RESUMO
The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the ß-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity.
Assuntos
Proteínas de Transporte , Proteínas de Escherichia coli , Escherichia coli , Chaperonas Moleculares , Peptidilprolil Isomerase , Trifosfato de Adenosina/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Peptidilprolil Isomerase/genética , Ligação Proteica , Domínios Proteicos , Dobramento de ProteínaRESUMO
Pathogenic spirochetes of the genus Leptospira are the causative agent of leptospirosis, a widely disseminated zoonosis that affects humans and animals. The ability of leptospires to quickly cross host barriers causing infection is not yet fully understood. Thus, understanding the mechanisms of pathogenicity is important to combat leptospiral infection. Outer membrane proteins are interesting targets to study as they are able to interact with host molecules. Proteins containing leucine-rich repeat (LRR) domains are characterized by the presence of multiple regions containing leucine residues and they have putative functions related to host-pathogen interactions. Hence, the present study aimed to clone and express the recombinant protein encoded by the LIC11098 gene, an LRR protein of L. interrogans serovar Copenhageni. In silico analyses predicted that the target protein is conserved among pathogenic strains of Leptospira, having a signal peptide and multiple LRR domains. The DNA sequence encoding the LRR protein was cloned in frame into the pAE vector, expressed without mutations in Escherichia coli and purified by His-tag chromatography. Circular dichroism (CD) spectrum showed that the recombinant protein was predominantly composed of ß-sheets. A dose-dependent interaction was observed with cellular and plasma fibronectins, laminin and the complement system component C9, suggesting a possible role of the protein encoded by LIC11098 gene at the initial stages of infection.
Assuntos
Leptospira interrogans , Proteínas de Repetições Ricas em Leucina , Proteínas Recombinantes , Leptospira interrogans/genética , Leptospira interrogans/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Simulação por Computador , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Clonagem Molecular , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Leptospirose/microbiologia , Animais , Interações Hospedeiro-Patógeno , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Dicroísmo Circular , Sequência de AminoácidosRESUMO
Gram-negative bacteria produce chaperone-usher pathway pili, which are extracellular protein fibers tipped with an adhesive protein that binds to a receptor with stereochemical specificity to determine host and tissue tropism. The outer-membrane usher protein, together with a periplasmic chaperone, assembles thousands of pilin subunits into a highly ordered pilus fiber. The tip adhesin in complex with its cognate chaperone activates the usher to allow extrusion across the outer membrane. The structural requirements to translocate the adhesin through the usher pore from the periplasm to the extracellular space remains incompletely understood. Here, we present a cryoelectron microscopy structure of a quaternary tip complex in the type 1 pilus system from Escherichia coli, which consists of the usher FimD, chaperone FimC, adhesin FimH, and the tip adapter FimF. In this structure, the usher FimD is caught in the act of secreting its cognate adhesin FimH. Comparison with previous structures depicting the adhesin either first entering or having completely exited the usher pore reveals remarkable structural plasticity of the two-domain adhesin during translocation. Moreover, a piliation assay demonstrated that the structural plasticity, enabled by a flexible linker between the two domains, is a prerequisite for adhesin translocation through the usher pore and thus pilus biogenesis. Overall, this study provides molecular details of adhesin translocation across the outer membrane and elucidates a unique conformational state adopted by the adhesin during stepwise secretion through the usher pore. This study elucidates fundamental aspects of FimH and usher dynamics critical in urinary tract infections and is leading to antibiotic-sparing therapeutics.
Assuntos
Adesinas de Escherichia coli , Microscopia Crioeletrônica , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Fímbrias , Fímbrias Bacterianas , Proteínas de Fímbrias/metabolismo , Proteínas de Fímbrias/química , Fímbrias Bacterianas/metabolismo , Adesinas de Escherichia coli/metabolismo , Adesinas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Modelos Moleculares , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/químicaRESUMO
The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded ß-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Escherichia coli/genética , Microscopia Crioeletrônica , Ligação Proteica , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/química , Mutação , Proteínas de Transporte , Peptidilprolil IsomeraseRESUMO
Efflux-mediated ß-lactam resistance is a major public health concern, reducing the effectiveness of ß-lactam antibiotics against many bacteria. Structural analyses show the efflux protein TolC in Gram-negative bacteria acts as a channel for antibiotics, impacting bacterial susceptibility and virulence. This study examines ß-lactam drug efflux mediated by TolC using experimental and computational methods. Molecular dynamics simulations of drug-free TolC reveal essential movements and key residues involved in TolC opening. A whole-gene-saturation mutagenesis assay, mutating each TolC residue and measuring fitness effects under ß-lactam selection, is performed. Here we show the TolC-mediated efflux of three antibiotics: oxacillin, piperacillin, and carbenicillin. Steered molecular dynamics simulations identify general and drug-specific efflux mechanisms, revealing key positions at TolC's periplasmic entry affecting efflux motions. Our findings provide insights into TolC's structural dynamics, aiding the design of new antibiotics to overcome bacterial efflux mechanisms.
Assuntos
Antibacterianos , Proteínas da Membrana Bacteriana Externa , Simulação de Dinâmica Molecular , Resistência beta-Lactâmica , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Antibacterianos/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Conformação ProteicaRESUMO
Lyme disease is the leading tick-borne infection in the United States, caused by the pathogenic spirochete Borreliella burgdorferi, formerly known as Borrelia burgdorferi. Diderms, or bacteria with dual-membrane ultrastructure, such as B. burgdorferi, have multiple methods of transporting and integrating outer membrane proteins (OMPs). Most integral OMPs are transported through the ß-barrel assembly machine (BAM) complex. This complex consists of the channel-forming OMP BamA and accessory lipoproteins that interact with the five periplasmic, polypeptide transport-associated (POTRA) domains of BamA. Another system, the translocation and assembly module (TAM) system, has also been implicated in OMP assembly and export. The TAM system consists of two proteins, the BamA paralog TamA which has three POTRA domains and the inner membrane protein TamB. TamB is characterized by a C-terminal DUF490 domain that interacts with the POTRA domains of TamA. Interestingly, while TamB is found in almost all diderms, including B. burgdorferi, TamA is found almost exclusively in Proteobacteria. This strongly suggests a TamA-independent role of TamB in most diderms. We previously demonstrated that BamA interacts with TamB in B. burgdorferi and hypothesized that this is facilitated by the BamA POTRA domains interacting with the TamB DUF490 domain. In this study, we utilized protein-protein co-purification assays to empirically demonstrate that the B. burgdorferi TamB DUF490 domain interacts with BamA POTRA2 and POTRA3. We also observed that the DUF490 domain of TamB interacts with the accessory lipoprotein BamB. To examine if the BamA-TamB interaction is more ubiquitous among diderms, we examined BamA-TamB interactions in Salmonella enterica serovar Typhimurium (St). Interestingly, even though St encodes a TamA protein that interacts with TamB, we observed that the TamB DUF490 of St interacts with BamA in this organism. Our combined findings strongly suggest that the TamB-BamA interaction occurs independent of the TamA component of the TAM protein export system.
Assuntos
Proteínas da Membrana Bacteriana Externa , Borrelia burgdorferi , Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/química , Domínios Proteicos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Ligação Proteica , Sequência de AminoácidosRESUMO
Autotransporter proteins are bacterial outer membrane proteins that display passenger domains with various functions through a ß-barrel shaped translocation domain. YeeJ is an autotransporter protein from E. coli MG1655. In contrast to most other autotransporter proteins, its passenger domain is located at the C-terminus of the translocation domain. Due to this inverted domain organization, YeeJ belongs to autotransporter proteins of type Ve. To investigate the assembly of YeeJ, the fluorescence of a heterologous mCherry passenger domain was measured to quantify its assembly. Based on AlphaFold2 models of 119 sequences similar to YeeJ, a sequence conservation logo for the ß1- and the ß12-strand of type Ve autotransporter proteins was generated. Then, the effect of mutations in these strands on the assembly of YeeJ were analyzed. Mutations of the N-terminal aromatic amino acid of the ß1-strand did not affect the assembly of the translocation domain and the display of the passenger domain. Likewise, exchange of the ß1-strand with the ß3-strand did not impair the assembly of the autotransporter fusion protein. Mutation of the C-terminal aromatic amino acid of the ß12-strand strongly impaired surface display of the mCherry passenger domain. This amino acid has been shown before as an essential feature of the ß-signals of classical autotransporter proteins and outer membrane ß-barrel proteins in general. We therefore propose that the ß12-strand of YeeJ acts as its ß-signal and that the assembly of the YeeJ ß-barrel is driven by its C-terminal ß-strand, like in most other autotransporter proteins, despite its inverted domain organization.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/química , Sistemas de Secreção Tipo V/metabolismo , Sistemas de Secreção Tipo V/genética , Sistemas de Secreção Tipo V/química , Modelos Moleculares , Mutação , Domínios Proteicos , Conformação Proteica em Folha beta , Sequência de AminoácidosRESUMO
Circular dichroism (CD) is a spectroscopic technique commonly used for the analysis of proteins. Particularly, it allows the determination of protein secondary structure content in various media, including the membrane environment. In this chapter, we present how CD applications can be used to analyze the interaction of proteins with bacterial outer membrane vesicles (OMVs). Most CD studies characterizing the structure of proteins inserted into membranes rely on artificial lipid bilayers, mimicking natural membranes. Nevertheless, these artificial models lack the important features of the true membrane, especially for the outer membrane of Gram-negative bacteria. These features include lipid diversity, glycosylation, and asymmetry. Here, we show how to analyze the interactions of proteins, either integral or peripheral, with OMVs in solution and with supported membranes of OMVs, using conventional CD and orientated circular dichroism (OCD). We explain how to decipher the spectroscopic signals to obtain information on the molecular structure of the protein upon its interaction with an OMV and through its potential insertion into an OMV membrane.
Assuntos
Proteínas da Membrana Bacteriana Externa , Dicroísmo Circular , Síncrotrons , Dicroísmo Circular/métodos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química , Estrutura Secundária de Proteína , Bicamadas Lipídicas/metabolismo , Bicamadas Lipídicas/químicaRESUMO
Multidrug efflux pumps, especially those belonging to the class of resistance-nodulation-division (RND), are the key contributors to the rapidly growing multidrug resistance in Gram-negative bacteria. Understanding the role of efflux pumps in real-time drug transport dynamics across the complex dual-cell membrane envelope of Gram-negative bacteria is thus crucial for developing efficient antibiotics against them. Here, we employ second harmonic generation-based nonlinear spectroscopy to study the role of the tripartite efflux pump and its individual components. We systematically investigate the effect of periplasmic adaptor protein AcrA, inner membrane transporter protein AcrB, and outer membrane channel TolC on the overall drug transport in live Acr-type Escherichia coli and its mutant strain cells. Our results reveal that when one of its components is missing, the tripartite AcrAB-TolC efflux pump machinery in Escherichia coli can effectively function as a bipartite system, a fact that has never been demonstrated in live Gram-negative bacteria.
Assuntos
Antibacterianos , Proteínas de Escherichia coli , Escherichia coli , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Análise Espectral/métodos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Transporte Biológico , LipoproteínasRESUMO
The global emergence of multidrug resistance (MDR) in gram-negative bacteria has become a matter of worldwide concern. MDR in these pathogens is closely linked to the overexpression of certain efflux pumps, particularly the resistance-nodulation-cell division (RND) efflux pumps. Inhibition of these pumps presents an attractive and promising strategy to combat antibiotic resistance, as the efflux pump inhibitors can effectively restore the potency of existing antibiotics. AcrAB-TolC is one well-studied RND efflux pump, which transports a variety of substrates, therefore providing resistance to a broad spectrum of antibiotics. To develop effective pump inhibitors, a comprehensive understanding of the structural aspect of the AcrAB-TolC efflux pump is imperative. Previous studies on this pump's structure have been limited to individual components or in vitro determination of fully assembled pumps. Recent advancements in cellular cryo-electron tomography (cryo-ET) have provided novel insights into this pump's assembly and functional mechanism within its native cell membrane environment. Here, we present a summary of the structural data regarding the AcrAB-TolC efflux pump, shedding light on its assembly pathway and operational mechanism.
Assuntos
Antibacterianos , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Farmacorresistência Bacteriana Múltipla , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Microscopia Crioeletrônica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/químicaRESUMO
The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic ß-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Escherichia coli , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Periplasma/metabolismo , Domínios Proteicos , Dobramento de ProteínaRESUMO
Ice-nucleating proteins (INpro) trigger the freezing of supercooled water droplets relevant to atmospheric, biological, and technological applications. The high ice nucleation activity of INpro isolated from the bacteria Pseudomonas syringae could be linked to the aggregation of proteins at the bacterial membrane or at the air-water interface (AWI) of droplets. Here, we imaged freezing onsets, providing direct evidence of these proposed mechanisms. High-speed cryo-microscopy identified the onset location of freezing in droplets between two protein-repellent glass slides. INpro from sterilized P. syringae (Snomax) statistically favored nucleation at the AWI of the droplets. Removing cellular fragments by filtration or adding surfactants increased the frequency of nucleation events at the AWI. On the other hand, cultivated intact bacteria cells or lipid-free droplets nucleated ice without an affinity to the AWI. Overall, we provide visual evidence that INpro from P. syringae trigger freezing at hydrophobic interfaces, such as the AWI or the bacterial membrane, with important mechanistic implications for applications of INpro.
Assuntos
Congelamento , Interações Hidrofóbicas e Hidrofílicas , Pseudomonas syringae , Pseudomonas syringae/metabolismo , Pseudomonas syringae/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Gelo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismoRESUMO
The work presents results of the in vitro and in silico study of formation of amyloid-like structures under harsh denaturing conditions by non-specific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with ß-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with acidic pH at elevated temperature followed by long-term incubation at room temperature is necessary. After heating at 95°C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of tertiary and secondary structure of the protein, which are accompanied by the increase in the content of total ß-structure and sharp decrease in the value of characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to the Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of the changes in intrinsic fluorescence during protein aggregation suggest that during formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only the areas with an internally disordered structure corresponding to the external loops of the porin, but also main framework of the molecule, which has a rigid spatial structure inherent to ß-barrel.
Assuntos
Porinas , Yersinia pseudotuberculosis , Porinas/química , Porinas/metabolismo , Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/química , Animais , Camundongos , Amiloide/metabolismo , Amiloide/química , Estrutura Secundária de Proteína , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Conformação ProteicaRESUMO
Antigen 43 (Ag43) proteins, found on the outer membrane of Escherichia coli, are ß-sheets that fold into a unique cylindrical structure known as a ß-barrel. There are several known structural similarities between bacterial Ag43 autotransporters and physical components; however, the factors that stabilize the barrel and the mechanism for Ag43 passenger domainmediated translocation across the pore of the ß-barrel remain unclear. In this study, we analyzed Ag43ß-enhanced green fluorescent protein chimeric variants to provide new insights into the autotransporter Ag43ß-barrel assembly, focusing on the impact of the α-helical linker domain. Among the chimeric variants, Ag43ß700 showed the highest surface display, which was confirmed through extracellular protease digestion, flow cytometry, and an evaluation of outer membrane vesicles (OMVs). The Ag43ß700 module offered reliable information on stable barrel folding and chimera expression at the exterior of the OMVs. [BMB Reports 2024; 57(8): 369-374].
Assuntos
Membrana Externa Bacteriana , Escherichia coli , Proteínas de Fluorescência Verde , Escherichia coli/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Adesinas de Escherichia coli/metabolismo , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/genética , Dobramento de Proteína , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/químicaRESUMO
Citrus Huanglongbing (HLB) is known as the cancer of citrus, where Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain causing HLB. In this study, we report a novel electrochemiluminescence (ECL) biosensor for the highly sensitive detection of the CLas outer membrane protein (Omp) gene by coupling rolling circle amplification (RCA) with a CRISPR/Cas12a-responsive smart DNA hydrogel. In the presence of the target, a large number of amplicons are generated through RCA. The amplicons activate the trans-cleavage activity of CRISPR/Cas12a through hybridizing with crRNA, triggering the response of smart DNA hydrogel to release the encapsulated AuAg nanoclusters (AuAg NCs) on the electrode and therefore leading to a decreased ECL signal. The ECL intensity change (I0 - I) is positively correlated with the concentration of the target in the range 50 fM to 5 nM, with a limit of detection of 40 fM. The performance of the sensor has also been evaluated with 10 samples of live citrus leaves (five HLB negative and five HLB positive), and the result is in excellent agreement with the gold standard qPCR result. The sensing strategy has expanded the ECL versatility for detecting varying levels of dsDNA or ssDNA in plants with high sensitivity.
Assuntos
Proteínas da Membrana Bacteriana Externa , Citrus , Técnicas Eletroquímicas , Medições Luminescentes , Técnicas Eletroquímicas/métodos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/química , Citrus/microbiologia , Citrus/química , Hidrogéis/química , Técnicas Biossensoriais/métodos , DNA/química , DNA/genética , Sistemas CRISPR-Cas/genética , Liberibacter/genética , Liberibacter/química , Técnicas de Amplificação de Ácido Nucleico , Doenças das Plantas/microbiologia , Ouro/química , Nanopartículas Metálicas/química , Limite de DetecçãoRESUMO
The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12-RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B12-btuB riboswitch recognition clearly beyond the known role in affinity.
Assuntos
Corrinoides , Riboswitch , Vitamina B 12 , Vitamina B 12/química , Vitamina B 12/metabolismo , Corrinoides/química , Corrinoides/metabolismo , Ligantes , Ligação de Hidrogênio , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Cobamidas/química , Cobamidas/metabolismo , Sítios de Ligação , Proteínas de Membrana TransportadorasRESUMO
Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.
Assuntos
Antígenos de Bactérias , Proteína de Ligação ao Complemento C4b , Streptococcus pyogenes , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/química , Proteína de Ligação ao Complemento C4b/metabolismo , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Humanos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/química , Ligação Proteica , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genéticaRESUMO
TolC is the outer membrane protein responsible for antibiotic efflux in E. coli. Compared to other outer membrane proteins it has an unusual fold and has been shown to fold independently of commonly used periplasmic chaperones, SurA and Skp. Here we find that the assembly of TolC involves the formation of two folded intermediates using circular dichroism, gel electrophoresis, site-specific disulfide bond formation and radioactive labeling. First the TolC monomer folds, and then TolC assembles into a trimer both in detergent-free buffer and in the presence of detergent micelles. We find that a TolC trimer also forms in the periplasm and is present in the periplasm before it inserts in the outer membrane. The monomeric and trimeric folding intermediates may be used in the future to develop a new approach to antibiotic efflux pump inhibition by targeting the assembly pathway of TolC.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Membrana Transportadoras , Dobramento de Proteína , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Dicroísmo Circular , Periplasma/metabolismo , Multimerização ProteicaRESUMO
Pseudomonas aeruginosa, a formidable pathogen renowned for its antimicrobial resistance, poses a significant threat to immunocompromised individuals. In this regard, the MexAB-OprM efflux pump acts as a pivotal line of defense by extruding antimicrobials from bacterial cells. The inner membrane homotrimeric protein MexB captures antibiotics and translocates them into the outer membrane OprM channel protein connected through the MexA adaptor protein. Despite extensive efforts, competitive inhibitors targeting the tight (T) protomer of the MexB protein have not received FDA approval for medical use. Over the past few years, allosteric inhibitors have become popular as alternatives to the classical competitive inhibitor-based approach because of their higher specificity, lower dosage, and reduced toxicological effects. Hence, in this study, we unveiled the existence of a transmembrane allosteric binding pocket of MexB inspired by the recent discovery of an important allosteric inhibitor, BDM88855, for the homolog AcrB protein. While repurposing BDM88855 proved ineffective in controlling the MexB loose (L) protomer, our investigation identified a promising alternative: a chlorine-containing variant of DB08385 (2-Cl DB08385 or Variant 1). Molecular dynamics simulations, including binding free energy estimation coupled with heterogeneous dielectric implicit membrane model (implicit-membrane MM/PBSA), interaction entropy (IE) analysis and potential of mean force (PMF) calculation, demonstrated Variant 1's superior binding affinity to the transmembrane pocket, displaying the highest energy barrier in the ligand unbinding process. To elucidate the allosteric crosstalk between the transmembrane and porter domain of MexB, we employed the 'eigenvector centrality' measure in the linear mutual information obtained from the protein correlation network. Notably, this study confirmed the presence of an allosteric transmembrane site in the MexB L protomer. In addition to this, Variant 1 emerged as a potent regulator of allosteric crosstalk, inducing an 'O-L intermediate state' in the MexB L protomer. This induced state might hold the potential to diminish substrate intake into the access pocket, leading to the ineffective efflux of antibiotics.
Assuntos
Antibacterianos , Proteínas da Membrana Bacteriana Externa , Simulação de Dinâmica Molecular , Pseudomonas aeruginosa , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Farmacorresistência Bacteriana/efeitos dos fármacosRESUMO
Safe and effective vaccine candidates are needed to address the limitations of existing vaccines against Brucellosis, a disease responsible for substantial economic losses in livestock. The present study aimed to encapsulate recombinant Omp25 and EipB proteins, knowledged antigen properties, into PLGA nanoparticles, characterize synthesized nanoparticles with different methods, and assessed theirin vitro/in vivoimmunostimulatory activities to develop new vaccine candidates. The recombinant Omp25 and EipB proteins produced with recombinant DNA technology were encapsulated into PLGA nanoparticles by double emulsion solvent evaporation technique. The nanoparticles were characterized using FE-SEM, Zeta-sizer, and FT-IR instruments to determine size, morphology, zeta potentials, and polydispersity index values, as well as to analyze functional groups chemically. Additionally, the release profiles and encapsulation efficiencies were assessed using UV-Vis spectroscopy. After loading with recombinant proteins, O-NPs reached sizes of 221.2 ± 5.21 nm, while E-NPs reached sizes of 274.4 ± 9.51 nm. The cumulative release rates of the antigens, monitored until the end of day 14, were determined to be 90.39% for O-NPs and 56.1% for E-NPs. Following the assessment of thein vitrocytotoxicity and immunostimulatory effects of both proteins and nanoparticles on the J774 murine macrophage cells,in vivoimmunization experiments were conducted using concentrations of 16µg ml-1for each protein. Both free antigens and antigen-containing nanoparticles excessively induced humoral immunity by increasing producedBrucella-specific IgG antibody levels for 3 times in contrast to control. Furthermore, it was also demonstrated that vaccine candidates stimulated Th1-mediated cellular immunity as well since they significantly raised IFN-gamma and IL-12 cytokine levels in murine splenocytes rather than IL-4 following to immunization. Additionally, the vaccine candidates conferred higher than 90% protection from the infection according to challenge results. Our findings reveal that PLGA nanoparticles constructed with the encapsulation of recombinant Omp25 or EipB proteins possess great potential to triggerBrucella-specific humoral and cellular immune response.