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Enterotoxigenic Escherichia coli (ETEC) strains, which produce the heat-stable enterotoxin (ST) either alone or in combination with the heat-labile enterotoxin, contribute to the bulk of the burden of child diarrheal disease in resource-limited countries and are associated with mortality. Developing an effective vaccine targeting ST presents challenges due to its potent enterotoxicity, non-immunogenicity, and the risk of autoimmune reaction stemming from its structural similarity to the human endogenous ligands, guanylin, and uroguanylin. This study aimed to assess a novel synthetic vaccine carrier platform employing a single chemical coupling step for making human ST (STh) immunogenic. Specifically, the method involved cross-linking STh to an 8-arm N-hydroxysuccinimide (NHS) ester-activated PEG cross-linker. A conjugate of STh with 8-arm structure was prepared, and its formation was confirmed through immunoblotting analysis. The impact of conjugation on STh epitopes was assessed using ELISAs with polyclonal and monoclonal antibodies targeting various epitopes of STh. Immunization of mice with the conjugate induced the production of anti-STh antibodies, exhibiting neutralizing activity against STh.
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Protein hydrolysates made from marine by-products are very nutritious but frequently contain trimethylamine (TMA), which has an unattractive fish-like smell. Bacterial trimethylamine monooxygenases can oxidize TMA into the odorless trimethylamine N-oxide (TMAO) and have been shown to reduce TMA levels in a salmon protein hydrolysate. To make the flavin-containing monooxygenase (FMO) Methylophaga aminisulfidivorans trimethylamine monooxygenase (mFMO) more suitable for industrial application, we engineered it using the Protein Repair One-Stop Shop (PROSS) algorithm. All seven mutant variants, containing 8 to 28 mutations, displayed increases in melting temperature of between 4.7°C and 9.0°C. The crystal structure of the most thermostable variant, mFMO_20, revealed the presence of four new stabilizing interhelical salt bridges, each involving a mutated residue. Finally, mFMO_20 significantly outperformed native mFMO in its ability to reduce TMA levels in a salmon protein hydrolysate at industrially relevant temperatures. IMPORTANCE Marine by-products are a high-quality source for peptide ingredients, but the unpleasant fishy odor caused by TMA limits their access to the food market. This problem can be mitigated by enzymatic conversion of TMA into the odorless TMAO. However, enzymes isolated from nature must be adapted to industrial requirements, such as the ability to tolerate high temperatures. This study has demonstrated that mFMO can be engineered to become more thermostable. Moreover, unlike the native enzyme, the best thermostable variant efficiently oxidized TMA in a salmon protein hydrolysate at industrial temperatures. Our results present an important next step toward the application of this novel and highly promising enzyme technology in marine biorefineries.
Assuntos
Metilaminas , Hidrolisados de Proteína , Animais , Metilaminas/metabolismoRESUMO
Although bacteria are commonly co-occurring in microalgal cultivation and production systems, little is known about their community structure and how it might be affected by specific microalgal groups or growth conditions. A better understanding about the underlying factors that determine the growth of specific bacterial populations is not only important for optimizing microalgal production processes, but also in the context of product quality when the algal biomass is to be used for future food or feed. We analyzed the bacterial community composition associated with nine microalgal strains in stock culture, maintained in two different growth media, to explore how specific taxonomic microalgal groups, microalgal origin, or the growth medium affect the bacterial community composition. Furthermore, we monitored the bacterial community composition for three Phaeodactylum strains during batch cultivation in bubble columns to examine if the bacterial composition alters during cultivation. Our results reveal that different microalgal genera, kept at the same cultivation conditions over many years, displayed separate and unique bacterial communities, and that different strains of the same genus had very similar bacterial community compositions, despite originating from different habitats. However, when maintained in a different growth medium, the bacterial composition changed for some. During batch cultivation, the bacterial community structure remained relatively stable for each Phaeodactylum strain. This indicates that microalgae seem to impact the development of the associated bacterial communities and that different microalgal genera could create distinct conditions that select for dominance of specific bacteria. However, other factors such as the composition of growth medium also affect the formation of the bacterial community structure.
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Entomopathogenic nematodes are used as biological control agents against a broad range of insect pests. We ascribed the pathogenicity of these organisms to the excretory/secretory products (ESP) released by the infective nematode. Our group characterized different virulence factors produced by Steinernema carpocapsae that underlie its success as an insect pathogen. A novel ShK-like peptide (ScK1) from this nematode that presents high sequence similarity with the ShK peptide from a sea anemone was successfully produced recombinantly in Escherichia coli. The secondary structure of ScK1 appeared redox-sensitive, exhibiting a far-UV circular dichroism spectrum consistent with an alpha-helical secondary structure. Thermal denaturation of the ScK1 allowed estimating the melting temperature to 59.2 ± 0.1 °C. The results from toxicity assays using Drosophila melanogaster as a model show that injection of this peptide can kill insects in a dose-dependent manner with an LD50 of 16.9 µM per adult within 24 h. Oral administration of the fusion protein significantly reduced the locomotor activity of insects after 48 h (p < 0.05, Tukey's test). These data show that this nematode expresses insecticidal peptides with potential as next-generation insecticides.
Assuntos
Drosophila melanogaster , Nematoides , Animais , Insetos , Agentes de Controle Biológico , VirulênciaRESUMO
Heat-stable enterotoxin (ST) producing enterotoxigenic Escherichia coli (ETEC) strains are among the top four enteropathogens associated with moderate-to-severe diarrhea in children under five years in low-to-middle income countries, thus making ST a target for an ETEC vaccine. However, ST must be mutated to abolish its enterotoxicity and to prevent a potential immunological cross-reaction due to its structural resemblance to the human peptides uroguanylin and guanylin. To reduce the risk of eliciting cross-reacting antibodies with our lead STh-A14T toxoid, L9 was chosen as an additional mutational target. A double mutant vaccine candidate immunogen, STh-L9A/A14T, was constructed by conjugation to the synthetic virus-like mi3 nanoparticle using the SpyTag/SpyCatcher technology. This immunogen elicited STh neutralizing antibodies in mice, but with less consistency than STh-A14T peptide control immunogens. Moreover, individual sera from mice immunized with both single and double mutant variants displayed varying levels of unwanted cross-reacting antibodies. The lowest levels of cross-reacting antibodies were observed with STh-L9K/A14T control immunogens, suggesting that it is indeed possible to reduce the risk of eliciting cross-reacting antibodies by mutation. However, mutant-specific antibodies were observed for most double mutant immunogens, demonstrating the delicate balancing act between disrupting cross-reacting epitopes, keeping protective ones, and avoiding the formation of neoepitopes.
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To support the bio-based industry in development of environment-friendly processes and products, an optimal toolbox of biocatalysts is key. Although functional screen of (meta)genomic libraries may potentially contribute to identifying new enzymes, the discovery of new enzymes meeting industry compliance demands is still challenging. This is particularly noticeable in the case of proteases, for which the reports of metagenome-derived proteases with industrial applicability are surprisingly limited. Indeed, proteolytic clones have been typically assessed by its sole activity on casein or skim milk and limited to mild screening conditions. Here, we demonstrate the use of six industry-relevant animal and plant by-products, namely bone, feather, blood meals, gelatin, gluten, and zein, as complementary substrates in functional screens and show the utility of temperature as a screening parameter to potentially discover new broad-substrate range and robust proteases for the biorefinery industry. By targeting 340,000 clones from two libraries of pooled isolates of mesophilic and thermophilic marine bacteria and two libraries of microbial communities inhabiting marine environments, we identified proteases in four of eleven selected clones that showed activity against all substrates herein tested after prolonged incubation at 55 °C. Following sequencing, in silico analysis and recombinant expression in Escherichia coli, one functional protease, 58% identical at sequence level to previously reported homologs, was found to readily hydrolyze highly insoluble zein at temperatures up to 50 °C and pH 9-11. It is derived from a bacterial group whose ability to degrade zein was unknown. This study reports a two-step screen resulting in identification of a new marine metagenome-derived protease with zein-hydrolytic properties at common biomass processing temperatures that could be useful for the modern biorefinery industry. KEY POINTS: ⢠A two-step multi-substrate strategy for discovery of robust proteases. ⢠Feasible approach for shortening enzyme optimization to industrial demands. ⢠A new temperature-tolerant protease efficiently hydrolyzes insoluble zein.
Assuntos
Metagenoma , Peptídeo Hidrolases , Animais , Bactérias/genética , Endopeptidases , Peptídeo Hidrolases/genética , TemperaturaRESUMO
Historically, algae have stimulated significant economic interest particularly as a source of fertilizers, feeds, foods and pharmaceutical precursors. However, there is increasing interest in exploiting algal diversity for their antiviral potential. Here, we present an overview of 50-years of scientific and technological developments in the field of algae antivirals. After bibliometric analysis of 999 scientific references, a survey of 16 clinical trials and analysis of 84 patents, it was possible to identify the dominant algae, molecules and viruses that have been shaping and driving this promising field of research. A description of the most promising discoveries is presented according to molecule class. We observed a diverse range of algae and respective molecules displaying significant antiviral effects against an equally diverse range of viruses. Some natural algae molecules, like carrageenan, cyanovirin or griffithsin, are now considered prime reference molecules for their outstanding antiviral capacity. Crucially, while many algae antiviral applications have already reached successful commercialization, the large spectrum of algae antiviral capacities already identified suggests a strong potential for future expansion of this field.
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Antivirais/farmacologia , Microalgas/metabolismo , Alga Marinha/metabolismo , Agricultura , Aquicultura , Proteínas de Bactérias/farmacologia , Ensaios Clínicos como Assunto , Diterpenos/farmacologia , Lectinas/farmacologia , Proteínas de Membrana/farmacologia , Lectinas de Plantas/farmacologia , Polissacarídeos/farmacologiaRESUMO
Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool for targeted genome editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial species. A collection of vectors compatible with high-throughput (HTP) fragment exchange (FX) cloning for heterologous expression in Escherichia coli and Bacillus was previously developed. This vector catalogue was through this work supplemented with editing plasmids for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to the stable integration of the gene into the amyE locus of a seven-protease-deficient KO7 strain. The newly engineered B. subtilis strain allowed the successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering, and contribute to a cloning platform for a quick transition from HTP enzyme expression to production through the fermentation of industrially relevant B. subtilis and related strains.IMPORTANCE We complemented a cloning platform with new editing plasmids that allow a quick transition from high-throughput cloning and the expression of new enzymes to the stable integration of genes for the production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome-editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus' capacity to sporulate and by introducing the stable expression of subtilisin. The tools should be useful to engineer B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry-relevant host.
Assuntos
Bacillus subtilis/genética , Sistemas CRISPR-Cas , Edição de Genes , Peptídeo Hidrolases/genética , Plasmídeos/genética , Bacillus subtilis/enzimologia , Peptídeo Hidrolases/metabolismo , Plasmídeos/metabolismoRESUMO
Enzymatic processing of fish by-products for recovery of peptides (hydrolysates) is a promising technology to reach food grade ingredients of high nutritional quality. Despite this, their bitter taste and "fish" odor block implementation in food products and limit their economic potential. Trimethylamine (TMA) is a known contributor to malodor in fish. Current strategies to mask or remove the odor either are not effective or give rise to undesirable side effects. As an alternative approach to remediate TMA, we propose a novel enzymatic strategy to convert TMA into the odorless trimethylamine N-oxide (TMAO) using TMA monooxygenases (Tmms). We identified a diverse set of bacterial Tmms using a sequence similarity network. Purified, recombinant enzymes were assessed for their biocatalytic capacity by monitoring NADPH consumption and TMAO generation. Selected Tmms were subjected to biochemical characterization and investigated for their ability to oxidize TMA in an industry-relevant substrate. From the 45 bacterial Tmm candidates investigated, eight enzymes from four different taxa were selected for their high activity toward TMA. The three most active enzymes were shown to vary in temperature optimum, with the highest being 45°C. Enzymatic activity dropped at high temperatures, likely due to structural unfolding. The enzymes were all active from pH 6.0 to 8.5, with functional stability being lowest around the optimal pH. All three Tmms, given sufficient NADPH cofactor, were found to generate TMAO in the TMA-rich salmon protein hydrolysate. The Tmms serve as unique starting points for engineering and should be useful for guiding process development for marine biorefineries.IMPORTANCE Enzyme-based conversion of marine biomass to high-quality peptide ingredients leaves a distinct smell of "fish" caused by the presence of trimethylamine, which limits their economic potential. We suggest an enzymatic solution for converting trimethylamine to the odorless trimethylamine N-oxide as a novel strategy to improve the smell quality of marine protein hydrolysates. Following a systematic investigation of 45 putative bacterial trimethylamine monooxygenases from several phyla, we expand the repertoire of known active trimethylamine monooxygenases. As a proof-of-concept, we demonstrate that three of these enzymes oxidized trimethylamine in an industry-relevant salmon protein hydrolysate. Our results add new oxidoreductases to the industrial biocatalytic toolbox and provide a new point of departure for enzyme process developments in marine biorefineries.
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Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Metilaminas/metabolismo , Oxigenases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Oxigenases/química , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Enterotoxigenic Escherichia coli (ETEC) causes diarrhoea by secreting enterotoxins into the small intestine. Human ETEC strains may secrete any combination of three enterotoxins: the heat-labile toxin (LT) and the heat-stable toxins (ST), of which there are two variants, called human ST (STh) and porcine ST (STp). Strains expressing STh, either alone or in combination with LT and/or STp, are among the four most important diarrhoea-causing pathogens affecting children in low- and middle-income countries. ST is therefore an attractive target for ETEC vaccine development. To produce a safe ST-based vaccine, several challenges must be solved. ST must be rendered immunogenic and non-toxic, and antibodies elicited by an ST vaccine should neutralize ST but not cross-react with the endogenous ligands uroguanylin and guanylin. Virus-like particles (VLPs) tend to be highly immunogenic and are increasingly being used as carriers for presenting heterologous antigens in new vaccines. In this study, we have coupled native STh and the STh-A14T toxoid to the coat protein of Acinetobacter phage AP205 by using the SpyCatcher system and immunized mice with these VLPs without the use of adjuvants. We found that both STs were efficiently coupled to the VLP, that both the STh and STh-A14T VLPs were immunogenic in mice, and that the resulting serum antibodies could completely neutralize the toxic activities of native STh. The serum antibodies showed a high degree of immunological cross-reaction to STp, while there was little or no unwanted cross-reaction to uroguanylin and guanylin. Moreover, compared to native STh, the STh-A14T mutation did not seem to negatively impact the immunogenicity of the construct or the neutralizing ability of the resulting sera. Taken together, these findings demonstrate that VLPs are suitable carriers for making STs immunogenic, and that the STh-A14T-coupled AP205 VLP represents a promising ETEC vaccine candidate.
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Anticorpos Antibacterianos/sangue , Escherichia coli Enterotoxigênica/imunologia , Vacinas contra Escherichia coli/imunologia , Toxoides/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Acinetobacter/virologia , Animais , Anticorpos Neutralizantes/sangue , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/imunologia , Bacteriófagos , Reações Cruzadas , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/administração & dosagem , Feminino , Hormônios Gastrointestinais/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos Natriuréticos/imunologia , Toxoides/administração & dosagem , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagemRESUMO
Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.
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Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Toxinas Bacterianas/imunologia , Escherichia coli Enterotoxigênica/imunologia , Enterotoxinas/imunologia , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Hormônios Gastrointestinais/imunologia , Peptídeos Natriuréticos/imunologia , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Reações Cruzadas , Escherichia coli Enterotoxigênica/genética , Enterotoxinas/administração & dosagem , Enterotoxinas/química , Enterotoxinas/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Vacinas contra Escherichia coli/administração & dosagem , Vacinas contra Escherichia coli/genética , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , SuínosRESUMO
Infection with enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea-related illness and death among children under 5 years of age in low- and middle-income countries (LMIC). Recent studies have found that it is the ETEC subtypes that produce the heat-stable enterotoxin (ST), irrespective of whether they also secrete the heat-labile enterotoxin (LT), which contribute most importantly to the disease burden in children from LMIC. Therefore, adding an ST toxoid would importantly complement ongoing ETEC vaccine development efforts. The ST's potent toxicity, its structural similarity to the endogenous peptides guanylin and uroguanylin, and its poor immunogenicity have all complicated the advancement of ST-based vaccine development. Recent remarkable progress, however, including the unprecedented screening for optimal ST mutants, mapping of cross-reacting ST epitopes and improved ST-carrier coupling strategies (bioconjugation and genetic fusion), enables the rational design of safe, immunogenic, and well-defined ST-based vaccine candidates.
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Toxinas Bacterianas/imunologia , Escherichia coli Enterotoxigênica/imunologia , Enterotoxinas/imunologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Reações Cruzadas , Escherichia coli Enterotoxigênica/genética , Enterotoxinas/administração & dosagem , Enterotoxinas/genética , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/genética , Humanos , CamundongosRESUMO
Sensitivity to environmental stressors largely depend on the genetic complement of the organism. Recent sequencing and assembly of teleost fish genomes enable us to trace the evolution of defense genes in the largest and most diverse group of vertebrates. Through genomic searches and in-depth analysis of gene loci in 76 teleost genomes, we show here that the xenosensor pregnane X receptor (Pxr, Nr1i2) is absent in more than half of these species. Notably, out of the 27 genome assemblies that belong to the Gadiformes order, the pxr gene was only retained in the Merluccidae family (hakes) and Pelagic cod (Melanonus zugmayeri). As an important receptor for a wide range of drugs and environmental pollutants, vertebrate PXR regulate the transcription of a number of genes involved in the biotransformation of xenobiotics, including cytochrome P450 enzymes (CYP). In the absence of Pxr, we suggest that the aryl hydrocarbon receptor (Ahr) have evolved an extended regulatory role by governing the expression of certain Pxr target genes, such as cyp3a, in Atlantic cod (Gadus morhua). However, as several independent losses of pxr have occurred during teleost evolution, other lineages and species may have adapted alternative compensating mechanisms for controlling crucial cellular defense mechanisms.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Evolução Molecular , Receptor de Pregnano X/genética , Receptores de Hidrocarboneto Arílico/genética , Animais , Gadiformes/genética , Genoma/genética , Filogenia , Xenobióticos/toxicidadeRESUMO
Enterotoxigenic Escherichia coli (ETEC), which secretes the heat-stable toxin (ST) is among the four most important enteropathogens that cause moderate-to-severe diarrhea in children in low- and middle-income countries. ST is an intestinal molecular antagonist causing diarrhea and hence an attractive vaccine target. A non-toxic and safe ST vaccine should include one or more detoxifying mutations, and rigorous characterization of such mutants requires structurally intact peptides. To this end, we established a system for purification of ST and ST mutants by fusing the sequence encoding the mature ST peptide to the disulfide isomerase DsbC. A Tobacco Etch Virus protease cleavage site facilitates the proteolytic release of free ST with no additional residues. The purified ST peptides have the expected molecular masses, the correct number of disulfide bridges, and have biological activities and antigenic properties comparable to ST isolated from ETEC. We also show that free DsbC can assist in refolding denatured and misfolded ST in vitro. Finally, we demonstrate that the purification system can be used to produce ST mutants with an intact neutralizing epitope, that two single mutations, L9S and A14T, reduce toxicity more than 100-fold, and that the L9S/A14T double mutant has no measurable residual toxicity.
Assuntos
Toxinas Bacterianas , Enterotoxinas , Proteínas de Escherichia coli , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Escherichia coli Enterotoxigênica , Enterotoxinas/genética , Enterotoxinas/isolamento & purificação , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Vacinas contra Escherichia coli , Mutação , Peptídeos/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Intracellular subtilisin proteases (ISPs) have important roles in protein processing during the stationary phase in bacteria. Their unregulated protein degrading activity may have adverse effects inside a cell, but little is known about their regulatory mechanism. Until now, ISPs have mostly been described from Bacillus species, with structural data from a single homolog. Here, we study a marine ISP originating from a phylogenetically distinct genus, Planococcus sp. The enzyme was successfully overexpressed in E. coli, and is active in presence of calcium, which is thought to have a role in minor, but essential, structural rearrangements needed for catalytic activity. The ISP operates at alkaline pH and at moderate temperatures, and has a corresponding melting temperature around 60 °C. The high-resolution 3-dimensional structure reported here, represents an ISP with an intact catalytic triad albeit in a configuration with an inhibitory pro-peptide bound. The pro-peptide is removed in other homologs, but the removal of the pro-peptide from the Planococcus sp. AW02J18 ISP appears to be different, and possibly involves several steps. A first processing step is described here as the removal of 2 immediate N-terminal residues. Furthermore, the pro-peptide contains a conserved LIPY/F-motif, which was found to be involved in inhibition of the catalytic activity.
Assuntos
Endopeptidases/genética , Peptídeos/genética , Planococcus (Bactéria)/enzimologia , Subtilisinas/genética , Organismos Aquáticos , Cálcio/química , Catálise , Endopeptidases/química , Endopeptidases/metabolismo , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Mutação , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Subtilisinas/metabolismo , TemperaturaRESUMO
BACKGROUND: Systems Biology Markup Language (SBML) is the standard model representation and description language in systems biology. Enriching and analysing systems biology models by integrating the multitude of available data, increases the predictive power of these models. This may be a daunting task, which commonly requires bioinformatic competence and scripting. RESULTS: We present SBMLmod, a Python-based web application and service, that automates integration of high throughput data into SBML models. Subsequent steady state analysis is readily accessible via the web service COPASIWS. We illustrate the utility of SBMLmod by integrating gene expression data from different healthy tissues as well as from a cancer dataset into a previously published model of mammalian tryptophan metabolism. CONCLUSION: SBMLmod is a user-friendly platform for model modification and simulation. The web application is available at http://sbmlmod.uit.no , whereas the WSDL definition file for the web service is accessible via http://sbmlmod.uit.no/SBMLmod.wsdl . Furthermore, the entire package can be downloaded from https://github.com/MolecularBioinformatics/sbml-mod-ws . We envision that SBMLmod will make automated model modification and simulation available to a broader research community.
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Modelos Teóricos , Interface Usuário-Computador , Linhagem Celular Tumoral , Humanos , Internet , Cinurenina/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Serotonina/metabolismo , Triptofano/metabolismoRESUMO
Subtilisins and other serine proteases are extensively used in the detergent, leather and food industry, and frequently under non-physiological conditions. New proteases with improved performance at extreme temperatures and in the presence of chemical additives may have great economical potential. The increasing availability of genetic sequences from different environments makes homology-based screening an attractive strategy for discovery of new proteases. A prerequisite for large-scale screening of protease-encoding sequences is an efficient screening procedure. We have developed and implemented a screening procedure that encompasses cloning of candidate sequences into multiple expression vectors, cytoplasmic expression in E. coli, and a casein-based functional screen. The procedure is plate-format compatible and can be completed in only four days, starting from the gene of interest in a suitable cloning vector. The expression vector suite includes six vectors with combinations of maltose-binding protein (MBP) or the small ubiquitin-related modifier (SUMO) for increased solubility, and polyhistidine tags for downstream purification. We used enhanced green fluorescent protein and four Bacilli subtilisins to validate the screening procedure and our results show that proteins were expressed, soluble and active. Interestingly, the highest activities were consistently achieved with either MBP or SUMO fusions, thus demonstrating the merit of including solubility tags. In conclusion, the results demonstrate that our approach can be used to efficiently screen for new subtilisins, and suggest that the approach may also be used to screen for proteins with other activities.
Assuntos
Proteínas de Bactérias/química , Clonagem Molecular/métodos , Escherichia coli/metabolismo , Proteínas Recombinantes/química , Subtilisinas/química , Bacillus licheniformis/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Escherichia coli/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Solubilidade , Subtilisinas/análise , Subtilisinas/metabolismoRESUMO
Enterotoxigenic Escherichia coli(ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity.
Assuntos
Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Escherichia coli/metabolismo , Toxoides/imunologia , Anticorpos Monoclonais , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos , Humanos , Modelos Moleculares , Mutagênese , Conformação ProteicaRESUMO
Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh.
Assuntos
Toxinas Bacterianas/metabolismo , Escherichia coli Enterotoxigênica/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Hormônios Gastrointestinais/metabolismo , Peptídeos Natriuréticos/metabolismo , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Clonagem Molecular , Escherichia coli Enterotoxigênica/genética , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/imunologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Hormônios Gastrointestinais/química , Hormônios Gastrointestinais/genética , Hormônios Gastrointestinais/imunologia , Regulação Bacteriana da Expressão Gênica/imunologia , Humanos , Modelos Moleculares , Peptídeos Natriuréticos/química , Peptídeos Natriuréticos/genética , Peptídeos Natriuréticos/imunologia , Ligação Proteica , Conformação ProteicaRESUMO
Tryptophan is utilized in various metabolic routes including protein synthesis, serotonin, and melatonin synthesis and the kynurenine pathway. Perturbations in these pathways have been associated with neurodegenerative diseases and cancer. Here we present a comprehensive kinetic model of the complex network of human tryptophan metabolism based upon existing kinetic data for all enzymatic conversions and transporters. By integrating tissue-specific expression data, modeling tryptophan metabolism in liver and brain returned intermediate metabolite concentrations in the physiological range. Sensitivity and metabolic control analyses identified expected key enzymes to govern fluxes in the branches of the network. Combining tissue-specific models revealed a considerable impact of the kynurenine pathway in liver on the concentrations of neuroactive derivatives in the brain. Moreover, using expression data from a cancer study predicted metabolite changes that resembled the experimental observations. We conclude that the combination of the kinetic model with expression data represents a powerful diagnostic tool to predict alterations in tryptophan metabolism. The model is readily scalable to include more tissues, thereby enabling assessment of organismal tryptophan metabolism in health and disease.