RESUMO
PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Regiões Promotoras Genéticas , Fenilacetatos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
A collection of repurposing drugs (Prestwick Chemical Library) containing 1200 compounds was screened to investigate the drugs' antimicrobial effects against planktonic cultures of the respiratory pathogen Streptococcus pneumoniae. After four discrimination rounds, a set of seven compounds was finally selected, namely (i) clofilium tosylate; (ii) vanoxerine; (iii) mitoxantrone dihydrochloride; (iv) amiodarone hydrochloride; (v) tamoxifen citrate; (vi) terfenadine; and (vii) clomiphene citrate (Z, E). These molecules arrested pneumococcal growth in a liquid medium and induced a decrease in bacterial viability between 90.0% and 99.9% at 25 µM concentration, with minimal inhibitory concentrations (MICs) also in the micromolar range. Moreover, all compounds but mitoxantrone caused a remarkable increase in the permeability of the bacterial membrane and share a common, minimal chemical structure consisting of an aliphatic amine linked to a phenyl moiety via a short carbon/oxygen linker. These results open new possibilities to tackle pneumococcal disease through drug repositioning and provide clues for the design of novel membrane-targeted antimicrobials with a related chemical structure.
Assuntos
Anti-Infecciosos , Infecções Pneumocócicas , Humanos , Streptococcus pneumoniae , Antibacterianos/farmacologia , Reposicionamento de Medicamentos , Mitoxantrona/farmacologia , Infecções Pneumocócicas/tratamento farmacológico , Anti-Infecciosos/farmacologia , Testes de Sensibilidade Microbiana , Membrana CelularRESUMO
Dynamic binding events are key to arrive at functionality in nature, and these events are often governed by electrostatic or hydrophobic interactions. Synthetic supramolecular polymers are promising candidates to obtain biomaterials that mimic this dynamicity. Here, we created four new functional monomers based on the benzene-1,3,5-tricarboxamide (BTA) motif. Choline or atropine groups were introduced to obtain functional monomers capable of competing with the cell wall of Streptococcus pneumoniae for binding of essential choline-binding proteins (CBPs). Atropine-functionalized monomers BTA-Atr and BTA-Atr3 were too hydrophobic to form homogeneous assemblies, while choline-functionalized monomers BTA-Chol and BTA-Chol3 were unable to form fibers due to charge repulsion. However, copolymerization of BTA-Chol3 with non-functionalized BTA-(OH)3 yielded dynamic fibers, similar to BTA-(OH)3. These copolymers showed an increased affinity toward CBPs compared to free choline due to multivalent effects. BTA-based supramolecular copolymers are therefore a versatile platform to design bioactive and dynamic supramolecular polymers with novel biotechnological properties.
Assuntos
Anti-Infecciosos , Streptococcus pneumoniae , Materiais Biocompatíveis/metabolismo , Colina/farmacologia , Polímeros/química , Streptococcus pneumoniae/metabolismoRESUMO
The ßß-solenoid structures are part of many proteins involved in the recognition of bacterial cell wall. They are elongated polypeptides consisting of repeated ß-hairpins connected by linker sequences and disposed around a superhelical axis stabilised by short-range interactions. Among the most studied ßß-solenoids are those belonging to the family of choline-binding modules (CBMs) from the respiratory pathogen Streptococcus pneumoniae (pneumococcus) and its bacteriophages, and their properties have been employed to develop several biotechnological and biomedical tools. We have carried out a theoretical, spectroscopic and thermodynamic study of the ßß-solenoid structure of the CBM from the pneumococcal LytA autolysin using peptides of increasing length containing 1-3 repeats of this structure. Our results show that hints of native-like tertiary structure are only observed with a minimum of three ß-hairpins, corresponding to one turn of the solenoid superhelix, and identify the linker sequences between hairpins as the major directors of the solenoid folding. This study paves the way for the rational structural engineering of ßß-solenoids aimed to find novel applications.
Assuntos
Proteínas de Bactérias/química , Colina/metabolismo , Streptococcus pneumoniae/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Fluorescência , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeos/metabolismo , Biossíntese de Proteínas , Estabilidade Proteica , Estrutura Secundária de Proteína , Desdobramento de Proteína , Soluções , TemperaturaRESUMO
Bacterial biopolymers are naturally occurring materials comprising a wide range of molecules with diverse chemical structures that can be produced from renewable sources following the principles of the circular economy. Over the last decades, they have gained substantial interest in the biomedical field as drug nanocarriers, implantable material coatings, and tissue-regeneration scaffolds or membranes due to their inherent biocompatibility, biodegradability into nonhazardous disintegration products, and their mechanical properties, which are similar to those of human tissues. The present review focuses upon three technologically advanced bacterial biopolymers, namely, bacterial cellulose (BC), polyhydroxyalkanoates (PHA), and γ-polyglutamic acid (PGA), as models of different carbon-backbone structures (polysaccharides, polyesters, and polyamides) produced by bacteria that are suitable for biomedical applications in nanoscale systems. This selection models evidence of the wide versatility of microorganisms to generate biopolymers by diverse metabolic strategies. We highlight the suitability for applied sustainable bioprocesses for the production of BC, PHA, and PGA based on renewable carbon sources and the singularity of each process driven by bacterial machinery. The inherent properties of each polymer can be fine-tuned by means of chemical and biotechnological approaches, such as metabolic engineering and peptide functionalization, to further expand their structural diversity and their applicability as nanomaterials in biomedicine.
RESUMO
D-amino acid oxidase (DAAO) is an enzyme that catalyzes the oxidation of D-amino acids generating H2O2. The enzymatic chimera formed by DAAO bound to the choline-binding domain of N-acetylmuramoyl-L-alanine amidase (CLytA) induces cytotoxicity in several pancreatic and colorectal carcinoma and glioblastoma cell models. In the current work, we determined whether the effect of CLytA-DAAO immobilized in magnetic nanoparticles, gold nanoparticles, and alginate capsules offered some advantages as compared to the free CLytA-DAAO. Results indicate that the immobilization of CLytA-DAAO in magnetic nanoparticles increases the stability of the enzyme, extending its time of action. Besides, we compared the effect induced by CLytA-DAAO with the direct addition of hydrogen peroxide, demonstrating that the progressive generation of reactive oxygen species by CLytA-DAAO is more effective in inducing cytotoxicity than the direct addition of H2O2. Furthermore, a pilot study has been initiated in biopsies obtained from pancreatic and colorectal carcinoma and glioblastoma patients to evaluate the expression of the main genes involved in resistance to CLytA-DAAO cytotoxicity. Based on our findings, we propose that CLytA-DAAO immobilized in magnetic nanoparticles could be effective in a high percentage of patients and, therefore, be used as an anti-cancer therapy for pancreatic and colorectal carcinoma and glioblastoma.
Assuntos
D-Aminoácido Oxidase/metabolismo , Nanopartículas de Magnetita/química , Neoplasias/terapia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/química , Linhagem Celular Tumoral , Neoplasias Colorretais/terapia , D-Aminoácido Oxidase/uso terapêutico , Glioblastoma/terapia , Humanos , Peróxido de Hidrogênio/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias Pancreáticas/terapia , Espécies Reativas de Oxigênio/toxicidade , Neoplasias PancreáticasRESUMO
The combination of the choline binding domain of the amidase N-acetylmuramoyl-L-alanine (CLytA)-D-amino acid oxidase (DAAO) (CLytA-DAAO) and D-Alanine induces cell death in several pancreatic and colorectal carcinoma and glioblastoma cell lines. In glioblastoma cell lines, CLytA-DAAO-induced cell death was inhibited by a pan-caspase inhibitor, suggesting a classical apoptotic cell death. Meanwhile, the cell death induced in pancreatic and colon carcinoma cell lines is some type of programmed necrosis. In this article, we studied the mechanisms that trigger CLytA-DAAO-induced cell death in pancreatic and colorectal carcinoma and glioblastoma cell lines and we acquire a further insight into the necrotic cell death induced in pancreatic and colorectal carcinoma cell lines. We have analyzed the intracellular calcium mobilization, mitochondrial membrane potential, PARP-1 participation and AIF translocation. Although the mitochondrial membrane depolarization plays a crucial role, our results suggest that CLytA-DAAO-induced cell death is context dependent. We have previously detected pancreatic and colorectal carcinoma cell lines (Hs766T and HT-29, respectively) that were resistant to CLytA-DAAO-induced cell death. In this study, we have examined the putative mechanism underlying the resistance in these cell lines, evaluating both detoxification mechanisms and the inflammatory and survival responses. Overall, our results provide a better understanding on the cell death mechanism induced by CLytA-DAAO, a promising therapy against cancer.
Assuntos
Fator de Indução de Apoptose/metabolismo , Neoplasias Colorretais/metabolismo , D-Aminoácido Oxidase/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Neoplasias Pancreáticas/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Antineoplásicos/farmacologia , Apoptose , Biópsia , Cálcio/metabolismo , Morte Celular , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Inflamação , Potencial da Membrana Mitocondrial , Subunidade p50 de NF-kappa B/metabolismo , Necrose , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for binding to the polyhydroxyalkanoate (PHA) granule. BioF has been exploited for biotechnological purposes as an affinity tag in the functionalization of PHA beads with fusion proteins both in vivo and in vitro The structural model of this domain suggests an amphipathic α-helical conformation with the hydrophobic residues facing the PHA granule. In this work, we analyzed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on PHA, stability on the surface of the PHA granule against pH, temperature, and ionic strength, and their possible influence on PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP) that retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing the functionality and stability of the fusion proteins MinP-ß-galactosidase and MinP-CueO from Escherichia coliIMPORTANCE Polyhydroxyalkanoates (PHAs) are biocompatible, nontoxic, and biodegradable biopolymers with exceptional applications in the industrial and medical fields. The complex structure of the PHA granule can be exploited as a toolbox to display molecules of interest on their surface. Phasins, the most abundant group of proteins on the granule, have been employed as anchoring tags to obtain functionalized PHA beads for high-affinity bioseparation, enzyme immobilization, diagnostics, or cell targeting. Here, a shorter module based on the previously designed BioF tag has been demonstrated to maintain the affinity for the PHA granule, with higher stability and similar functionalization efficiency. The use of a 67% shorter peptide, which maintains the binding properties of the entire protein, constitutes an advantage for the immobilization of recombinant proteins on the PHA surface both in vitro and in vivo.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas putida/metabolismo , Biotecnologia , Enzimas Imobilizadas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes/metabolismoRESUMO
Choline-binding proteins (CBPs) from Streptococcus pneumoniae comprise a family of modular polypeptides involved in essential events of this pathogen. They recognize the choline residues present in the teichoic and lipoteichoic acids of the cell wall using the so-called choline-binding modules (CBMs). The importance of CBPs in pneumococcal physiology points to them as novel targets to combat antimicrobial resistances shown by this organism. In this work we have tested the ability of exogenously added CBMs to act as CBP inhibitors by competing with the latter for the binding to the choline molecules in the bacterial surface. First, we carried out a thorough physicochemical characterization of three native CBMs, namely C-LytA, C-Cpl1, and C-CbpD, and assessed their affinity for choline and macromolecular, pneumococcal cell-wall mimics. The interaction with these substrates was evaluated by molecular modeling, analytical ultracentrifugation, surface plasmon resonance, and fluorescence and circular dichroism spectroscopies. Van't Hoff thermal analyses unveiled the existence of one noncanonical choline binding site in each of the C-Cpl1 and C-CbpD proteins, leading in total to 5 ligand-binding sites per dimer and 4 sites per monomer, respectively. Remarkably, the binding affinities of the CBMs do not directly correlate with their native oligomeric state or with the number of choline-binding sites, suggesting that choline recognition by these modules is a complex phenomenon. On the other hand, the exogenous addition of CBMs to pneumococcal planktonic cultures caused extensive cell-chaining probably as a consequence of the inhibition of CBP attachment to the cell wall. This was accompanied by bacterial aggregation and sedimentation, causing an enhancement of bacterial phagocytosis by peritoneal macrophages. In addition, the rational design of an oligomeric variant of a native CBM led to a substantial increase in its antibacterial activity by multivalency effects. These results suggest that CBMs might constitute promising nonlytic antimicrobial candidates based on the natural induction of the host defense system.
Assuntos
Amidoidrolases , Proteínas de Bactérias , Colina , Macrófagos Peritoneais/citologia , Fagocitose , Streptococcus pneumoniae , Animais , Sítios de Ligação , Camundongos , Modelos MolecularesRESUMO
D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids generating hydrogen peroxide, a potential producer of reactive oxygen species. In this study, we used a CLytA-DAAO chimera, both free and bound to magnetic nanoparticles, against colon carcinoma, pancreatic adenocarcinoma, and glioblastoma cell lines. We found that the enzyme induces cell death in most of the cell lines tested and its efficiency increases significantly when it is immobilized in nanoparticles. We also tested this enzyme therapy in non-tumor cells, and we found that there is not cell death induction, or it is significantly lower than in tumor cells. The mechanism triggering cell death is apparently a classical apoptosis pathway in the glioblastoma cell lines, while in colon and pancreatic carcinoma cell lines, CLytA-DAAO-induced cell death is a necrosis. Our results constitute a proof of concept that an enzymatic therapy, based on magnetic nanoparticles-delivering CLytA-DAAO, could constitute a useful therapy against cancer and besides it could be used as an enhancer of other treatments such as epigenetic therapy, radiotherapy, and treatments based on DNA repair.
Assuntos
Apoptose , Colina/química , D-Aminoácido Oxidase/química , Nanopartículas de Magnetita/química , N-Acetil-Muramil-L-Alanina Amidase/química , Necrose , Células 3T3-L1 , Adenocarcinoma/patologia , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias do Colo/patologia , Dano ao DNA , Reparo do DNA , Glioblastoma/patologia , Humanos , Concentração Inibidora 50 , Camundongos , Neoplasias Pancreáticas/patologia , Espécies Reativas de Oxigênio/químicaRESUMO
A common interpretation of Anfinsen's hypothesis states that one amino acid sequence should fold into a single, native, ordered state, or a highly similar set thereof, coinciding with the global minimum in the folding-energy landscape, which, in turn, is responsible for the function of the protein. However, this classical view is challenged by many proteins and peptide sequences, which can adopt exchangeable, significantly dissimilar conformations that even fulfill different biological roles. The similarities and differences of concepts related to these proteins, mainly chameleon sequences, metamorphic proteins, and switch peptides, which are all denoted herein "turncoat" polypeptides, are reviewed. As well as adding a twist to the conventional view of protein folding, the lack of structural definition adds clear versatility to the activity of proteins and can be used as a tool for protein design and further application in biotechnology and biomedicine.
Assuntos
Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Sequência de Aminoácidos , Modelos Moleculares , TermodinâmicaRESUMO
BACKGROUND: Phasins are low molecular mass proteins that accumulate strongly in bacterial cells in response to the intracellular storage of polyhydroxyalkanoates (PHA). Although lacking catalytic activity, phasins are the major components of the surface of the PHA granules and could be potentially involved in the formation of a network-like protein layer surrounding the polyester inclusions. Structural models revealed phasins to possess coiled-coil regions that might be important in the establishment of protein-protein interactions. However, there is not experimental evidence of a coiled-coil mediated oligomerization in these proteins. METHODS: Structure prediction analyses were used to characterize the coiled-coil motifs of phasins PhaF and PhaI -produced by the model bacterium Pseudomonas putida KT2440-. Their oligomerization was evaluated by biolayer interferometry and the in vivo two-hybrid (BACTH) system. The interaction ability of a series of coiled-coil mutated derivatives was also measured. RESULTS: The formation of PhaF and PhaI complexes was detected. A predicted short leucine zipper-like coiled-coil (ZIP), containing "ideal" residues located within the hydrophobic core, was shown responsible for the oligomers stability. The substitution of key residues (leucines or valines) in PhaI ZIP (ZIPI) for alanine reduced by four fold the oligomerization efficiency. CONCLUSIONS: These results indicate that coiled-coil motifs are essential for phasin interactions. Correct oligomerization requires the formation of a stable hydrophobic interface between both phasins. GENERAL SIGNIFICANCE: Our findings elucidate the oligomerization motif of PhaF and PhaI. This motif is present in most phasins from PHA-accumulating bacteria and offers a potentially important target for modulating the PHA granules stability.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Zíper de Leucina , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas putida/metabolismo , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Interações Hidrofóbicas e Hidrofílicas , Poli-Hidroxialcanoatos/química , Poli-Hidroxialcanoatos/genéticaRESUMO
Antibiotic resistance is a global current threat of increasing importance. Moreover, biofilms represent a medical challenge since the inherent antibiotic resistance of their producers demands the use of high doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence, therefore demanding the development of novel antimicrobials. Esters of bicyclic amines (EBAs), which are strong inhibitors of Streptococcus pneumoniae growth, were initially designed as inhibitors of pneumococcal choline-binding proteins on the basis of their structural analogy to the choline residues in the cell wall. However, instead of mimicking the characteristic cell chaining phenotype caused by exogenously added choline on planktonic cultures of pneumococcal cells, EBAs showed an unexpected lytic activity. In this work we demonstrate that EBAs display a second, and even more important, function as cell membrane destabilizers. We then assayed the inhibitory and disintegrating activity of these molecules on pneumococcal biofilms. The selected compound (EBA 31) produced the highest effect on S. pneumoniae (encapsulated and non-encapsulated) biofilms at very low concentrations. EBA 31 was also effective on mixed biofilms of non-encapsulated S. pneumoniae plus non-typeable Haemophilus influenzae, two pathogens frequently forming a self-produced biofilm in the human nasopharynx. These results support the role of EBAs as a promising alternative for the development of novel, broad-range antimicrobial drugs encompassing both Gram-positive and Gram-negative pathogens.
Assuntos
Anti-Infecciosos/farmacologia , Biofilmes , Ésteres/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Streptococcus pneumoniae/efeitos dos fármacos , Aminas/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Farmacorresistência Bacteriana , N-Acetil-Muramil-L-Alanina Amidase/química , Permeabilidade/efeitos dos fármacosRESUMO
The presence of synthetic dyes in wastewaters generated by the textile industry constitutes a serious environmental and health problem that urges the scientific community on an appropriate action. As a proof-of-concept, we have developed a novel approach to design enzymatic bioreactors with the ability to decolorize dye solutions through the immobilization of the bacterial CueO laccase-like multicopper oxidase from Escherichia coli on polyhydroxybutyrate (PHB) beads by making use of the BioF affinity tag. The decolorization efficiency of the system was characterized by a series of parameters, namely maximum enzyme adsorption capacity, pH profile, kinetic constants, substrate range, temperature and bioreactor recycling. Depending on the tested dye, immobilization increased the catalytic activity of CueO by up to 40-fold with respect to the soluble enzyme, reaching decolorization efficiencies of 45-90%. Our results indicate that oxidase bioreactors based on polyhydroxyalkanoates are a promising alternative for the treatment of coloured industrial wastewaters.
Assuntos
Corantes/metabolismo , Enzimas Imobilizadas/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidroxibutiratos/metabolismo , Oxirredutases/metabolismo , Poliésteres/metabolismo , Águas Residuárias/química , Poluentes Químicos da Água/metabolismo , Reatores Biológicos , Concentração de Íons de Hidrogênio , Cinética , Ligação Proteica , Especificidade por Substrato , TemperaturaRESUMO
Choline-binding repeats (CBRs) are ubiquitous sequences with a ß-hairpin core that are found in the surface proteins of several microorganisms such as S. pneumoniae (pneumococcus). Previous studies on a 14-mer CBR sequence derived from the pneumoccal LytA autolysin (LytA239-252 peptide) have demonstrated a switch behaviour for this peptide, so that it acquires a stable, native-like ß-hairpin conformation in aqueous solution but is reversibly transformed into an amphipathic α-helix in the presence of detergent micelles. With the aim of understanding the factors responsible for this unusual ß-hairpin to α-helix transition, and to specifically assess the role of peptide hydrophobicity and helical amphipathicity in the process, we designed a series of LytA239-252 variants affecting these two parameters and studied their interaction with dodecylphosphocholine (DPC) micelles by solution NMR, circular dichroism and fluorescence spectroscopies. Our results indicate that stabilising cross-strand interactions become essential for ß-hairpin stability in the absence of optimal turn sequences. Moreover, both amphipathicity and hydrophobicity display comparable importance for helix stabilisation of CBR-derived peptides in micelles, indicating that these sequences represent a novel class of micelle/membrane-interacting peptides.
Assuntos
Colina/metabolismo , Micelas , Peptídeos/química , Colina/química , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Ressonância Magnética Nuclear BiomolecularRESUMO
Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that accumulate in the cytoplasm of certain bacteria. One promising biotechnological application utilizes these biopolymers as supports for protein immobilization. Here, the PHA-binding domain of the Pseudomonas putida KT2440 PhaF phasin (BioF polypeptide) was investigated as an affinity tag for the in vitro functionalization of poly-3-hydroxybutyrate (PHB) particles with recombinant proteins, namely, full-length PhaF and two fusion proteins tagged to BioF (BioF-C-LytA and BioF-ß-galactosidase, containing the choline-binding module C-LytA and the ß-galactosidase enzyme, respectively). The protein-biopolyester interaction was strong and stable at a wide range of pHs and temperatures, and the bound protein was highly protected from self-degradation, while the binding strength could be modulated by coating with amphiphilic compounds. Finally, BioF-ß-galactosidase displayed very stable enzymatic activity after several continuous activity-plus-washing cycles when immobilized in a minibioreactor. Our results demonstrate the potentialities of PHA and the BioF tag for the construction of novel bioactive materials.IMPORTANCE Our results confirm the biotechnological potential of the BioF affinity tag as a versatile tool for functionalizing PHA supports with recombinant proteins, leading to novel bioactive materials. The wide substrate range of the BioF tag presumably enables protein immobilization in vitro of virtually all natural PHAs as well as blends, copolymers, or artificial chemically modified derivatives with novel physicochemical properties. Moreover, the strength of protein adsorption may be easily modulated by varying the coating of the support, providing new perspectives for the engineering of bioactive materials that require a tight control of protein loading.
Assuntos
Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas , Lectinas de Plantas/química , Pseudomonas putida/metabolismo , Proteínas Recombinantes de Fusão/químicaRESUMO
Polyhydroxyalkanoates (PHAs) are natural polyesters of increasing biotechnological importance that are synthesized by many prokaryotic organisms as carbon and energy storage compounds in limiting growth conditions. PHAs accumulate intracellularly in form of inclusion bodies that are covered with a proteinaceous surface layer (granule-associated proteins or GAPs) conforming a network-like surface of structural, metabolic and regulatory polypeptides, and configuring the PHA granules as complex and well-organized subcellular structures that have been designated as 'carbonosomes'. GAPs include several enzymes related to PHA metabolism (synthases, depolymerases and hydroxylases) together with the so-called phasins, an heterogeneous group of small-size proteins that cover most of the PHA granule and that are devoid of catalytic functions but nevertheless play an essential role in granule structure and PHA metabolism. Structurally, phasins are amphiphilic proteins that shield the hydrophobic polymer from the cytoplasm. Here, we summarize the characteristics of the different phasins identified so far from PHA producer organisms and highlight the diverse opportunities that they offer in the Biotechnology field.
Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Filogenia , Lectinas de Plantas/genética , Poli-Hidroxialcanoatos/metabolismo , Bactérias/química , Bactérias/classificação , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/metabolismoRESUMO
Walk on the small side. Nanotechnology meets Microbiology thanks to the high versatility of synthetic routes in microorganisms, leading to the production of nanoparticles of biotechnological and biomedical interest.
Assuntos
Bactérias/metabolismo , Biotecnologia/métodos , Nanopartículas/metabolismo , Biotecnologia/tendências , Redes e Vias MetabólicasRESUMO
Streptococcus pneumoniae (pneumococcus) is an important pathogen responsible for acute invasive and non-invasive infections such as meningitis, sepsis and otitis media, being the major cause of community-acquired pneumonia. The fight against pneumococcus is currently hampered both by insufficient vaccine coverage and by rising antimicrobial resistances to traditional antibiotics, making necessary the research on novel targets. Choline binding proteins (CBPs) are a family of polypeptides found in pneumococcus and related species, as well as in some of their associated bacteriophages. They are characterized by a structural organization in two modules: a functional module (FM), and a choline-binding module (CBM) that anchors the protein to the choline residues present in the cell wall through non-covalent interactions. Pneumococcal CBPs include cell wall hydrolases, adhesins and other virulence factors, all playing relevant physiological roles for bacterial viability and virulence. Moreover, many pneumococcal phages also make use of hydrolytic CBPs to fulfill their infectivity cycle. Consequently, CBPs may play a dual role for the development of novel antipneumococcal drugs, both as targets for inhibitors of their binding to the cell wall and as active cell lytic agents (enzybiotics). In this article, we review the current state of knowledge about host- and phage-encoded pneumococcal CBPs, with a special focus on structural issues, together with their perspectives for effective anti-infectious treatments.
RESUMO
A double approach was followed in the search of novel inhibitors of the surface choline-binding proteins (CBPs) of Streptococcus pneumoniae (pneumococcus) with antimicrobial properties. First, a library of 49 rationally-designed esters of alkyl amines was screened for their specific binding to CBPs. The best binders, being esters of bicyclic amines (EBAs), were then tested for their inâ vitro effect on pneumococcal growth and morphology. Second, the efficiency of EBA-induced CBP inhibition was enhanced about 45,000-fold by multivalency effects upon synthesizing a poly(propylene imine) dendrimer containing eight copies of an atropine derivative. Both approaches led to compounds that arrest bacterial growth, dramatically decrease cell viability, and exhibit a protection effect in animal disease models, demonstrating that the pneumococcal CBPs are adequate targets for the discovery of novel antimicrobials that overcome the currently increasing antimicrobial resistance issues.