/biossíntese , Compostagem , Peptídeo Hidrolases/biossíntese , Bacillus/classificação , Bacillus/genética , Bacillus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Brasil , Peptídeo Hidrolases/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo
10.
Cancer Lett ; 440-441: 1-10, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30312729

RESUMO

Multiple myeloma remains an incurable disease, and continued efforts are required to develop novel agents and novel drug combinations with more effective anti-myeloma activity. Here, we show that the pan-PIM kinase inhibitors SGI1776 and CX6258 exhibit significant anti-myeloma activity and that combining a pan-PIM kinase inhibitor with the immunomodulatory agent lenalidomide in an in vivo myeloma xenograft mouse model resulted in synergistic myeloma cell killing without additional hematologic or hepatic toxicities. Further investigations indicated that treatment with a pan-PIM kinase inhibitor promoted increased ubiquitination and subsequent degradation of IKZF1 and IKZF3, two transcription factors crucial for survival of myeloma cells. Combining a pan-PIM kinase inhibitor with lenalidomide led to more effective degradation of IKZF1 and IKZF3 in multiple myeloma cell lines as well as xenografts of myeloma tumors. We also demonstrated that treatment with a pan-PIM kinase inhibitor resulted in increased expression of cereblon, and that knockdown of cereblon via a shRNA lentivirus abolished the effects of PIM kinase inhibition on the degradation of IKZF1 and IKZF3 and myeloma cell apoptosis, demonstrating a central role of cereblon in pan-PIM kinase inhibitor-mediated down-regulation of IKZF1 and IKZF3 and myeloma cell killing. These data elucidate the mechanism of pan-PIM kinase inhibitor mediated anti-myeloma effect and the rationale for the synergy observed with lenalidomide co-treatment, and provide justification for a clinical trial of the combination of pan-PIM kinase inhibitors and lenalidomide for the treatment of multiple myeloma.


Assuntos
Fator de Transcrição Ikaros/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Animais , Regulação para Baixo/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Lenalidomida/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/enzimologia , Peptídeo Hidrolases/biossíntese , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Piridazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Prep Biochem Biotechnol ; 48(8): 725-733, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30303449

RESUMO

The hydrolysates of soy protein and milk protein are nutritional and functional food ingredients. Aspergillus pseudoglaucus aspergillopepsin I (App) is an acidic protease, including signal peptide, propeptide, and catalytic domain. Here, we cloned the catalytic domain App with or without propeptide in Escherichia coli. The results showed that the App without propeptide was not expressed or did not exhibit activity and App with propeptide (proApp) was highly expressed with a specific activity of 903 U/mg. Moreover, the denaturation temperature of proApp was 4.1 °C higher than App's. The proApp showed 104 U/mg and 252 U/mg hydrolysis activities towards soy protein and milk protein under acidic conditions. By RP-HPLC analysis, the peptides obtained from the hydrolysates of soy protein and milk protein were hydrophilic peptides. This work first demonstrates efficient proteolysis of soy protein and milk protein through the functional expression of full-length proApp, which will likely have valuable industrial applications.


Assuntos
Aspergillus/genética , Escherichia coli/metabolismo , Proteínas Fúngicas , Proteínas do Leite/química , Peptídeo Hidrolases , Proteólise , Proteínas de Soja/química , Aspergillus/enzimologia , Escherichia coli/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
12.
Sci Rep ; 8(1): 10157, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29976981

RESUMO

The study show usefulness of rapeseed cake, rich in fats and proteins byproduct generated after oil production, which may be used as a microbial medium for lipase and protease biosynthesis. Of 26 different filamentous fungi screened by solid-state fermentation, Penicillium camemberti AM83 was found to abundantly produce lipase and protease. Various process parameters were then optimized to maximize lipase and protease secretion, including carbon and nitrogen source, C/N ratio, metal ions, temperature, moisture content, initial pH, and inoculum size. Lipase production increased approximately 11.2-fold in solid-state cultures on rapeseed cake supplemented with lactose and calcium chloride, alkalinized to pH 8, hydrated to 80%, and inoculated with 1.2 × 106 spores/mL. Similarly, protease production increased approximately 8.4-fold in optimized cultures inoculated with 3.2 × 108 spores/mL, and grown on rapeseed cake with lactose and ammonium sulfate at pH 9 and moisture content 60%. The results highlight the potential economic value of solid-state fermentation on rapeseed cake to produce industrial hydrolases.


Assuntos
Brassica rapa/metabolismo , Fermentação , Hidrolases/biossíntese , Penicillium/metabolismo , Carbono/farmacologia , Umidade , Íons , Lipase/biossíntese , Lipase/metabolismo , Metais/farmacologia , Nitrogênio/farmacologia , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/metabolismo , Temperatura
13.
Protein Expr Purif ; 152: 46-55, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30055246

RESUMO

In this study, protease Pph_Pro1 from Pseudoalteromonas phenolica, possessing extracellular proteolytic activity and salt tolerance, was investigated for cloning, expression, and purification purposes. Through optimization, it was determined that optimum soluble recombinant expression was achieved when Pph_Pro1 was co-expressed with the pTf16 vector chaperone in LB medium supplemented with CaCl2. Pph_Pro1 was purified using osmotic shock and immobilized metal-affinity chromatography (IMAC). Isolated Pph_Pro1 activity was measured as 0.44 U/mg using casein as a substrate. Interestingly, Pph_Pro1 displayed halophilic, alkaliphilic, and unexpected thermostable properties. Furthermore, it was resistant to several hydrophilic and hydrophobic organic solvents. Substrate specificity and kinetic values such as Km and Vmax were determined with casein, bovine serum albumin (BSA), and algal waste protein as substrates, indicating that the Pph_Pro1 protease enzyme had a greater affinity for casein. Based on the remarkable characteristics of this Pph_Pro1 protease enzyme, it can potentially be utilized in many biotechnological industries.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Peptídeo Hidrolases/genética , Pseudoalteromonas/enzimologia , Proteínas Recombinantes de Fusão/genética , Proteínas de Algas/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Cloreto de Cálcio/farmacologia , Caseínas/química , Cromatografia de Afinidade , Clonagem Molecular , Meios de Cultura/química , Meios de Cultura/farmacologia , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/isolamento & purificação , Proteólise , Pseudoalteromonas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Salinidade , Tolerância ao Sal/fisiologia , Soroalbumina Bovina/química , Especificidade por Substrato
14.
J Fish Dis ; 41(9): 1395-1402, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29893005

RESUMO

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)-containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha-D-mannose/alpha-D-glucose >N-acetyl-beta-D-glucosamine >N-acetyl-beta-D-glucosamine/N-acetylneuraminic acid >N-acetyl-D-galactosamine >alpha-D-galactose/N-acetyl-alpha-D-galactosamine >beta-D-galactose = alpha-L-fucose. Virulence studies demonstrated isolate AL-02-36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%-100% versus 60% for isolate ALG-00-530 at equivalent doses (~3 × 106  CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2-3 logs) and survived (28 days) in formulated water-containing catfish mucus. Highly virulent isolate AL-02-36 possessed at least 2.5- to fivefold higher protease activity following growth in mucus than the less virulent ALG-00-530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.


Assuntos
Peixes-Gato/microbiologia , Flavobacterium/enzimologia , Flavobacterium/crescimento & desenvolvimento , Muco/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Peixes-Gato/metabolismo , Meios de Cultura/química , Meios de Cultura/farmacologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/mortalidade , Infecções por Flavobacteriaceae/microbiologia , Infecções por Flavobacteriaceae/mortalidade , Flavobacterium/efeitos dos fármacos , Flavobacterium/patogenicidade , Galactose/metabolismo , Brânquias/microbiologia , Glicosilação , Lectinas/metabolismo , Muco/química , Peptídeo Hidrolases/biossíntese , Proteólise , Virulência
15.
Vet Microbiol ; 220: 47-52, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29885800

RESUMO

The yeast Malassezia pachydermatis is a component of the microbiota of dogs and cats, however it can cause otitis and seborrheic dermatitis in these animals. The objective of this study was to determine the antifungal susceptibility, and evaluate virulence and pathogenicity of 25 M. pachydermatis strains from animals. Susceptibility to ketoconazole, fluconazole, itraconazole, voriconazole, terbinafine, and amphotericin B was evaluated by broth microdilution assay. In addition, biofilm-forming ability, protease, phospholipase, hemolysin and melanin production and adhesion to epithelial cells by this yeast species were assessed. Finally, strain pathogenicity was investigated using the nematode Caenorhabditis elegans. Concerning the planktonic susceptibility, minimum inhibitory concentrations varied from <0.03 to>64 µg/mL for azole derivatives, 1 to >16 µg/mL for amphotericin B and 0.03 to 0.25 µg/mL for terbinafine. All strains were classified as strong biofilm producers, and ketoconazole, fluconazole and amphotericin B presented the best inhibitory effect against mature biofilms. All fungal isolates produced proteases, whereas 14/25 strains were positive for phospholipase production. Hemolytic activity was not observed and 18/25 strains showed dark pigmentation in the presence of L-DOPA. Regarding adhesion to epithelial cells, a low adhesion rate was observed in 10/12 evaluated strains. C. elegans mortality rate reached 95.9% after 96 h of exposure of the worms to M. pachydermatis. This yeast species produces important virulence factors and presents high pathogenicity, corroborating its clinical importance.


Assuntos
Antifúngicos/farmacologia , Dermatomicoses/veterinária , Malassezia/efeitos dos fármacos , Malassezia/patogenicidade , Animais , Aderência Bacteriana , Biofilmes/efeitos dos fármacos , Caenorhabditis elegans , Doenças do Gato/microbiologia , Gatos , Dermatomicoses/microbiologia , Doenças do Cão/microbiologia , Cães , Células Epiteliais/microbiologia , Fluconazol/farmacologia , Raposas/microbiologia , Itraconazol/farmacologia , Cetoconazol/farmacologia , Malassezia/enzimologia , Malassezia/isolamento & purificação , Testes de Sensibilidade Microbiana/métodos , Peptídeo Hidrolases/biossíntese , Fosfolipases/biossíntese , Virulência
16.
J Basic Microbiol ; 58(9): 730-738, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29938805

RESUMO

Twelve actinobacterial strains were isolated from tomato rhizospheric soil from Manipur, a state in North East Indian Himalayan Region and screened for keratinolytic and plant growth promoting traits. Nine promising isolates were identified as Streptomyces species using partial 16S rRNA gene sequencing. Among the seven isolates showing chicken feather degradation activity, three keratinolytic strains RCM-SSR-2, -6, and -12 were found to be the most efficient feather degrading strains achieving 90% feather weight loss within 48 h of incubation. They also showed maximum keratinase and soluble peptide production. Strain RCM-SSR-2, -5, -6, -8, and -11 showed positive results for all plant growth promoting traits tested. Maximum indole-3-acetic acid production was exhibited by RCM-SSR-6. Strain RCM-SSR-1, -2, -5, -6, -9, and -11 showed antagonistic activity against three important plant pathogens. Feather hydrolysate of RCM-SSR-6 was also evaluated for in vitro seed germination test using garden pea seeds. Higher concentration of feather protein hydrolysate (3 mg ml-1 ) inhibited shoot and root length of the germinating embryo. However, lower concentration (0.01 mg ml-1 ) of feather protein hydrolysate promoted seed germination. Among the 12 strains, four isolates namely RCM-SSR-1, -2, -5, and -6 were found to be promising as multi-traits plant growth promoting rhizobacteria for development of organic fertilizer, phytostimulator, and biocontrol agents.


Assuntos
Antibiose/fisiologia , Plumas/metabolismo , Lycopersicon esculentum/microbiologia , Reguladores de Crescimento de Planta/metabolismo , Microbiologia do Solo , Streptomyces/isolamento & purificação , Streptomyces/metabolismo , Animais , Biodegradação Ambiental , Agentes de Controle Biológico/farmacologia , DNA Bacteriano/genética , Fungos/efeitos dos fármacos , Germinação , Índia , Ácidos Indolacéticos/metabolismo , Queratinas/metabolismo , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/metabolismo , Fosfatos/metabolismo , Reguladores de Crescimento de Planta/biossíntese , RNA Ribossômico 16S/genética , Rizosfera , Sideróforos/metabolismo , Streptomyces/classificação , Streptomyces/genética
17.
Eur Rev Med Pharmacol Sci ; 22(9): 2778-2786, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29771430

RESUMO

OBJECTIVE: To investigate whether SENP3 protects H9C2 cells from apoptosis triggered by H/R through the signal transducer and activator of transcription 3 (STAT3) pathway. MATERIALS AND METHODS: Male C57BL mice were cultured and mouse models of myocardial I/RI were established. At the same time, cardiomyoblast H9C2 cell line of rat embryo was cultured. Reactive oxygen species (ROS) level was detected during H/R using 2',7'-dichlorofluorescein diacetate (DCFH) kit. Apoptotic cells were checked by flow cytometry. The expressions of p-JAK2, JAK2, STAT3, p-STAT3, cleaved-caspase3 (c-caspase3), and Bcl/Bax were detected using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: We revealed that SENP3 rose in mice of I/R group and in H9C2 cells following H/R with an increase in p-STAT3. Furthermore, increased expression of SENP3 was found to be dependent on the generation of ROS, as the SENP3 accumulation was inhibited by antioxidant (NAC). Inhibition of SENP3 suppressed the p-STAT3 expression, but promoted cell apoptosis, c-caspase3 expression, and Bcl/Bax ratio. Besides, SENP3 overexpression alleviated the cell apoptosis, which was abrogated by AG490. CONCLUSIONS: SENP3 could protect H9C2 against H/R through enhancing JAK2/STAT3 pathway.


Assuntos
Apoptose/fisiologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Peptídeo Hidrolases/biossíntese , Fator de Transcrição STAT3/biossíntese , Transdução de Sinais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologia
18.
World J Microbiol Biotechnol ; 34(5): 68, 2018 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-29752585

RESUMO

Vibrio parahaemolyticus, a Gram-negative bacterium, inhabits marine and estuarine environments and it is a major pathogen responsible globally for most cases of seafood-associated gastroenteritis in humans and acute hepatopancreatic necrosis syndrome in shrimps. There has been a dramatic worldwide increase in V. parahaemolyticus infections over the last two decades. The pathogenicity of V. parahaemolyticus has been linked to the expression of different kinds of virulence factors including extracellular proteases, such as metalloproteases and serine proteases. V. parahaemolyticus expresses the metalloproteases; PrtV, VppC, VPM and the serine proteases; VPP1/Protease A, VpSP37, PrtA. Extracellular proteases have been identified as potential virulence factors which directly digest many kinds of host proteins or indirectly are involved in the processing of other toxic protein factors. This review summarizes findings on the metalloproteases and serine proteases produced by V. parahaemolyticus and their roles in infections. Identifying the role of V. parahaemolyticus virulence-associated extracellular proteases deepens our understanding of diseases caused by this bacterium.


Assuntos
Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/classificação , Vibrio parahaemolyticus/enzimologia , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Metaloproteases/biossíntese , Metaloproteases/genética , Metaloproteases/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Alimentos Marinhos/microbiologia , Serina Proteases/biossíntese , Serina Proteases/genética , Serina Proteases/metabolismo , Vibrioses/microbiologia , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/patogenicidade , Virulência , Fatores de Virulência/genética
19.
J Basic Microbiol ; 58(6): 492-500, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29566274

RESUMO

In the present study, Serratia marcescens EGD-HP20 strain was demonstrated to utilize poultry waste comprising of both white non-melanized and dark/brown melanized poultry feathers. The potential of the isolate to hydrolyze diverse keratinous wastes was further corroborated by comparative genomics which indicated the presence of genes for broad substrate specific proteases including metallo-proteases, serine endoprotease, dipeptidase, oligopeptidase, etc. Multiple gene sequence alignments of above genes showed 99-100% sequence identities with that of closely related strains of S. marcescens. The secondary structure, 3D structures and energy models suggested the stable nature of all the observed enzymes. Comparative genomics and hydrolysis of mixed feather waste indicated that the above potential of the isolate was associated with synergistic action of various types of proteases.


Assuntos
Queratinas/metabolismo , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/genética , Serratia marcescens/enzimologia , Serratia marcescens/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Plumas/metabolismo , Genes Bacterianos/genética , Genoma Bacteriano , Hibridização In Situ , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/classificação , Aves Domésticas , Conformação Proteica , RNA Ribossômico 16S/genética , Alinhamento de Sequência , Serratia marcescens/isolamento & purificação , Especificidade por Substrato , Resíduos , Sequenciamento Completo do Genoma
20.
Mol Oral Microbiol ; 33(3): 240-248, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29498485

RESUMO

Porphyromonas gingivalis and Tannerella forsythia secrete proteases, gingipains and KLIKK-proteases. In addition, T. forsythia produces a serpin (miropin) with broad inhibitory spectrum. The aim of this pilot study was to determine the level of expression of miropin and individual proteases in vivo in periodontal and peri-implant health and disease conditions. Biofilm and gingival crevicular fluid (GCF)/ peri-implant sulcular fluid (PISF) samples were taken from healthy tooth and implant sites (n = 10), gingivitis and mucositis sites (n = 12), and periodontitis and peri-implantitis sites (n = 10). Concentration of interleukin-8 (IL-8), IL-1ß and IL-10 in GCF was determined by enzyme-linked immunosorbent assay. Loads of P. gingivalis and T. forsythia and the presence of proteases and miropin genes were assessed in biofilm by quantitative PCR, whereas gene expression was estimated by quantitative RT-PCR. The presence of P. gingivalis and T. forsythia, as well as the level of IL-8 and IL-1ß, were associated with disease severity in the periodontal and peri-implant tissues. In biofilm samples harboring T. forsythia, genes encoding proteases were found to be present at 72.4% for karilysin and 100% for other KLIKK-protease genes and miropin. At the same time, detectable mRNA expression of individual genes ranged from 20.7% to 58.6% of samples (for forsylisin and miropsin-1, respectively). In comparison with the T. forsythia proteases, miropin and the gingipains were highly expressed. The level of expression of gingipains was associated with those of miropin and certain T. forsythia proteases around teeth but not implants. Cumulatively, KLIKK-proteases and especially miropin, might play a role in pathogenesis of both periodontal and peri-implant diseases.


Assuntos
Peptídeo Hidrolases/biossíntese , Peri-Implantite/metabolismo , Periodontite/metabolismo , Porphyromonas gingivalis/enzimologia , Inibidores de Proteases/metabolismo , Serpinas/biossíntese , Tannerella forsythia/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Biofilmes , Biomarcadores , Implantes Dentários/microbiologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Líquido do Sulco Gengival/química , Gengivite/metabolismo , Gengivite/microbiologia , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Mucosite/metabolismo , Mucosite/microbiologia , Peptídeo Hidrolases/genética , Peri-Implantite/microbiologia , Periodontite/microbiologia , Projetos Piloto , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/patogenicidade , RNA Mensageiro/metabolismo , Serpinas/genética , Suécia , Tannerella forsythia/genética , Tannerella forsythia/patogenicidade
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