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1.
J Agric Food Chem ; 67(42): 11703-11709, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31578056

RESUMO

Astaxanthin is a carotenoid of high commercial value because of its excellent antioxidative, anti-inflammatory, and anticancer properties. Here, we developed a novel strategy for improving the production of astaxanthin via morphology and oxidative stress engineering. First, we identified the morphology-/membrane- and oxidative stress-related genes, which should be knocked down, using the CRISPRi system. Deleting the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) generated longer and larger cells with higher reactive oxygen species (ROS) levels, thus enhancing the production of astaxanthin and decreasing cell growth. To not only improve cell growth but also obtain longer and larger cells with higher ROS levels, a complementary expression system using a temperature-sensitive plasmid was established. Complementarily expressing the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) further improved the production of astaxanthin to 11.92 mg/g dry cell weight in shake flask cultures.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/citologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Engenharia Metabólica , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Xantofilas/biossíntese
2.
J Med Microbiol ; 68(10): 1540-1543, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31483245

RESUMO

Four group A streptococcus (GAS) bacteraemia occurred in a small burn unit within 2 weeks. The GAS patient isolates, characterized as emm89, shared the same PFGE pulsotype with two other strains isolated 2 months later. The outbreak investigation revealed that a nurse was the most likely source of GAS transmission, as she was confirmed to carry the same outbreak strain in her throat and had direct and regular contact with the six outbreak patients in the unit. The outbreak was controlled after the nurse had undergone eradication treatment. This report highlights the emergence of the emm89 clone and its capacity to elicit invasive GAS outbreaks.


Assuntos
Unidades de Queimados/estatística & dados numéricos , Infecção Hospitalar/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/isolamento & purificação , Adulto , Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Infecção Hospitalar/epidemiologia , Surtos de Doenças , Feminino , Humanos , Pessoa de Meia-Idade , Epidemiologia Molecular , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Tunísia , Adulto Jovem
3.
Chemistry ; 25(50): 11635-11640, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31368214

RESUMO

Disulfide-containing detergents (DCDs) are introduced, which contain a disulfide bond in the hydrophobic tail. DCDs form smaller micelles than corresponding detergents with linear hydrocarbon chains, while providing good solubilization and reconstitution of membrane proteins. The use of this new class of detergents in structural biology is illustrated with solution NMR spectra of the human G protein-coupled receptor A2A AR, which is an α-helical protein, and the ß-barrel protein OmpX from E. coli.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Detergentes/química , Proteínas de Escherichia coli/química , Hidrolases/química , Receptor A2A de Adenosina/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Dissulfetos/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Hidrolases/metabolismo , Micelas , Ressonância Magnética Nuclear Biomolecular , Estabilidade Proteica , Receptor A2A de Adenosina/metabolismo
4.
Nat Commun ; 10(1): 3073, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300643

RESUMO

Many organisms regulate their social life through kin recognition, but the underlying mechanisms are poorly understood. Here, we use a social bacterium, Myxococcus xanthus, to investigate kin recognition at the molecular level. By direct visualization of a cell surface receptor, TraA, we show how these myxobacteria identify kin and transition towards multicellularity. TraA is fluid on the cell surface, and homotypic interactions between TraA from juxtaposed cells trigger the receptors to coalesce, representing a 'molecular handshake'. Polymorphisms within TraA govern social recognition such that receptors cluster only between individuals bearing compatible alleles. TraA clusters, which resemble eukaryotic gap junctions, direct the robust exchange of cellular goods that allows heterogeneous populations to transition towards homeostasis. This work provides a conceptual framework for how microbes use a fluid outer membrane receptor to recognize and assemble kin cells into a cooperative multicellular community that resembles a tissue.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Myxococcus xanthus/fisiologia , Percepção de Quorum/fisiologia , Receptores de Superfície Celular/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Polimorfismo Genético , Receptores de Superfície Celular/genética
5.
Biochemistry (Mosc) ; 84(6): 672-685, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238867

RESUMO

Mature pore-forming OmpF protein from the outer membrane of Yersinia pseudotuberculosis was expressed in Escherichia coli in the form of inclusion bodies (IBs) under different cultivation conditions. The properties and structural organization of the IBs as well as the structure of the recombinant porin (rOmpF) solubilized from the IBs were investigated using electron microscopy, dynamic light scattering, optical spectroscopy, and specific hydrophobic dyes. The size, shape, and stability of the IBs under denaturing solutions were determined. It was found that the IBs were readily soluble in SDS and more resistant to urea. Dissolution of the IBs in both denaturing agents led to formation of a heterogeneous in size population of oligomeric particles. The IBs contained an intermediate form of the rOmpF with native-like secondary structure and elements of tertiary structure, which was able to penetrate a lipid bilayer and adopt a functionally active conformation. There were no significant differences in the properties and structure between the examined IBs formed at different concentrations of the inducer (IPTG). However, the content of amyloids in the IBs increased with increasing concentration of the inducer. These results contribute to the development of new approaches for the production of active proteins from IBs, as well as biologically and functionally active IBs.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Corpos de Inclusão/metabolismo , Porinas/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Microscopia Eletrônica de Varredura , Porinas/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
6.
Microbiol Res ; 223-225: 79-87, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178055

RESUMO

Vibrio parahaemolyticus is a seafood-borne Gram-negative bacteria causing diarrheal diseases in humans world wide. ToxR is a membrane-associated transcriptional factor which plays an important role in acid stress tolerance and regulates the expression of virulence genes including type III secretion system 1 (T3SS1) and type VI secretion system 1 (T6SS1) in V. parahaemolyticus. However, possible mechanisms of ToxR mediating virulence gene expression have not been fully understood. In this study, we demonstrated that ToxR is essential for V. parahaemolyticus to tolerate acid stress by constructing a ToxR deletion mutant (ΔtoxR) and its complemented strain (toxR+). Quantitative PCR showed that the expression of toxR was up regulated under acid stress condition. RNA-seq analysis showed that ompU encoding one of outer membrane proteins was dramatically down regulated in ΔtoxR. Furthermore, the mutation of ompU also led to a significant reduction in tolerating acid stress indicating that ToxR mediated acid stress through regulating ompU expression. RNA-seq results further confirmed that acid stress condition could alter multiple signaling pathways either depending on ToxR (e.g., quorum sensing, fatty acid metabolism) or independent of ToxR (e.g., biosynthesis of secondary metabolites, microbial metabolism in diverse environment, biosynthesis of antibiotics, biosynthesis of amino acids and carbon metabolism pathways). We also for the first time demonstrated that ToxR positively regulated the expression of T6SS2 gene and the interbacteria killing activity. Our study provides comprehensive understanding of signaling pathways which are regulated by both acid stress and ToxR.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Vibrio parahaemolyticus/metabolismo , Adesinas Bacterianas/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Ácidos Graxos/metabolismo , Percepção de Quorum , Metabolismo Secundário , Análise de Sequência de RNA , Deleção de Sequência , Fatores de Transcrição/genética , Transcriptoma , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo VI/genética , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/crescimento & desenvolvimento , Virulência/genética
7.
Subcell Biochem ; 92: 39-77, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214984

RESUMO

The Lpp lipoprotein of Escherichia coli is the first identified protein with a covalently linked lipid. It is chemically bound by its C-terminus to murein (peptidoglycan) and inserts by the lipid at the N-terminus into the outer membrane. As the most abundant protein in E. coli (106 molecules per cell) it plays an important role for the integrity of the cell envelope. Lpp represents the type protein of a large variety of lipoproteins found in Gram-negative and Gram-positive bacteria and in archaea that have in common the lipid structure for anchoring the proteins to membranes but otherwise strongly vary in sequence, structure, and function. Predicted lipoproteins in known prokaryotic genomes comprise 2.7% of all proteins. Lipoproteins are modified by a unique phospholipid pathway and transferred from the cytoplasmic membrane into the outer membrane by a special system. They are involved in protein incorporation into the outer membrane, protein secretion across the cytoplasmic membrane, periplasm and outer membrane, signal transduction, conjugation, cell wall metabolism, antibiotic resistance, biofilm formation, and adhesion to host tissues. They are only found in bacteria and function as signal molecules for the innate immune system of vertebrates, where they cause inflammation and elicit innate and adaptive immune response through Toll-like receptors. This review discusses various aspects of Lpp and other lipoproteins of Gram-negative and Gram-positive bacteria and archaea.


Assuntos
Archaea , Bactérias , Lipoproteínas/química , Lipoproteínas/metabolismo , Animais , Archaea/química , Archaea/metabolismo , Bactérias/química , Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/biossíntese , Peptidoglicano/química , Peptidoglicano/metabolismo
8.
Nat Commun ; 10(1): 2635, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201302

RESUMO

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Antibacterianos/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica/métodos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Tomografia com Microscopia Eletrônica/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/ultraestrutura , Microscopia Intravital/métodos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Peptidoglicano/metabolismo , Periplasma/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos
9.
Microbiol Immunol ; 63(7): 261-268, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31209918

RESUMO

Pasteurella multocida is one of the most important bacteria responsible for diseases of animals. Crude extracts from sonicated P. multocida strain Dainai-1, which is serotype A isolated from bovine pneumonia, were found to inhibit proliferation of mouse spleen cells stimulated with Con A. The crude extract was purified by cation and anion exchange chromatography and hydroxyapatite chromatography. Its molecular weight was 27 kDa by SDS-PAGE and it was named PM27. PM27 was found to inhibit proliferation of mouse spleen cells stimulated with Con A as effectively as did the crude extract; however, its activity was lost after heating to 100°C for 20 min. PM27 did not directly inhibit proliferation of HT-2 cells, which are an IL-2-dependent T cell line, nor did it modify IL-2 production by Con A-stimulated mouse spleen cells. The N-terminal amino acid sequence of PM27 was determined and BLAST analysis revealed its identity to uridine phosphorylase (UPase) from P. multocida. UPase gene from P. multocida Dainai-1 was cloned into expression vector pQE-60 in Escherichia coli XL-1 Blue. Recombinant UPase (rUPase) tagged with His at the C-terminal amino acid was purified with Ni affinity chromatography. rUPase was found to inhibit proliferation of mouse spleen cells stimulated with Con A; however, as was true for PM27, its activity was lost after heating to 100°C for 20 min. Thus, PM27/UPase purified from P. multocida has significant antiproliferative activity against Con A-stimulated mouse spleen cells and may be a virulence factor.


Assuntos
Proteínas da Membrana Bacteriana Externa/farmacologia , Proliferação de Células/efeitos dos fármacos , Pasteurella multocida/metabolismo , Uridina Fosforilase/isolamento & purificação , Uridina Fosforilase/farmacologia , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bovinos , Linhagem Celular/efeitos dos fármacos , Escherichia coli/genética , Humanos , Interleucina-2/metabolismo , Camundongos , Peso Molecular , Pasteurella multocida/genética , Fosforilases , Proteínas Recombinantes , Baço , Linfócitos T/efeitos dos fármacos , Uridina Fosforilase/genética , Uridina Fosforilase/metabolismo
10.
Enzyme Microb Technol ; 128: 1-8, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31186105

RESUMO

α2,3-Sialyltransferase from Pasteurella multocida (PmST1) is an enzyme that transfers a sialyl group of donor substrates to an acceptor substrate called N-acetyl-d-lactosamine (LacNAc). In this study PmST1 was expressed on the outer membrane of wildtype Escherichia coli (BL21) with lipopolysaccharide (LPS) and ClearColi with no LPS, and then the enzyme activity and expression level of PmST1 were compared. As the first step, the expression levels of PmST1 on the outer membranes of wildtype E. coli (BL21) and ClearColi were compared according to the IPTG induction time, and the absolute amount of surface-displayed PmST1 was calculated using densitometry of SDS-PAGE. As the next step, the influence of LPS on the PmST1 activity was estimated by analyzing Michaelis-Menten plot. The enzyme activity of PmST1 was analyzed by measuring the concentration of CMP, which was a by-product after the transfer of the sialyl group of donor compounds to the acceptor compounds. From a Michaelis-Menten plot, the enzyme activity of the surface-displayed PmST1 and the maximum rate (Vmax) of ClearColi were higher than those of wildtype E. coli (BL21). However, the KM value, which represented the concentration of substrate to reach half the maximum rate (Vmax), was similar for both enzymes. These results represented such a difference in enzyme activity was occurred from the interference of LPS on the mass transport of the donor and acceptor to PmST1 for the sialyl group transfer.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Técnicas de Visualização da Superfície Celular/métodos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Sialiltransferases/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Perfilação da Expressão Gênica , Cinética , Pasteurella multocida/enzimologia , Pasteurella multocida/genética , Proteínas Recombinantes/genética , Sialiltransferases/genética
11.
Curr Microbiol ; 76(7): 879-887, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31089795

RESUMO

The goal of this study was to elucidate the role of the outer membrane protein A (ompA) gene of Xanthomonas axonopodis pv. glycines in bacterial pustule pathogenesis of soybean. An ompA mutant of X. axonopodis pv. glycines KU-P-SW005 was shown to significantly decrease cellulase, pectate lyase, and polysaccharide production. The production of these proteins in the ompA mutant was approximately five times lower than that of the wildtype. The ompA mutant also exhibited modified biofilm development. More importantly, the mutant reduced disease severity to the soybean. Ten days after inoculation, the virulence rating of the susceptible soybean cv. SJ4 inoculated with the ompA mutant was 11.23%, compared with 87.98% for the complemented ompA mutant. Production of cellulase, pectate lyase, polysaccharide was restored, biofilm, and pustule numbers were restored in the complemented ompA mutant that did not differ from the wild type. Taken together, these data suggest that OmpA-mediated invasion plays an important role in protein secretion during pathogenesis to soybean.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Doenças das Plantas/microbiologia , Soja/microbiologia , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/patogenicidade , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Celulase/metabolismo , Teste de Complementação Genética , Mutação , Folhas de Planta/microbiologia , Polissacarídeo-Liase/metabolismo , Polissacarídeos Bacterianos/metabolismo , Virulência/genética
12.
Ecotoxicol Environ Saf ; 180: 208-214, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31096126

RESUMO

Dimethyl phthalate (DMP), a phthalate ester (PAE), is a ubiquitous and organic pollutant. In this study, the toxicity of DMP to Escherichia coli K12 and its underlying mechanism were investigated. The results showed that DMP inhibited the growth of E. coli K12 and induced cell inactivation and/or death. DMP caused serious damage to the cell membrane of E. coli K12, and the damage increased with higher DMP concentrations. DMP exposure disrupted cell membranes, as evidenced by dose-dependent variations of cell structures, surface properties, and membrane compositions. Increases in the malondialdehyde (MDA) content indicated an increase in oxidative stress induced by DMP in E. coli K12. The activity of succinic dehydrogenase (SDH) was changed by DMP, which could affect energy metabolism in the membrane of E. coli K12. The expression levels of OmpA and OmpX were increased, and the expression levels of OmpF and OmpW were decreased, in E. coli K12 exposed to DMP. The toxicities of DMP to E. coli K12 could be ascribed to membrane disruption and oxidative stress-induced cell inactivation and/or death. The outcomes will shed new light on the assessment of the ecological effects of DMP.


Assuntos
Poluentes Ambientais/toxicidade , Escherichia coli K12/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/efeitos dos fármacos , Escherichia coli K12/metabolismo , Malondialdeído/análise , Estresse Oxidativo
13.
PLoS Pathog ; 15(5): e1007731, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31083688

RESUMO

The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Secretina/química , Sistemas de Secreção Tipo II/química , Vibrio vulnificus/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Lipoproteínas/química , Modelos Moleculares , Conformação Proteica , Secretina/metabolismo , Homologia de Sequência , Sistemas de Secreção Tipo II/metabolismo , Vibrio vulnificus/crescimento & desenvolvimento
14.
J Microbiol ; 57(6): 498-508, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31054137

RESUMO

Outer membrane vesicles (OMVs) are nanostructures of 20-200 nm diameter deriving from the surface of several Gram-negative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and characterized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on samples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular matrix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gradient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, proteomic and fatty acid analysis of purified OMVs revealed a specific biochemical "fingerprint", suggesting interesting details concerning their biogenesis and physiological role. Moreover, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Vesículas Secretórias/química , Vesículas Secretórias/enzimologia , Sphingomonadaceae/metabolismo , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Exocitose , Ácidos Graxos/análise , Humanos , Queratinócitos/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Nanopartículas , Peptídeo Hidrolases/metabolismo , Proteômica/métodos , Sphingomonadaceae/citologia
15.
Nat Commun ; 10(1): 1967, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036849

RESUMO

Autotransporters are the largest family of outer membrane and secreted proteins in Gram-negative bacteria. Most autotransporters are localised to the bacterial surface where they promote colonisation of host epithelial surfaces. Here we present the crystal structure of UpaB, an autotransporter that is known to contribute to uropathogenic E. coli (UPEC) colonisation of the urinary tract. We provide evidence that UpaB can interact with glycosaminoglycans and host fibronectin. Unique modifications to its core ß-helical structure create a groove on one side of the protein for interaction with glycosaminoglycans, while the opposite face can bind fibronectin. Our findings reveal far greater diversity in the autotransporter ß-helix than previously thought, and suggest that this domain can interact with host macromolecules. The relevance of these interactions during infection remains unclear.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glicosaminoglicanos/metabolismo , Escherichia coli Uropatogênica/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fatores de Virulência/química , Fatores de Virulência/metabolismo
16.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 368-376, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045566

RESUMO

The bacterial periplasmic protein LpoA is an outer membrane lipoprotein and an activator for the cross-linking activity of PBP1A, a bifunctional peptidoglycan synthase. Previous structures of the amino-terminal (N) domain of LpoA showed it to consist entirely of helices and loops, with at least four tetratricopeptide-like repeats. Although the previously determined orthorhombic crystal structure of the N domain of Haemophilus influenzae LpoA showed a typical curved structure with a concave groove, an NMR structure of the same domain from Escherichia coli was relatively flat. Here, a crystal structure of the N domain of E. coli LpoA was determined to a resolution of 2.1 Šand was found to be more similar to the H. influenzae crystal structure than to the E. coli NMR structure. To provide a quantitative description for these comparisons, the various structures were superimposed pairwise by fitting the first half of each structure to its pairwise partner and then calculating the rotation axis that would optimally superimpose the second half. Differences in both the magnitude of the rotation and the direction of the rotation axis were observed between different pairs of structures. A 1.35 Šresolution structure of a monoclinic crystal form of the N domain of H. influenzae LpoA was also determined. In this structure, the subdomains rotate 10° relative to those in the original orthorhombic H. influenzae crystal structure to further narrow the groove between the subdomains. To accommodate this, a bound chloride ion (in place of sulfate) allowed the closer approach of a helix that forms one side of the groove.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Cloretos/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Haemophilus influenzae/química , Lipoproteínas/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína
17.
Comp Immunol Microbiol Infect Dis ; 63: 131-135, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30961808

RESUMO

The aim of the study was to determine whether the presence of the Yersinia virulence plasmid could affect the production of enterotoxin YstA by Y. enterocolitica strains isolated from pigs which are the main source of infection for humans. The phenotypic features characteristic for the Yersinia virulence plasmid were detected on CRMOX agar in 8 out of 12 strains producing enterotoxin YstA, in 5 out of 12 doubtful strains, and in 11 out of 12 strains not producing YstA. Autoagglutination ability was detected in all 12 Y. enterocolitica strains that were positive in the suckling mice bioassay, in 11 doubtful strains and 10 negative strains. CRMOX+ colonies were generally ystA, myfA, virF and yadA positive, while CRMOX- colonies were only ystA and myfA positive. The amplicons of yadA were not detected in 2 (8.3%) out of 24 CRMOX+ and virF positive strains. The results of this study indicate that the presence of pYV does not affect the enterotoxin-producing ability of Y. enterocolitica strains.


Assuntos
Toxinas Bacterianas/biossíntese , Enterotoxinas/biossíntese , Plasmídeos/genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Adesinas Bacterianas/metabolismo , Animais , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Meios de Cultura/farmacologia , DNA Bacteriano/genética , Humanos , Camundongos , Suínos , Doenças dos Suínos/microbiologia , Yersinia enterocolitica/patogenicidade
18.
Microb Pathog ; 132: 38-44, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30986451

RESUMO

Live attenuated bacteria is a promising candidate vector for the delivery of vaccines in clinic trials. In the field of aquaculture industry, live vector vaccine also could provide long-term and effective protection against fish bacterial diseases. In our previous work, we demonstrated attenuated Listeria monocytogenes (Lm) had the potential to be an aquaculture vaccine vector in cellular level and zebrafish model. To further investigate the potential application of attenuated Lm in aquaculture vaccines, the outer membrane protein K (OmpK) from Vibrio parahaemolyticus (V. parahaemolyticus), as a conservative protective antigen, was fused to a new antigen-delivery system, and introduced into double-gene attenuated Lm strain (EGDe-ΔactA/inlB, Lmdd) to get live-vector vaccine strain Lmdd-OmpK. The strain Lmdd-OmpK showed the stable secrete efficacy of OmpK and was tested the cross-protective immunity against Vibrio species. After intraperitoneal administration in zebrafish, Lmdd and Lmdd-OmpK strain both improved the survival rates of zebrafish infected by V. parahaemolyticus, Vibrio alginolyticus (V. alginolyticus) and Vibrio anguillarum (V. anguillarum), respectively. In summary, attenuated Lm is able to protect zebrafish against Vibrio species challenge, illustrating its potential value for further aquaculture vaccines development.


Assuntos
Vacinas Bacterianas/imunologia , Doenças dos Peixes/prevenção & controle , Listeria monocytogenes/imunologia , Vacinas Atenuadas/imunologia , Vibrioses/prevenção & controle , Vibrio/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Clonagem Molecular , Modelos Animais de Doenças , Doenças dos Peixes/microbiologia , Listeria monocytogenes/genética , Alinhamento de Sequência , Vibrio alginolyticus , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/imunologia , Peixe-Zebra
19.
Nat Commun ; 10(1): 1520, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944318

RESUMO

In Pseudomonas aeruginosa, MexAB-OprM plays a central role in multidrug resistance by ejecting various drug compounds, which is one of the causes of serious nosocomial infections. Although the structures of the components of MexAB-OprM have been solved individually by X-ray crystallography, no structural information for fully assembled pumps from P. aeruginosa were previously available. In this study, we present the structure of wild-type MexAB-OprM in the presence or absence of drugs at near-atomic resolution. The structure reveals that OprM does not interact with MexB directly, and that it opens its periplasmic gate by forming a complex. Furthermore, we confirm the residues essential for complex formation and observed a movement of the drug entrance gate. Based on these results, we propose mechanisms for complex formation and drug efflux.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Membrana Transportadoras/química , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Relação Estrutura-Atividade
20.
Chem Biol Interact ; 305: 195-202, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30940451

RESUMO

A significant risk to the food chain is the presence of noxious pollutants in the feeds of animals whose products are used in human nutrition. Consequently, analytical methods and biosensors have been developed to detect these types of contaminates in feeds. Here we have evaluated whether the expression of TolC, a promiscuous component of several ATP-dependent efflux pumps in E. coli, up-regulated in response to chemical stress, could be a useful biomarker for this aim. Changes in TolC expression in response to toxic compounds, with different abilities to induce DNA damage, were determined using two E. coli strains with (DH5α) and without (BL21(DE3)) inactivating mutation in RecA gene. Deoxycholic acid and potassium dichromate up-regulated TolC in both strains. In contrast, cisplatin-induced TolC up-regulation was abolished in the absence of a functional RecA. When the effect of several insecticides, herbicides, antibiotics and common soil pollutants on TolC expression was analyzed, a relationship between toxicity and their ability to up-regulate TolC was observed. However, this was not a general event because the insecticide α-cipermetrin induced a reduction in cell viability, which was not accompanied by TolC up-regulation. In contrast, the soil pollutant benzene was able to stimulate TolC expression at non-toxic concentrations. When this test was used to analyze aqueous extracts from different feedstuffs, up-regulation of TolC was found in the absence of cell toxicity and was even accompanied by enhanced cell viability. In conclusion, TolC expression is partly dependent on the integrity of the RecA/LexaA system. Although toxic compounds up-regulate TolC in a dose-dependent manner, this response is also activated by non-toxic agents. Thus, owing to its poor specificity regardless of its sensitivity, the use of TolC up-regulation in E. coli to detect the presence of toxic pollutants in conventional and unconventional sources of nutrients for ruminant feeding requires supplementary biomarkers.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Biomarcadores/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Poluentes do Solo/toxicidade , Regulação para Cima/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Cisplatino/farmacologia , Ácido Desoxicólico/toxicidade , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Compostos Organoplatínicos/toxicidade , Dicromato de Potássio/toxicidade , RNA Mensageiro/metabolismo , Poluentes do Solo/química
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