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1.
Methods Mol Biol ; 2498: 413-424, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35727561

RESUMO

The bifunctional enzyme acylpeptide hydrolase (APEH) is involved in important metabolic processes both as an exopeptidase and as an endopeptidase. Hence, the growing interest in the study of this protein and the need to set up in vitro assays for its characterization. This chapter describes two in vitro assays able to detect the activities of APEH, one for the exopeptidase activity and one for the endopeptidase activity. In particular, these assays have been set up on the two APEH isoforms from Antarctic fish, characterized by a distinct functionality and marked exo- and endopeptidase activities.


Assuntos
Peixes , Peptídeo Hidrolases , Animais , Regiões Antárticas , Endopeptidases/metabolismo , Exopeptidases/metabolismo , Peixes/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise
2.
Nat Prod Res ; 36(12): 3069-3077, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34315292

RESUMO

An undescribed tricyclic spiroketal compound clathriketal was purified from the solvent extract of the Microcionidae sponge Clathria prolifera, and was characterised as 7-(hydroxymethyl)-13-methoxy-3,11-dimethyl-4-oxo-octahydrospiro[chromene-9,13-pyran]-11-yl propionate by spectroscopic experiments. Clathriketal exhibited significant anti-hyperglycemic property by attenuating serine protease dipeptidyl peptidase-IV (IC50 0.37 mM), and its activity was comparable with the standard diprotin A (IC50 0.31 mM). The spiroketal also exhibited significant inhibitory potentials against carbolytic enzymes α-glucosidase (IC50 0.43 mM) and α-amylase (IC50 0.41 mM). Superior antioxidant properties of clathriketal against the oxidants, 2, 2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid and 2, 2-diphenyl-1-picrylhydrazyl (IC50 ∼1.2 mM) also reinforced its promising anti-hyperglycemic activity. Considerably greater topological surface area (91.29) coupled with lesser steric parameters of clathriketal, as elucidated from the structure-activity relationship analyses could further ascribe the improved ligand-receptor interactions resulting in its prospective anti-hyperglycemic activity. Molecular docking analysis of clathriketal with dipeptidyl peptidase-IV recorded lesser binding energy (-9.63 kcal/mol), which further corroborated its prospective antihyperglycemic activity.


Assuntos
Poríferos , Serina , Animais , Exopeptidases , Furanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Simulação de Acoplamento Molecular , Compostos de Espiro
3.
Molecules ; 26(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201554

RESUMO

The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. Within this work, a systematic review on the enzymatic preparation of Angiotensin-I-Converting Enzyme inhibitory (ACEi) peptides is presented. The systematic review is conducted by following PRISMA guidelines. Soybeans and velvet beans are known to have high protein contents that make them suitable as sources of parent proteins for the production of ACEi peptides. Endopeptidase is commonly used in the preparation of soybean-based ACEi peptides, whereas for velvet bean, a combination of both endo- and exopeptidase is frequently used. Soybean glycinin is the preferred substrate for the preparation of ACEi peptides. It contains proline as one of its major amino acids, which exhibits a potent significance in inhibiting ACE. The best enzymatic treatments for producing ACEi peptides from soybean are as follows: proteolytic activity by Protease P (Amano-P from Aspergillus sp.), a temperature of 37 °C, a reaction time of 18 h, pH 8.2, and an E/S ratio of 2%. On the other hand, the best enzymatic conditions for producing peptide hydrolysates with high ACEi activity are through sequential hydrolytic activity by the combination of pepsin-pancreatic, an E/S ratio for each enzyme is 10%, the temperature and reaction time for each proteolysis are 37 °C and 0.74 h, respectively, pH for pepsin is 2.0, whereas for pancreatin it is 7.0. As an underutilized pulse, the studies on the enzymatic hydrolysis of velvet bean proteins in producing ACEi peptides are limited. Conclusively, the activity of soybean-based ACEi peptides is found to depend on their molecular sizes, the amino acid residues, and positions. Hydrophobic amino acids with nonpolar side chains, positively charged, branched, and cyclic or aromatic residues are generally preferred for ACEi peptides.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Mucuna/metabolismo , Soja/metabolismo , Aminoácidos/química , Inibidores da Enzima Conversora de Angiotensina/química , Aspergillus/enzimologia , Endopeptidases/química , Exopeptidases/química , Globulinas/química , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Pancreatina/química , Peptídeo Hidrolases/química , Peptídeos/química , Prolina/química , Proteínas de Soja/química , Temperatura
4.
Food Chem ; 360: 130026, 2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34023711

RESUMO

The proteolytic activity of some soybean endogenous proteases have been clarified in the previous studies, but the information concerning the roles of these proteases and some other unknown ones during soybean processing are scarce. Herein, 16 endopeptidases, 13 exopeptidases, 24 inhibitors (two serpin-ZX and one subtilisin inhibitor firstly identified), and one glutamate decarboxylase were identified in the soybean water extract by the liquid chromatography tandem mass spectrometry analysis. Amongst the identified endopeptidases, just the aspartic endopeptidases (optimal at pH 2.5-3 and 35-45 °C) showed the detectable proteolytic activity by the tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and protease inhibitor assay analyses, whereas serine, cysteine, and metallo- endopeptidases (except P34 probable thiol protease) did not. Free amino acid analysis showed that the exopeptidases and glutamate decarboxylase were optimal at pH 6 and 45 °C, and by 6 h incubation, the free amino acids and γ-aminobutyric acid almost doubled.


Assuntos
Endopeptidases/metabolismo , Exopeptidases/metabolismo , Glutamato Descarboxilase/metabolismo , Soja/enzimologia , Água/química , Alérgenos/metabolismo , Inibidores de Proteases/farmacologia , Proteólise
5.
ISME J ; 15(8): 2379-2389, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33654265

RESUMO

Pseudomonas aeruginosa is a primary bacterial model to study cooperative behaviors because it yields exoproducts such as siderophores and exoproteases that act as public goods and can be exploited by selfish nonproducers behaving as social cheaters. Iron-limited growth medium, mainly casamino acids medium supplemented with transferrin, is typically used to isolate and study nonproducer mutants of the siderophore pyoverdine. However, using a protein as the iron chelator could inadvertently select mutants unable to produce exoproteases, since these enzymes can degrade the transferrin to facilitate iron release. Here we investigated the evolutionary dynamics of pyoverdine and exoprotease production in media in which iron was limited by using either transferrin or a cation chelating resin. We show that concomitant loss of pyoverdine and exoprotease production readily develops in media containing transferrin, whereas only pyoverdine loss emerges in medium treated with the resin. Characterization of exoprotease- and pyoverdine-less mutants revealed loss in motility, different mutations, and large genome deletions (13-33 kb) including Quorum Sensing (lasR, rsal, and lasI) and flagellar genes. Our work shows that using transferrin as an iron chelator imposes simultaneous selective pressure for the loss of pyoverdine and exoprotease production. The unintended effect of transferrin uncovered by our experiments can help to inform the design of similar studies.


Assuntos
Ferro , Pseudomonas aeruginosa , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Exopeptidases , Ferro/metabolismo , Oligopeptídeos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Sideróforos , Transferrina
6.
Talanta ; 222: 121429, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33167196

RESUMO

To have information on the proteolytic activity of convertases and exo-peptidases on human salivary proteins, this study investigated the relative amounts of the truncated proteoforms in the saliva of preterm newborns and compared them with the relative amounts measured in saliva of at-term newborns, of babies (0-10 years old) and of adults. Results indicated that convertase(s), acting on acidic proline-rich proteins and histatin 3, and carboxypeptidase(s) acting on acidic proline-rich proteins, P-C peptide, histatin 6 and statherin were many folds more active in preterm newborns than in the other groups. Conversely, the aminopeptidase responsible for the removal of the N-terminal Asp residue of statherin was not active in preterm newborns, becoming active only several months after the normal term of delivery. The high activity of convertases determined in preterm newborns suggests that it is required for the molecular events connected to the fetus development, and encourages further studies devoted to the characterization of their specific substrates.


Assuntos
Saliva , Proteínas e Peptídeos Salivares , Adulto , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Exopeptidases , Desenvolvimento Fetal , Humanos , Lactente , Recém-Nascido
7.
Food Chem ; 345: 128764, 2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-33310254

RESUMO

Research concerning the utilization of oilseed endogenous proteases is scarce. Herein, we investigated the peanut proteases and their effects on peanut proteins. Liquid chromatography tandem mass spectrometry analysis showed that peanut contained several endopeptidases and exopeptidases. Protease inhibitor assay and analysis of cleavage sites showed that the obvious proteolytic activity at pH 2-5 and 20-60 °C was from aspartic endopeptidases (optimal at pH 3) and one legumain (pH 4). The above endopeptidases destroyed five and six IgE-binding epitopes of Ara h 1 at pH 3 and 4, respectively. Ara h 1 (>95%) and arachin (50-60%) could be hydrolyzed to generate 10-20 kDa and <4 kDa peptides at pH 3, which was enhanced by the pH 3 â†’ 4 incubation. Further, the limited hydrolysis improved the gel-forming ability and in vitro digestibility (approximately 15%) of peanut proteins. Free amino acid analysis showed that the activity of exopeptidases was low at pH 2-5.


Assuntos
Arachis/metabolismo , Endopeptidases/metabolismo , Exopeptidases/metabolismo , Alérgenos/metabolismo , Antígenos de Plantas/química , Epitopos/metabolismo , Hidrólise , Hipersensibilidade a Amendoim , Peptídeos/metabolismo , Proteólise
8.
Anal Chem ; 92(7): 5023-5032, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32167277

RESUMO

Due to mechanisms such as proteolytic processing or alternative translation starts, in vivo proteoforms do not necessarily correspond directly to those encoded in the genome. Therefore, the knowledge of protein termini is an indispensable prerequisite to understand protein functions. So far, sequencing of protein N- and C-termini has been limited to single purified protein species, while the proteome-wide identification of N- and C-termini relies on the generation of single, terminal proteotypic peptides followed by chemical enrichment or depletion strategies to facilitate their detection via mass spectrometry (MS). To overcome the numerous limitations in such approaches, we present an alternative concept that readily enables unbiased ladder sequencing of protein N- and C-termini. The approach combines exopeptidase digestions of the proteome with two-dimensional chromatographic separation and tandem-MS. We demonstrate the potential of the methodology by analyzing the N- and C-terminome of S. cerevisiae, identifying 2190 N-termini and 1562 C-termini. In conclusion, the presented method largely expands the proteomics toolbox enabling N- and C-terminal sequential characterization of entire proteomes.


Assuntos
Exopeptidases/metabolismo , Proteoma/metabolismo , Proteômica , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Proteoma/análise , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo
9.
Food Chem ; 306: 125613, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31610331

RESUMO

Reduction of bitter taste in protein hydrolysates is a challenging task. The aim of this study was to apply a simple two-step approach to prepare low bitter hydrolysates and investigate the influence of peptide modifications on taste characteristics. Protein hydrolysates were prepared from porcine muscle and plasma through simultaneous hydrolysis using endo- and exo-peptidases combined with peptide glycation by glucosamine (GlcN). Spectroscopic analysis and quantification of major alpha-dicarbonyl compounds (α-DCs) indicated the relatively low extent of Maillard reaction in GlcN-glycated protein hydrolysates. Thermal degradation of high MW peptides (>10 kDa) might play a major role in Maillard reaction, reflected by the formation of more Maillard reacted peptides (1-5 kDa), especially in plasma samples. Sensory evaluation indicated that glycation by GlcN can alter taste profiles of protein hydrolysates, which may be attributed to the formation of Maillard reacted peptides and peptide modifications revealed by LC-MS/MS analysis.


Assuntos
Exopeptidases/química , Músculo Esquelético/química , Paladar , Animais , Aspergillus oryzae/enzimologia , Exopeptidases/metabolismo , Glucosamina/química , Glucosamina/metabolismo , Glicosilação , Hidrólise , Reação de Maillard , Músculo Esquelético/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Hidrolisados de Proteína/química , Suínos
10.
Anal Chem ; 91(24): 15890-15898, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31774262

RESUMO

Mass spectrometry (MS)-based identification of ubiquitinated sites requires trypsin digestion prior to MS analysis, and a signature peptide was produced with a diglycine residue attached to the ubiquitinated lysine (K-ε-GG peptide). However, the missed cleavage of modified lysines by trypsin results in modified peptides with increased length and charge, whose detection by MS analysis is suppressed by the vast majority of internally unmodified peptides. LysargiNase, the mirrored trypsin, is reported to cleave before lysine and arginine residues and to be favorable for the identification of methylation and phosphorylation, but its digestive characteristics related to ubiquitination are unclear. Herein, we tested the capacity of the in-house developed acetylated LysargiNase (Ac-LysargiNase) with high activity and stability, for cleaving ubiquitinated sites in both the seven types of ubiquitin chains and their corresponding K-ε-GG peptides. Interestingly, Ac-LysargiNase could efficiently cleave the K63-linked chain but had little effect on the other types of chains. Additionally, Ac-LysargiNase had higher exopeptidase activity than trypsin. Utilizing these features of the paired mirror proteases, a workflow of trypsin and Ac-LysargiNase tandem digestion was developed for the identification of ubiquitinated proteins. Through this method, the charge states and ionization capacity of the unmodified peptides were efficiently reduced, and the identification of modified sites was consequently increased by 30% to 50%. Strikingly, approximately 15% of the modified sites were cleaved by Ac-LysargiNase, resulting in shorter K-ε-GG peptides for better identification. The enzyme Ac-LysargiNase is expected to serve as an option for increasing the efficiency of modified site identification in ubiquitome research.


Assuntos
Lisina/análise , Peptídeos/metabolismo , Espectrometria de Massas em Tandem , Tripsina/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Exopeptidases/metabolismo , Lisina/metabolismo , Peptídeos/química , Ubiquitinação
11.
J Agric Food Chem ; 67(43): 11948-11954, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31577435

RESUMO

Corn gluten hydrolysate (CGH) was prepared by food-grade bacterial proteases, alcalase and neutral protease. Digestion of CGH with carboxypeptidase A and leucine aminopeptidase extensively changed the elution patterns of peptides as observed from reversed phase high performance liquid chromatography-mass spectrometry (LC-MS), whereas digestion with pepsin and trypsin hardly affected the elution patterns. Twenty-five major peptides in CGH were identified. After digestion with exopeptidases, only prolyl dipeptides and pyroglutamyl di- and tripeptides remained, whereas the other 17 peptides completely disappeared. On the other hand, all 25 peptides remained after digestion with pepsin and trypsin. These facts suggest that a majority of short-chain peptides in food protein hydrolysates are degraded by exopeptidases during digestion and absorption processes. Thus, susceptibility to exopeptidases should be considered for prediction of bioactive peptide upon ingestion, which has not been considered in most of previous studies on food-derived bioactive peptides.


Assuntos
Proteínas de Bactérias/química , Glutens/química , Peptídeo Hidrolases/química , Peptídeos/química , Zea mays/química , Bacillus/enzimologia , Biocatálise , Exopeptidases/química , Espectrometria de Massas , Hidrolisados de Proteína/química
12.
Food Res Int ; 121: 28-38, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31108750

RESUMO

The objective of this study was to investigate the impact of endo- and exo-peptidase treatment on certain structural characteristics of peptides and volatile compounds of porcine hemoglobin and whole blood hydrolysates. Porcine hemoglobin and whole blood were hydrolyzed by endo- and exo-peptidases. The presence of exopeptidases reduced the bitterness and altered the volatile profiles of protein hydrolysates. Exopeptidase treatment can release terminal amino acids from peptides, which in turn may contribute to formation of volatile compounds by Maillard reactions. In contrast, endopeptidases conferred a slightly bitter taste and different volatile profiles. For hemoglobin hydrolysates, principal component analysis revealed that proteases were categorized into three groups based on endo- or exo-peptidase activity. Whole blood is a more complex raw material, yet the proteases were still categorized in a similar fashion. This work contributes to understanding structural characteristics responsible for taste and volatile profiles of protein hydrolysates.


Assuntos
Proteínas Sanguíneas , Odorantes/análise , Hidrolisados de Proteína , Compostos Orgânicos Voláteis , Animais , Sangue/metabolismo , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Exopeptidases/metabolismo , Feminino , Hemoglobinas , Humanos , Masculino , Hidrolisados de Proteína/análise , Hidrolisados de Proteína/química , Hidrolisados de Proteína/metabolismo , Suínos , Paladar , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismo
13.
Anal Biochem ; 573: 1-7, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30849379

RESUMO

Given that the biological functions of proteins may decrease or even be lost due to degradation by proteases, it is of great significance to identify potential proteases that degrade protein drugs during systemic circulation. In this work, we describe a method based on high-performance liquid chromatography (HPLC) to identify key proteases that degrade therapeutic proteins in blood, including endopeptidases and exopeptidases. Here, the degradation of proteins was detected by competition with standard substrates of proteases and is shown as the relative residue rate. Four protein drugs were subjected to this method, and the results suggested that growth hormone was degraded by aminopeptidase N and kallikrein-related peptidase 5, pertuzumab was hardly degraded by the proteases, factor VII was degraded by carboxypeptidase B, neprilysin, dipeptidyl peptidase-4 and peptidyl dipeptidase A, and fibrinogen was degraded by carboxypeptidase B and kallikrein-related peptidase 5, findings consistent with the literature. The results were confirmed by microscale thermophoresis; additionally, activity detection in vitro substantiated that the degradation of factor VII decreased its activity. We demonstrate that this method can be used to identify key proteases of proteins with high accuracy, precision and durability.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Peptídeo Hidrolases/análise , Anticorpos Monoclonais Humanizados/metabolismo , Endopeptidases/análise , Endopeptidases/metabolismo , Exopeptidases/análise , Exopeptidases/metabolismo , Hormônio do Crescimento/metabolismo , Hidrólise , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo
14.
PLoS One ; 14(2): e0212429, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30807611

RESUMO

The exoproteome of parasitic protists constitutes extracellular proteins that play a fundamental role in host-parasite interactions. Lytic factors, especially secreted proteases, are capable of modulating tissue invasion, thereby aggravating host susceptibility. Despite the important role of exoproteins during infection, the exoproteomic data on Histomonas meleagridis are non-existent. The present study employed traditional 1D-in-gel-zymography (1D-IGZ) and micro-LC-ESI-MS/MS (shotgun proteomics), to investigate H. meleagridis exoproteomes, obtained from a clonal virulent and an attenuated strain. Both strains were maintained as mono-eukaryotic monoxenic cultures with Escherichia coli. We demonstrated active in vitro secretion kinetics of proteases by both parasite strains, with a widespread proteolytic activity ranging from 17 kDa to 120 kDa. Based on protease inhibitor susceptibility assay, the majority of proteases present in both exoproteomes belonged to the family of cysteine proteases and showed stronger activity in the exoproteome of a virulent H. meleagridis. Shotgun proteomics, aided by customized database search, identified 176 proteins including actin, potential moonlighting glycolytic enzymes, lytic molecules such as pore-forming proteins (PFPs) and proteases like cathepsin-L like cysteine protease. To quantify the exoproteomic differences between the virulent and the attenuated H. meleagridis cultures, a sequential window acquisition of all theoretical spectra mass spectrometric (SWATH-MS) approach was applied. Surprisingly, results showed most of the exoproteomic differences to be of bacterial origin, especially targeting metabolism and locomotion. By deciphering such molecular signatures, novel insights into a complex in vitro protozoan- bacteria relationship were elucidated.


Assuntos
Parabasalídeos/genética , Parabasalídeos/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Exopeptidases/genética , Exopeptidases/metabolismo , Interações Hospedeiro-Parasita/genética , Parabasalídeos/patogenicidade , Aves Domésticas , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/parasitologia , Mapas de Interação de Proteínas , Proteoma/genética , Proteômica , Infecções Protozoárias em Animais/microbiologia , Infecções Protozoárias em Animais/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Virulência/genética
15.
PLoS One ; 14(1): e0211290, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682135

RESUMO

Tobramycin is commonly used to treat Pseudomonas aeruginosa lung infections in patients with Cystic Fibrosis (CF). Tobramycin treatment leads to increased lung function and fewer clinical exacerbations in CF patients, and modestly reduces the density of P. aeruginosa in the lungs. P. aeruginosa resides primarily in the mucus overlying lung epithelial cells and secretes outer membrane vesicles (OMVs) that diffuse through the mucus and fuse with airway epithelial cells, thus delivering virulence factors into the cytoplasm that modify the innate immune response. The goal of this study was to test the hypothesis that Tobramycin reduces the abundance of virulence factors in OMVs secreted by P. aeruginosa. Characterization of the proteome of OMVs isolated from control or Tobramycin-exposed P. aeruginosa strain PAO1 revealed that Tobramycin reduced several OMV-associated virulence determinants, including AprA, an alkaline protease that enhances P. aeruginosa survival in the lung, and is predicted to contribute to the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion by primary human bronchial epithelial cells. Deletion of the gene encoding AprA reduced the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion. Moreover, as predicted by our proteomic analysis, OMVs isolated from Tobramycin treated P. aeruginosa had a diminished inhibitory effect on Phe508del-CFTR Cl- secretion compared to OMVs isolated from control P. aeruginosa. Taken together, our proteomic analysis of OMVs and biological validation suggest that Tobramycin may improve lung function in CF patients infected with P. aeruginosa by reducing several key virulence factors in OMVs that reduce CFTR Cl- secretion, which is essential for bacterial clearance from the lungs.


Assuntos
Exopeptidases/metabolismo , Proteômica/métodos , Pseudomonas aeruginosa/patogenicidade , Vesículas Secretórias/microbiologia , Tobramicina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Brônquios/citologia , Brônquios/metabolismo , Brônquios/microbiologia , Células Cultivadas , Fibrose Cística/genética , Fibrose Cística/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Exopeptidases/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Virulência/efeitos dos fármacos
16.
Microb Pathog ; 126: 379-392, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30476580

RESUMO

The emerging prevalence of multidrug-resistance in Gram-negative pathogens, due to conventional antimicrobial therapeutics, has led the researchers to emphasize on development of alternative novel strategies to suppress the bacterial virulence and pathogenicity through inhibition of quorum sensing (QS) and biofilms. QS is a bacterial communication system to produce density-dependent response via chemical signalling that controls pathogenesis and biofilms formation. Leaves of green tea are used worldwide as beverage which is also known for its broad-spectrum therapeutic efficacy. In this work, we have identified and characterized the most bioactive faction of green tea extract and evaluated the anti-QS and antibiofilm activity of green tea ethyl acetate fraction (GTEF) i.e. most active fraction, on three different Gram-negative bacterial pathogens. GTEF inhibited the violacein production by >75% in C. violaceum 12472. Many virulence factors of P. aeruginosa PAO1 viz. pyocyanin, pyoverdin, exoprotease, elastase, rhamnolipid production, and swimming motility were remarkably reduced in presence of sub-MICs of GTEF. Moreover, prodigiosin, protease activity, cell surface hydrophobicity, and swimming of S. marcescens MTCC 97 were also decreased significantly by the supplementation of GTEF in culture media. GTEF exhibited broad-spectrum antibiofilm action with >80% reduction in biofilm formation of test pathogens. In silico studies gave a mechanistic insight of action of GTEF. Molecular modelling revealed that phytoconstituents detected by GC/MS exhibited affinity (in order of 104 M-1) towards AHL synthases (LasI and EsaI). The molecular binding between phytocompounds and receptor proteins (LasR, RhlR, and PqsR) of QS circuit was also energetically favourable (ΔG°≥ 5.0 kcal mol-1) and supported by hydrogen bonds and hydrophobic interactions. These compounds were found to be docked in ligand binding domain of CviR and occupied same cavity as that of its antagonist. Squalene and thunbergol interacted with LasA at tartaric acid binding pocket and the complex was strengthened with binding energy -5.9 kcal mol-1. Moreover, interaction of thunbergol with biofilm-associated proteins viz. PilT and PilY1, might be disabling the pilus assembly and consequently inhibiting biofilm formation. In vivo validation of results suggested the protective role GTEF against QS-mediated pathogenicity and it might become a novel non-antibiotic QS inhibitor to control bacterial infection.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Modelos Moleculares , Extratos Vegetais/farmacologia , Percepção de Quorum/efeitos dos fármacos , Chá/química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Exopeptidases/metabolismo , Glicolipídeos/metabolismo , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Indóis/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Oligopeptídeos/metabolismo , Peptídeo Hidrolases/efeitos dos fármacos , Extratos Vegetais/química , Folhas de Planta/química , Prodigiosina/metabolismo , Piocianina/metabolismo , Fatores de Virulência/metabolismo
17.
Food Funct ; 9(11): 5989-5998, 2018 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-30379169

RESUMO

Enzymatic hydrolysis with endopeptidases can be used to modify the colloidal properties of food proteins. In this study, sodium caseinate was hydrolyzed with Sternzym BP 25201, containing a thermolysin-like endopeptidase from Geobacillus stearothermophilus as the only peptidase, to a DH of 2.3 ± 1%. The hydrolysate (pre-hydrolysate) obtained was increased in its foam (+35%) and emulsion stability (+200%) compared to untreated sodium caseinate but showed a bitter taste. This hydrolysate was further treated with the exopeptidases PepN, PepX or PepA, acting on the N-terminus of peptides. Depending on the specificity of the exopeptidase used, changes regarding the hydrolysate properties (hydrophobicity, size), colloidal behavior (emulsions, foams) and taste were observed. No changes regarding the bitterness but further improvements regarding the colloidal stability (foam: +69%, emulsion: +29%) were determined after the application of PepA, which is specific for the hydrophilic amino acids Asp, Glu and Ser. By contrast, treatment with the general aminopeptidase PepN resulted in a non-bitter product, with no significant changes regarding the colloidal properties compared to the pre-hydrolysate (p < 0.05). Similar results to those for PepN (reduced bitterness compared to the pre-hydrolysate, enhanced colloidal stability compared to sodium caseinate) were also obtained using commercial Flavourzyme, which was reduced in its endopeptidase activity (exo-flavourzyme). In conclusion, the modifications obtained with the applied exopeptidases offer a potent tool for researchers and the industry to produce non-bitter protein hydrolysates with increased colloidal properties.


Assuntos
Caseínas/química , Exopeptidases/metabolismo , Hidrolisados de Proteína/química , Paladar , Adulto , Aminopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Pessoa de Meia-Idade , Peso Molecular , Peptídeo Hidrolases/metabolismo , Peptídeos , Adulto Jovem
18.
Biofouling ; 34(10): 1079-1092, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30698028

RESUMO

The effects of dual species interactions on biofilm formation by Aeromonas hydrophila in the presence of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pectobacterium carotovorum, Salmonella Typhimurium, and Listeria monocytogenes were examined. High-performance liquid chromatography and liquid-chromatography-mass spectrometry were performed to identify N-acyl homoserine lactone (AHL) molecules secreted by monocultures and dual cultures grown in crab broth. Field emission scanning electron microscopy was performed to observe attachment and biofilm formation. P. aeruginosa and P. fluorescens inhibited biofilm formation by A. hydrophila on the crab surface, without affecting their own biofilm-forming abilities. Dual biofilms of S. Typhimurium, L. monocytogenes, or P. carotovorum did not affect A. hydrophila biofilm formation. Exoprotease, AHL, and AI-2 levels were significantly reduced in dual cultures of P. aeruginosa and P. fluorescens with A. hydrophila, supporting the relationship between quorum sensing and biofilm formation. Dual-species biofilms were studied in their natural environment and in the laboratory.


Assuntos
Aeromonas hydrophila/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Braquiúros/microbiologia , Exopeptidases/metabolismo , Microbiota/fisiologia , Percepção de Quorum/fisiologia , Alimentos Marinhos/microbiologia , Acil-Butirolactonas/metabolismo , Aeromonas hydrophila/enzimologia , Aeromonas hydrophila/fisiologia , Animais , Aderência Bacteriana/fisiologia , Técnicas de Cocultura
19.
J Med Food ; 21(4): 356-363, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29172966

RESUMO

Seed oils from oleaginous plants are rich in fatty acids (FAs) that play important roles in the health of the consumers. Recent studies indicate that FA also can play an important role in communication and regulation of virulence in bacteria. Nevertheless, evidence demonstrating protection against bacterial infections mediated by their quorum sensing inhibition (QSI) activity is scarce. In this study, sunflower, chia, and amaranth oils, were assayed for their QSI capacity by inhibiting violacein production and alkaline exoprotease activity of Chromobacterium violaceum. In vitro assays revealed that the oils exhibited QSI activities, whereas in vivo they delayed death of mice inoculated intraperitoneally with the bacterium. Gas chromatography coupled with mass spectrometry analysis of the oils indicated the presence of saturated FA (SAFA) and unsaturated FA as main components. Through a structure-activity relationship study of free FAs, bactericidal effect was identified mainly for polyunsaturated FAs, whereas QSI activity was restricted to SAFA of chains 12-18 carbon atoms in length. These data correlate with a possible interaction suggested by molecular docking analysis of lauric, myristic, and stearic acids with the CviR protein. Our study highlights the antiquorum sensing potential of SAFA, which may be future antivirulence therapeutic agents for the treatment of bacterial infections.


Assuntos
Antibacterianos/farmacologia , Chromobacterium/efeitos dos fármacos , Ácidos Graxos/farmacologia , Magnoliopsida/química , Óleos Vegetais/farmacologia , Percepção de Quorum/efeitos dos fármacos , Sementes/química , Amaranthus/química , Animais , Antibacterianos/química , Antibacterianos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Chromobacterium/metabolismo , Chromobacterium/patogenicidade , Exopeptidases/metabolismo , Ácidos Graxos/química , Ácidos Graxos/uso terapêutico , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/farmacologia , Ácidos Graxos Insaturados/uso terapêutico , Cromatografia Gasosa-Espectrometria de Massas , Helianthus/química , Indóis/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Óleos Vegetais/química , Óleos Vegetais/uso terapêutico , Salvia/química , Relação Estrutura-Atividade , Fatores de Virulência/metabolismo
20.
Dev Comp Immunol ; 81: 116-126, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29174605

RESUMO

The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (Riptortus pedestris) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila, we used an aprA-deficient P. entomophila mutant strain (ΔaprA). When ΔaprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the ΔaprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses.


Assuntos
Heterópteros/imunologia , Inseticidas/metabolismo , Metaloproteases/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas/fisiologia , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Exopeptidases/genética , Engenharia Genética , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Imunidade Celular , Imunidade Humoral , Terapia de Imunossupressão , Metaloproteases/genética , Mutação/genética , Infecções por Pseudomonas/microbiologia , Fatores de Virulência/genética
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