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1.
J Pept Sci ; : e3647, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-39091086

RESUMEN

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.

2.
Appl Environ Microbiol ; 89(6): e0039023, 2023 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-37222584

RESUMEN

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.


Asunto(s)
Metilaminas , Hidrolisados de Proteína , Animales , Metilaminas/metabolismo
3.
Appl Microbiol Biotechnol ; 105(8): 3195-3209, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33770243

RESUMEN

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.


Asunto(s)
Metagenoma , Péptido Hidrolasas , Animales , Bacterias/genética , Endopeptidasas , Péptido Hidrolasas/genética , Temperatura
4.
Mar Drugs ; 19(2)2021 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-33562153

RESUMEN

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.


Asunto(s)
Antivirales/farmacología , Microalgas/metabolismo , Algas Marinas/metabolismo , Agricultura , Acuicultura , Proteínas Bacterianas/farmacología , Ensayos Clínicos como Asunto , Diterpenos/farmacología , Lectinas/farmacología , Proteínas de la Membrana/farmacología , Lectinas de Plantas/farmacología , Polisacáridos/farmacología
5.
Appl Environ Microbiol ; 87(1)2020 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-33097498

RESUMEN

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.


Asunto(s)
Bacillus subtilis/genética , Sistemas CRISPR-Cas , Edición Génica , Péptido Hidrolasas/genética , Plásmidos/genética , Bacillus subtilis/enzimología , Péptido Hidrolasas/metabolismo , Plásmidos/metabolismo
6.
Appl Environ Microbiol ; 86(24)2020 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-32978141

RESUMEN

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.


Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Metilaminas/metabolismo , Oxigenasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Oxigenasas/química , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia
7.
Infect Immun ; 87(7)2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31061144

RESUMEN

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.


Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Toxinas Bacterianas/inmunología , Escherichia coli Enterotoxigénica/inmunología , Enterotoxinas/inmunología , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Hormonas Gastrointestinales/inmunología , Péptidos Natriuréticos/inmunología , Animales , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Reacciones Cruzadas , Escherichia coli Enterotoxigénica/genética , Enterotoxinas/administración & dosificación , Enterotoxinas/química , Enterotoxinas/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/genética , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Porcinos
8.
Proteins ; 86(9): 965-977, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29907987

RESUMEN

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.


Asunto(s)
Endopeptidasas/genética , Péptidos/genética , Planococcus (Bacteria)/enzimología , Subtilisinas/genética , Organismos Acuáticos , Calcio/química , Catálisis , Endopeptidasas/química , Endopeptidasas/metabolismo , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Mutación , Péptidos/química , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Subtilisinas/metabolismo , Temperatura
9.
BMC Bioinformatics ; 18(1): 314, 2017 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-28646877

RESUMEN

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.


Asunto(s)
Modelos Teóricos , Interfaz Usuario-Computador , Línea Celular Tumoral , Humanos , Internet , Quinurenina/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Serotonina/metabolismo , Triptófano/metabolismo
10.
Infect Immun ; 84(4): 1239-1249, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26883587

RESUMEN

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.


Asunto(s)
Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Escherichia coli/metabolismo , Toxoides/inmunología , Anticuerpos Monoclonales , Línea Celular Tumoral , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos , Humanos , Modelos Moleculares , Mutagénesis , Conformación Proteica
11.
Am J Hum Genet ; 90(4): 727-33, 2012 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-22444669

RESUMEN

Genome-wide association studies (GWASs) are critically dependent on detailed knowledge of the pattern of linkage disequilibrium (LD) in the human genome. GWASs generate lists of variants, usually SNPs, ranked according to the significance of their association to a trait. Downstream analyses generally focus on the gene or genes that are physically closest to these SNPs and ignore their LD profile with other SNPs. We have developed a flexible R package (LDsnpR) that efficiently assigns SNPs to genes on the basis of both their physical position and their pairwise LD with other SNPs. We used the positional-binning and LD-based-binning approaches to investigate whether including these "LD-based" SNPs would affect the interpretation of three published GWASs on bipolar affective disorder (BP) and of the imputed versions of two of these GWASs. We show how including LD can be important for interpreting and comparing GWASs. In the published, unimputed GWASs, LD-based binning effectively "recovered" 6.1%-8.3% of Ensembl-defined genes. It altered the ranks of the genes and resulted in nonnegligible differences between the lists of the top 2,000 genes emerging from the two binning approaches. It also improved the overall gene-based concordance between independent BP studies. In the imputed datasets, although the increases in coverage (>0.4%) and rank changes were more modest, even greater concordance between the studies was observed, attesting to the potential of LD-based binning on imputed data as well. Thus, ignoring LD can result in the misinterpretation of the GWAS findings and have an impact on subsequent genetic and functional studies.


Asunto(s)
Estudio de Asociación del Genoma Completo/estadística & datos numéricos , Desequilibrio de Ligamiento/genética , Trastorno Bipolar/genética , Interpretación Estadística de Datos , Humanos , Polimorfismo de Nucleótido Simple , Programas Informáticos/estadística & datos numéricos
12.
J Biol Chem ; 288(48): 34555-66, 2013 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-24129579

RESUMEN

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.


Asunto(s)
Hígado/enzimología , Redes y Vías Metabólicas/genética , Modelos Teóricos , Triptófano/metabolismo , Humanos , Cinética , Quinurenina/metabolismo , Hígado/metabolismo , Especificidad de Órganos , Transcriptoma , Triptófano/genética
13.
Infect Immun ; 82(7): 2913-22, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24778111

RESUMEN

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.


Asunto(s)
Toxinas Bacterianas/metabolismo , Escherichia coli Enterotoxigénica/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Hormonas Gastrointestinales/metabolismo , Péptidos Natriuréticos/metabolismo , Secuencia de Aminoácidos , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Clonación Molecular , Escherichia coli Enterotoxigénica/genética , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/inmunología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Hormonas Gastrointestinales/química , Hormonas Gastrointestinales/genética , Hormonas Gastrointestinales/inmunología , Regulación Bacteriana de la Expresión Génica/inmunología , Humanos , Modelos Moleculares , Péptidos Natriuréticos/química , Péptidos Natriuréticos/genética , Péptidos Natriuréticos/inmunología , Unión Proteica , Conformación Proteica
14.
Front Microbiol ; 14: 1108018, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36778846

RESUMEN

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.

15.
Nucleic Acids Res ; 38(Database issue): D167-80, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19920119

RESUMEN

Linear motifs are short segments of multidomain proteins that provide regulatory functions independently of protein tertiary structure. Much of intracellular signalling passes through protein modifications at linear motifs. Many thousands of linear motif instances, most notably phosphorylation sites, have now been reported. Although clearly very abundant, linear motifs are difficult to predict de novo in protein sequences due to the difficulty of obtaining robust statistical assessments. The ELM resource at http://elm.eu.org/ provides an expanding knowledge base, currently covering 146 known motifs, with annotation that includes >1300 experimentally reported instances. ELM is also an exploratory tool for suggesting new candidates of known linear motifs in proteins of interest. Information about protein domains, protein structure and native disorder, cellular and taxonomic contexts is used to reduce or deprecate false positive matches. Results are graphically displayed in a 'Bar Code' format, which also displays known instances from homologous proteins through a novel 'Instance Mapper' protocol based on PHI-BLAST. ELM server output provides links to the ELM annotation as well as to a number of remote resources. Using the links, researchers can explore the motifs, proteins, complex structures and associated literature to evaluate whether candidate motifs might be worth experimental investigation.


Asunto(s)
Secuencias de Aminoácidos/genética , Biología Computacional/métodos , Bases de Datos Genéticas , Bases de Datos de Ácidos Nucleicos , Células Eucariotas/química , Secuencia de Aminoácidos , Animales , Biología Computacional/tendencias , Bases de Datos de Proteínas , Humanos , Almacenamiento y Recuperación de la Información/métodos , Internet , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Programas Informáticos
16.
Vaccines (Basel) ; 10(2)2022 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-35214698

RESUMEN

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.

17.
Toxins (Basel) ; 14(11)2022 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-36356004

RESUMEN

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.


Asunto(s)
Drosophila melanogaster , Nematodos , Animales , Insectos , Agentes de Control Biológico , Virulencia
18.
Bioinformatics ; 26(18): i540-6, 2010 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-20823319

RESUMEN

MOTIVATION: The world-wide community of life scientists has access to a large number of public bioinformatics databases and tools, which are developed and deployed using diverse technologies and designs. More and more of the resources offer programmatic web-service interface. However, efficient use of the resources is hampered by the lack of widely used, standard data-exchange formats for the basic, everyday bioinformatics data types. RESULTS: BioXSD has been developed as a candidate for standard, canonical exchange format for basic bioinformatics data. BioXSD is represented by a dedicated XML Schema and defines syntax for biological sequences, sequence annotations, alignments and references to resources. We have adapted a set of web services to use BioXSD as the input and output format, and implemented a test-case workflow. This demonstrates that the approach is feasible and provides smooth interoperability. Semantics for BioXSD is provided by annotation with the EDAM ontology. We discuss in a separate section how BioXSD relates to other initiatives and approaches, including existing standards and the Semantic Web. AVAILABILITY: The BioXSD 1.0 XML Schema is freely available at http://www.bioxsd.org/BioXSD-1.0.xsd under the Creative Commons BY-ND 3.0 license. The http://bioxsd.org web page offers documentation, examples of data in BioXSD format, example workflows with source codes in common programming languages, an updated list of compatible web services and tools and a repository of feature requests from the community.


Asunto(s)
Biología Computacional/métodos , Almacenamiento y Recuperación de la Información , Internet , Lenguajes de Programación , Secuencia de Aminoácidos , Almacenamiento y Recuperación de la Información/normas , Datos de Secuencia Molecular , Semántica , Programas Informáticos , Flujo de Trabajo
19.
Infect Immun ; 78(5): 1824-31, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20231404

RESUMEN

Enterotoxigenic Escherichia coli (ETEC) is responsible for 280 million to 400 million episodes of diarrhea and about 380,000 deaths annually. Epidemiological data suggest that ETEC strains which secrete heat-stable toxin (ST), alone or in combination with heat-labile toxin (LT), induce the most severe disease among children in developing countries. This makes ST an attractive target for inclusion in an ETEC vaccine. ST is released upon colonization of the small intestine and activates the guanylate cyclase C receptor, causing profuse diarrhea. To generate a successful toxoid, ST must be made immunogenic and nontoxic. Due to its small size, ST is nonimmunogenic in its natural form but becomes immunogenic when coupled to an appropriate large-molecular-weight carrier. This has been successfully achieved with several carriers, using either chemical conjugation or recombinant fusion techniques. Coupling of ST to a carrier may reduce toxicity, but further reduction by mutagenesis is desired to obtain a safe vaccine. More than 30 ST mutants with effects on toxicity have been reported. Some of these mutants, however, have lost the ability to elicit neutralizing immune responses to the native toxin. Due to the small size of ST, separating toxicity from antigenicity is a particular challenge that must be met. Another obstacle to vaccine development is possible cross-reactivity between anti-ST antibodies and the endogenous ligands guanylin and uroguanylin, caused by structural similarity to ST. Here we review the molecular and biological properties of ST and discuss strategies for developing an ETEC vaccine that incorporates immunogenic and nontoxic derivatives of the ST toxin.


Asunto(s)
Toxinas Bacterianas/inmunología , Escherichia coli Enterotoxigénica/inmunología , Enterotoxinas/inmunología , Vacunas contra Escherichia coli/inmunología , Secuencia de Aminoácidos , Toxinas Bacterianas/toxicidad , Enterotoxinas/toxicidad , Proteínas de Escherichia coli , Vacunas contra Escherichia coli/efectos adversos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína
20.
Mol Cancer ; 9: 173, 2010 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-20594292

RESUMEN

BACKGROUND: The tumor suppressor pRb plays a key role regulating cell cycle arrest, and disturbances in the RB1 gene have been reported in different cancer forms. However, the literature reports contradictory findings with respect to a pro--versus anti--apoptotic role of pRb, and the consequence of alterations in RB1 to chemotherapy sensitivity remains unclear. This study is part of a project investigating alterations in pivotal genes as predictive factors to chemotherapy sensitivity in breast cancer. RESULTS: Analyzing 73 locally advanced (stage III) breast cancers, we identified two somatic and one germline single nucleotide changes, each leading to amino acid substitution in the pRb protein (Leu607Ile, Arg698Trp, and Arg621Cys, respectively). This is the first study reporting point mutations affecting RB1 in breast cancer tissue. In addition, MLPA analysis revealed two large multiexon deletions (exons 13 to 27 and exons 21 to 23) with the exons 21-23 deletion occurring in the tumor also harboring the Leu607Ile mutation. Interestingly, Leu607Ile and Arg621Cys point mutations both localize to the spacer region of the pRb protein, a region previously shown to harbor somatic and germline mutations. Multiple sequence alignment across species indicates the spacer to be evolutionary conserved. All three RB1 point mutations encoded nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro. Notably, three out of four tumors harboring RB1 mutations displayed primary resistance to treatment with either 5-FU/mitomycin or doxorubicin while only 14 out of 64 tumors without mutations were resistant (p = 0.046). CONCLUSIONS: Although rare, our findings suggest RB1 mutations to be of pathological importance potentially affecting sensitivity to mitomycin/anthracycline treatment in breast cancer.


Asunto(s)
Apoptosis/genética , Neoplasias de la Mama/genética , Genes de Retinoblastoma , Mutación Puntual , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Neoplasias de la Mama/patología , Exones , Femenino , Humanos , Pérdida de Heterocigocidad , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Homología de Secuencia de Aminoácido
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