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1.
Nat Commun ; 11(1): 769, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034139

RESUMO

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH.


Assuntos
Histidina Quinase/química , Histidina Quinase/metabolismo , Thermotoga maritima/enzimologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Histidina/metabolismo , Histidina Quinase/genética , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação , Fosforilação , Conformação Proteica , Thermotoga maritima/genética , Transativadores/química , Transativadores/metabolismo
2.
Nat Commun ; 11(1): 851, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051408

RESUMO

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.


Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Imunização , Antígenos O/imunologia , Salmonella typhimurium/imunologia , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteção Cruzada , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Imunoglobulina G/sangue , Camundongos , Modelos Moleculares , Antígenos O/química , Antígenos O/genética , Porinas/química , Porinas/genética , Porinas/imunologia , Conformação Proteica , Salmonelose Animal/imunologia , Salmonelose Animal/prevenção & controle , Análise de Sequência de Proteína
3.
Nat Commun ; 11(1): 564, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992701

RESUMO

Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Detergentes/química , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Espectrometria de Massas , Lipídeos de Membrana , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Redobramento de Proteína , Solubilidade
4.
Biochim Biophys Acta Biomembr ; 1862(1): 183025, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31351059

RESUMO

Lipopolysaccharides (LPS) provide the outer membrane (OM) of Gram-negative bacteria with a strong protective barrier. The periplasm-spanning Lpt machinery is responsible for the transport of LPS molecules across the periplasm, culminating in insertion by the outer-membrane proteins LptD and LptE. In order to elucidate the mechanisms of LPS insertion by LptDE, we performed over 14 microseconds of equilibrium molecular dynamics simulations. Bilayer-dependent differences in the fluctuations and secondary structure of LptD's extracellular loops are observed for a pure DMPE membrane vs. a model of the OM. Furthermore, LptD's periplasmic N-terminal domain is highly dynamic, which may help to maintain the integrity of the periplasm-spanning complex amidst relative motion of the inner-membrane and outer-membrane anchored domains. In addition, our simulations demonstrate that binding of LPS substrate activates a switching between the associated and dissociated states of two lumenal loops at the interface between the ß-barrel and the N-terminal domain as well as LptD's lateral gate on the microsecond timescale, neither of which is observed for the apo state. Placement of a substrate LPS molecule also causes an increase in the average separation of the LptD lateral gate strands and a lowering of the energetic barrier to lateral gate opening.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Simulação de Dinâmica Molecular , Proteínas da Membrana Bacteriana Externa/química , Transporte Biológico , Escherichia coli/química , Proteínas de Escherichia coli/química , Bicamadas Lipídicas/farmacologia , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína/efeitos dos fármacos
5.
Biochim Biophys Acta Biomembr ; 1862(1): 183031, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374213

RESUMO

Electrostatic side chain contacts can contribute substantial interaction energy terms to the stability of proteins. The impact of electrostatic interactions on the structure and architecture of outer membrane proteins is however not well studied compared to soluble proteins. Here, we report the results of a systematic study of all charged side chains of the E. coli outer membrane protein X (OmpX). The data identify three distinct salt-bridge clusters in the core of OmpX that contribute significantly to protein stability in dodecylphosphocholine detergent micelles. The three clusters form an "electrostatic core" of the membrane protein OmpX, corresponding in its architectural role to the hydrophobic core of soluble proteins. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Hidrolases/química , Eletricidade Estática , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Escherichia coli/fisiologia , Hidrolases/fisiologia , Interações Hidrofóbicas e Hidrofílicas , Micelas , Modelos Moleculares , Conformação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína
6.
Biochim Biophys Acta Biomembr ; 1862(1): 183062, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31520605

RESUMO

The ß-barrel assembly machinery (BAM) is responsible for the biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria. These OMPs have a membrane-embedded domain consisting of a ß-barrel fold which can vary from 8 to 36 ß-strands, with each serving a diverse role in the cell such as nutrient uptake and virulence. BAM was first identified nearly two decades ago, but only recently has the molecular structure of the full complex been reported. Together with many years of functional characterization, we have a significantly clearer depiction of BAM's structure, the intra-complex interactions, conformational changes that BAM may undergo during OMP biogenesis, and the role chaperones may play. But still, despite advances over the past two decades, the mechanism for BAM-mediated OMP biogenesis remains elusive. Over the years, several theories have been proposed that have varying degrees of support from the literature, but none has of yet been conclusive enough to be widely accepted as the sole mechanism. We will present a brief history of BAM, the recent work on the structures of BAM, and a critical analysis of the current theories for how it may function.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Conformação Proteica em Folha beta , Proteínas da Membrana Bacteriana Externa/biossíntese , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/ultraestrutura , Estrutura Secundária de Proteína
7.
PLoS One ; 14(12): e0226088, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31887111

RESUMO

Feral pigeons, common wood pigeons and Eurasian collared doves are the most common representatives of the Columbidae family in Switzerland and are mostly present in highly populated, urban areas. Pigeons may carry various members of the obligate intracellular Chlamydiaceae family, particularly Chlamydia (C.) psittaci, a known zoonotic agent, and C. avium. The objective of the study was to identify the infection rates of common free-roaming pigeons for different Chlamydia species with the overall aim to assess the risk pigeons pose to public health. In this study, 431 pigeons (323 feral pigeons, 34 domestic pigeons, 39 Eurasian collared doves, 35 common wood pigeons) from several geographic locations in Switzerland were investigated for the presence of Chlamydiaceae. Samples consisted of pooled choanal-cloacal swabs (n = 174), liver samples (n = 52), and paired swab and liver samples from 205 pigeons (n = 410). All 636 samples were screened using a Chlamydiaceae family-specific 23S rRNA real-time PCR (qPCR). Subsequent species identification was performed by DNA-microarray assay, sequencing of a 16S rRNA gene fragment and a C. psittaci specific qPCR. In total, 73 of the 431 pigeons tested positive for Chlamydiaceae, of which 68 were positive for C. psittaci, four were C. avium-positive and one pigeon was co-infected with C. avium and C. psittaci. The highest infection rates were detected in feral (64/323) and domestic pigeons (5/34). Common wood pigeons (2/35) and Eurasian collared doves (2/39) revealed lower infection rates. Additionally, multilocus sequence typing of twelve selected C. psittaci-positive samples revealed closely related sequence types (ST) between and within different Swiss cities. Furthermore, liver and corresponding swab samples from the same bird were colonized by the same ST. Considering the high infection rates of C. psittaci in domestic and feral pigeons, close or frequent contact to these birds poses a human health risk.


Assuntos
Doenças das Aves/microbiologia , Chlamydiaceae/genética , Chlamydophila psittaci/genética , Psitacose/microbiologia , Animais , Animais Domésticos , Animais Selvagens , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/classificação , Proteínas da Membrana Bacteriana Externa/genética , Doenças das Aves/diagnóstico , Chlamydiaceae/classificação , Chlamydiaceae/isolamento & purificação , Chlamydophila psittaci/isolamento & purificação , Columbidae , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Tipagem de Sequências Multilocus , Filogenia , Dinâmica Populacional , Psitacose/diagnóstico , RNA Ribossômico 16S/química , RNA Ribossômico 16S/isolamento & purificação , RNA Ribossômico 16S/metabolismo , Suíça
8.
J Chem Theory Comput ; 15(12): 6551-6561, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31665603

RESUMO

The plasticity of membranes plays an important functional role in cells, cell components, and micelles, where bending, budding, and remodeling implement numerous recognition and communication processes. Comparatively, molecular simulation methods to induce, control, and quantitatively characterize such deformations remain scarce. This work defines a novel collective coordinate associated with membrane bending, which strives to combine realism (by preserving the notion of local atomic curvatures) and low computational cost (allowing its evaluation at every time step of a molecular dynamics simulation). Enhanced sampling simulations along this conformational coordinate provide convenient access to the underlying bending free energy landscape. To showcase its potential, the method is applied to three state-of-the-art problems: the determination of the bending free energy landscape of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) bilayer, the formation of a POPE liposome, and the study of the influence of the Pseudomonas quinolone signal on the budding of Gram-negative bacterial outer membranes.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Fosfatidiletanolaminas/química , Lipossomos/química , Conformação Molecular , Quinolonas/química
9.
Parasit Vectors ; 12(1): 497, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640755

RESUMO

BACKGROUND: Mass drug administration (MDA) with azithromycin is a cornerstone of the trachoma elimination strategy. Although the global prevalence of active trachoma has declined considerably, prevalence persists or even increases in some communities and districts. To increase understanding of MDA impact, we investigated the prevalence of active trachoma and ocular C. trachomatis prevalence, organism load, and circulating strains at baseline and one-year post-MDA in The Gambia and Senegal. METHODS: Pre- and one-year post-MDA, children aged 0-9 years were examined for clinical signs of trachoma in six Gambian and 12 Senegalese villages. Ocular swabs from each child's right conjunctiva were tested for evidence of ocular C. trachomatis infection and organism load (ompA copy number), and ompA and multi-locus sequence typing (MLST) was performed. RESULTS: A total of 1171 children were examined at baseline and follow-up in The Gambia. Active trachoma prevalence decreased from 23.9% to 17.7%, whereas ocular C. trachomatis prevalence increased from 3.0% to 3.8%. In Senegal, 1613 and 1771 children were examined at baseline and follow-up, respectively. Active trachoma prevalence decreased from 14.9% to 8.0%, whereas ocular C. trachomatis prevalence increased from 1.8% to 3.6%. Higher organism load was associated with having active trachoma and severe inflammation. Sequence typing demonstrated that all Senegalese samples were genovar A, whereas Gambian samples were a mix of genovars A and B. MLST provided evidence of clustering at village and household levels and demonstrated differences of strain variant frequencies in Senegal, indicative of an "outbreak". MLST, including partial ompA typing, provided greater discriminatory power than complete ompA typing. CONCLUSIONS: We found that one round of MDA led to an overall decline in active trachoma prevalence but no impact on ocular C. trachomatis infection, with heterogeneity observed between villages studied. This could not be explained by MDA coverage or number of different circulating strains pre- and post-MDA. The poor correlation between active trachoma and infection prevalence supports the need for further work on alternative indicators to clinical signs for diagnosing ocular C. trachomatis infection. MLST typing has potential molecular epidemiology utility, including better understanding of transmission dynamics, although relationship to whole-genome sequence variability requires further exploration.


Assuntos
Antibacterianos/administração & dosagem , Azitromicina/administração & dosagem , Tracoma/epidemiologia , Tracoma/prevenção & controle , Antibacterianos/uso terapêutico , Azitromicina/uso terapêutico , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Criança , Pré-Escolar , Chlamydia trachomatis/classificação , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/genética , Gâmbia/epidemiologia , Genótipo , Humanos , Lactente , Administração Massiva de Medicamentos , Tipagem de Sequências Multilocus , Filogenia , Testes Imediatos , Polimorfismo Genético , Prevalência , Senegal/epidemiologia , Tracoma/tratamento farmacológico , Sequenciamento Completo do Genoma
10.
Int J Nanomedicine ; 14: 6601-6613, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496701

RESUMO

Purpose: The primary goal of the present study was to explore and evaluate the highly conserved Neisserial surface protein A (NspA) molecule, fused with truncated HBV virus-like particles (VLPs), as a candidate vaccine against the virulent Neisseria meningitidis serogroup B (NMB). Methods: NspA was inserted into the major immunodominant region of the truncated hepatitis B virus core protein (HBc; amino acids 1-144). The chimeric protein, HBc-N144-NspA, was expressed from a prokaryotic vector and generated HBc-like particles, as determined by transmission electron microscopy. Further, the chimeric protein and control proteins were used to immunize mice and the resulting immune responses evaluated by flow cytometry, enzyme-linked immunosorbent assay, and analysis of serum bactericidal activity (SBA) titer. Results: Evaluation of the immunogenicity of the recombinant HBc-N144-NspA protein showed that it elicited the production of high levels of NspA-specific total IgG. The SBA titer of HBc-N144-NspA/F reached 1:16 2 weeks after the last immunization in BALB/c mice, when human serum complement was included in the vaccine. Immunization of HBc-N144-NspA, even without adjuvant, induced high levels of IL-4 and a high IgG1 to IgG2a ratio, confirming induction of an intense Th2 immune response. Levels of IL-17A increased rapidly in mice after the first immunization with HBc-N144-NspA, indicating the potential for this vaccine to induce a mucosal immune response. Meanwhile, the immunization of HBc-N144-NspA without adjuvant induced only mild inflammatory infiltration into the mouse muscle tissue. Conclusion: This study demonstrates that modification using HBc renders NspA a candidate vaccine, which can trigger protective immunity against NMB.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vírus da Hepatite B/metabolismo , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis/patogenicidade , Sorogrupo , Vírion/metabolismo , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Citocinas/metabolismo , Escherichia coli/metabolismo , Feminino , Imunidade , Imunização , Inflamação/patologia , Ativação Linfocitária/imunologia , Infecções Meningocócicas/patologia , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Teste Bactericida do Soro , Baço/microbiologia , Linfócitos T/imunologia , Vacinação , Virulência
11.
APMIS ; 127(12): 753-763, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31512768

RESUMO

Iron uptake system is expressed in early stages of Acinetobacter baumannii infections under iron-restricted conditions. This study is aimed at the evaluation of immuno-protectivity of BfnH in comparison with BauA in both mature and selected fragmental proteins. The study was designed in single and combined forms of antigens. BfnH is presented in 3472 strains of A. baumannii with more than 97% identity. The preliminary immune-informatics analysis of this protein indicated a region from the ß-barrel domain including exposed loops 2-5, with antigenic score comparable to that of BfnH. There was a significant rise in the specific IgG response in all test groups. The bacterial challenge with a lethal dose of A. baumannii demonstrated partial protection of whole proteins which coincides with a significant reduction in the bacterial population colonized in the main organs and an increase in the survival level. Passive immunization of the mice brought about 50% survival in the mice groups immunized with BfnH and with a combination of BfnH and BauA. The protectivity of siderophore receptors suggests their potential immunogenic role that could be considered as a component of multivalent subunit vaccine candidates against A. baumannii.


Assuntos
Infecções por Acinetobacter/prevenção & controle , Acinetobacter baumannii/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Imunização , Receptores de Superfície Celular/imunologia , Infecções por Acinetobacter/imunologia , Infecções por Acinetobacter/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Epitopos de Linfócito B , Feminino , Imunogenicidade da Vacina , Camundongos Endogâmicos BALB C , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética
12.
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
13.
Nat Commun ; 10(1): 3673, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31413254

RESUMO

Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe3+ complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Enterobactina/metabolismo , Ferro/metabolismo , Pseudomonas aeruginosa/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias , Sítios de Ligação , Proteínas de Transporte/química , Cristalização , Cristalografia por Raios X , Enterobactina/química , Escherichia coli , Técnicas In Vitro , Radioisótopos de Ferro , Proteínas de Membrana , Receptores de Superfície Celular/química , Sideróforos/química , Sideróforos/metabolismo
14.
Bioelectrochemistry ; 130: 107336, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31377396

RESUMO

Cytokinin oxidase from Nipponbare (OsCKX4) was successfully displayed on the surface of E. coli cells by an ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif and a maltodextrin-binding protein(MBP) from E. coli as a solubility enhancer. The OsCKX4-displayed bacteria can be directly immobilized onto an electrode to selectively detect cytokinins, thus eliminating the need for enzyme extraction and purification. Direct electrochemistry of the cofactor FADH2 in OsCKX4 has been achieved on an edge-plane pyrolytic graphite electrode (PGE) with a formal potential (E0') of -0.45 V at pH 7.0 in phosphate buffer. With the addition of isopentenyladenine, the reduction peak current for FADH2 decreased, and the oxidative peak current increased gradually. Therefore, a bacteria-OsCKX4-modified PGE has been developed for the detection of isopentenyladenine with a linear range of 1.0-11.0 µM and a lower limit of detection of 0.7 µM (S/N = 3). Slight interference was observed in the presence of other phytohormones, including brassinosteroid, abscisic acid, methylene jasminate and gibberellin. The proposed bacterial biosensor is stable, specific and simple and has great potential for applications that require the detection of cytokinins.


Assuntos
Técnicas Biossensoriais/métodos , Citocininas/análise , Enzimas Imobilizadas/química , Oryza/enzimologia , Oxirredutases/química , Proteínas da Membrana Bacteriana Externa/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas Periplásmicas de Ligação/química , Pseudomonas/química , Proteínas Recombinantes de Fusão/química
15.
Elife ; 82019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31385808

RESUMO

Many microbes and fungi acquire the essential ion Fe3+ through the synthesis and secretion of high-affinity chelators termed siderophores. In Gram-negative bacteria, these ferric-siderophore complexes are actively taken up using highly specific TonB-dependent transporters (TBDTs) located in the outer bacterial membrane (OM). However, the detailed mechanism of how the inner-membrane protein TonB connects to the transporters in the OM as well as the interplay between siderophore- and TonB-binding to the transporter is still poorly understood. Here, we present three crystal structures of the TBDT FoxA from Pseudomonas aeruginosa (containing a signalling domain) in complex with the siderophore ferrioxamine B and TonB and combine them with a detailed analysis of binding constants. The structures show that both siderophore and TonB-binding is required to form a translocation-competent state of the FoxA transporter in a two-step TonB-binding mechanism. The complex structure also indicates how TonB-binding influences the orientation of the signalling domain.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Desferroxamina/química , Desferroxamina/metabolismo , Compostos Férricos/química , Compostos Férricos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Pseudomonas aeruginosa/enzimologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Transporte Biológico , Quelantes de Ferro/química , Quelantes de Ferro/metabolismo , Estrutura Quaternária de Proteína
16.
Int J Mol Sci ; 20(17)2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31443507

RESUMO

The effectors of the type IV secretion system (T4SS) of bacteria play important roles in mediating bacterial intracellular proliferation and manipulating host-related pathway responses to bacterial infection. Brucella Spp. inhibit the apoptosis of host cells to benefit their own intracellular proliferation. However, the underlying mechanisms between T4SS effectors and Brucella-inhibited apoptosis in goat trophoblast cells remain unclear. Here, based on Brucella suis vaccine strain 2, the VceC was deleted by allelic exchange. We show that ΔVceC was able to infect and proliferate to high titers in goat trophoblast cells (GTCs) and increase C/EBP-homologous protein (CHOP)-mediated apoptosis. GRP78 expression decreased upon ΔVceC infection. In addition, we discovered that the inositolrequiring enzyme 1 (IRE1) pathway was inhibited in this process. Changing endoplasmic reticulum (ER) stress affected Brucella intracellular replication in GTCs. The replication of ΔVceC was more sensitive under the different ERstress conditions in the GTC line after treatment with ER stress inhibitors 4 phenyl butyric acid (4-PBA) or ER stress activator Tm. Together, our findings show that VceC has a protective effect on the intracellular persistence of Brucella infection, and inhibits ER stress-induced apoptosis in the CHOP pathway. The present work provides new insights for understanding the mechanism of VceC in the establishment of chronic Brucella infection.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Brucella/fisiologia , Brucelose/veterinária , Proteínas Serina-Treonina Quinases/metabolismo , Trofoblastos/metabolismo , Trofoblastos/microbiologia , Sequência de Aminoácidos , Animais , Apoptose , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Estresse do Retículo Endoplasmático/genética , Cabras , Interações Hospedeiro-Patógeno , Humanos , Viabilidade Microbiana , Mutação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Ovinos , Doenças dos Ovinos/metabolismo , Doenças dos Ovinos/microbiologia , Transdução de Sinais
17.
ACS Appl Mater Interfaces ; 11(32): 29276-29289, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31329408

RESUMO

Synthosomes are polymer vesicles with transmembrane proteins incorporated into block copolymer membranes. They have been used for selective transport in or out of the vesicles as well as catalysis inside the compartments. However, both the insertion process of the membrane protein, forming nanopores, and the spreading of the vesicles on planar substrates to form solid-supported biomimetic membranes have been rarely studied yet. Herein, we address these two points and, first, shed light on the real-time monitoring of protein insertion via isothermal titration calorimetry. Second, the spreading process on different solid supports, namely, SiO2, glass, and gold, via different techniques like spin- and dip-coating as well as a completely new approach of potential-assisted spreading on gold surfaces was studied. While inhomogeneous layers occur via traditional methods, our proposed potential-assisted strategy to induce adsorption of positively charged vesicles by applying negative potential on the electrode leads to remarkable vesicle spreading and their further fusion to form more homogeneous planar copolymer films on gold. The polymer vesicles in our study are formed from amphiphilic copolymers poly(2-methyl oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl oxazoline) (PMOXA-b-PDMS-b-PMOXA). Engineered variants of the transmembrane protein ferric hydroxamate uptake protein component A (FhuA), one of the largest ß-barrel channel proteins, are used as model nanopores. The incorporation of FhuA Δ1-160 is shown to facilitate the vesicle spreading process further. Moreover, high accessibility of cysteine inside the channel was proven by linkage of a fluorescent dye inside the engineered variant FhuA ΔCVFtev and hence preserved functionality of the channels after spreading. The porosity and functionality of the spread synthosomes on the gold plates have been examined by studying the passive ion transport response in the presence of Li+ and ClO4- ions and electrochemical impedance spectroscopy analysis. Our approach to form solid-supported biomimetic membranes via the potential-assisted strategy could be important for the development of new (bio-) sensors and membranes.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Membranas Artificiais , Nanoporos , Transporte de Íons , Propriedades de Superfície
18.
Int J Med Microbiol ; 309(7): 151322, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31262642

RESUMO

In Gram-negative bacteria, secretion of toxins ensure the survival of the bacterium. Such toxins are secreted by sophisticated multiprotein systems. The most conserved part in some of these secretion systems are components, called secretins, which form the outer membrane ring in these systems. Recent structural studies shed some light on the oligomeric organization of secretins. However, the mechanisms by which these proteins are targeted to the outer membrane and assemble there into ring structures are still not fully understood. This review discusses the various species-specific targeting and assembly pathways that are taken by secretins in order to form their functional oligomers.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , /química , Sistemas de Secreção Bacterianos/química , Sistemas de Secreção Bacterianos/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/metabolismo , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Multimerização Proteica , Transporte Proteico
19.
Nat Commun ; 10(1): 3358, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350400

RESUMO

The integration of ß-barrel proteins into the bacterial outer membrane (OM) is catalysed by the ß-barrel assembly machinery (BAM). The central BAM subunit (BamA) itself contains a ß-barrel domain that is essential for OM protein biogenesis, but its mechanism of action is unknown. To elucidate its function, here we develop a method to trap a native Escherichia coli ß-barrel protein bound stably to BamA at a late stage of assembly in vivo. Using disulfide-bond crosslinking, we find that the first ß-strand of a laterally 'open' form of the BamA ß-barrel forms a rigid interface with the C-terminal ß-strand of the substrate. In contrast, the lipid-facing surface of the last two BamA ß-strands forms weaker, conformationally heterogeneous interactions with the first ß-strand of the substrate that likely represent intermediate assembly states. Based on our results, we propose that BamA promotes the membrane integration of partially folded ß-barrels by a 'swing' mechanism.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/química , Membrana Celular/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ligação Proteica , Domínios Proteicos
20.
Nat Protoc ; 14(8): 2344-2369, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31278399

RESUMO

Observation of structure and conformational dynamics of membrane proteins at high resolution in their native environments is challenging because of the lack of suitable techniques. We have developed an approach for high-precision distance measurements in the nanometer range for outer-membrane proteins (OMPs) in intact Escherichia coli and native membranes. OMPs in Gram-negative bacteria rarely have reactive cysteines. This enables in situ labeling of engineered cysteines with a methanethiosulfonate spin label (MTSL) with minimal background signals. Following overexpression of the target protein, spin labeling is performed with E. coli or isolated outer membranes (OMs) under selective conditions. The interspin distances are measured in situ, using pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. The residual background signals, which are problematic for in situ structural biology, contribute specifically to the intermolecular part of the signal and can be selectively removed to extract the desired interspin distance distribution. The initial cloning stage can take 5-7 d, and the subsequent protein expression, OM isolation, spin labeling, PELDOR experiment, and data analysis typically take 4-5 d. The described protocol provides a general strategy for observing protein ligand-substrate interactions, oligomerization, and conformational dynamics of OMPs in their native OM and intact E. coli.


Assuntos
Proteínas da Membrana Bacteriana Externa , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Cisteína/química , Cisteína/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Mesilatos/química , Mesilatos/metabolismo , Conformação Proteica , Marcadores de Spin
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