RESUMO
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
Under specific conditions, some proteins can self-assemble into fibrillar structures called amyloids. Initially, these proteins were associated with neurodegenerative diseases in eucaryotes. Nevertheless, they have now been identified in the three domains of life. In bacteria, they are involved in diverse biological processes and are usually useful for the cell. For this reason, they are classified as "functional amyloids". In this work, we focus our analysis on a bacterial functional amyloid called Hfq. Hfq is a pleiotropic regulator that mediates several aspects of genetic expression, mainly via the use of small noncoding RNAs. Our previous work showed that Hfq amyloid-fibrils interact with membranes. This interaction influences Hfq amyloid structure formation and stability, but the specifics of the lipid on the dynamics of this process is unknown. Here, we show, using spectroscopic methods, how lipids specifically drive and modulate Hfq amyloid assembly or, conversely, its disassembly. The reported effects are discussed in light of the consequences for bacterial cell life.
Assuntos
Amiloide , Pequeno RNA não Traduzido , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Pequeno RNA não Traduzido/genética , Bactérias/metabolismo , Lipídeos , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/genética , Regulação Bacteriana da Expressão GênicaRESUMO
Useful structural information about the conformation of nucleic acids can be quickly acquired by circular and linear dichroism (CD/LD) spectroscopy. These techniques, rely on the differential absorption of polarised light and are indeed extremely sensitive to subtle changes in the structure of chiral biomolecules. Many CD analyses of DNA or DNA:protein complexes have been conducted with substantial data acquisitions. Conversely, CD RNA analysis are still scarce, despite the fact that RNA plays a wide cellular function. This chapter seeks to introduce the reader to the use of circular, linear dichroism and in particular the use of Synchrotron Radiation for such samples. The use of these techniques on small noncoding RNA (sRNA) will be exemplified by analyzing changes in base stacking and/or helical parameters for the understanding of sRNA structure and function, especially by translating the dynamics of RNA:RNA annealing but also to access RNA stability or RNA:RNA alignment. The effect of RNA remodeling proteins will also be addressed. These analyses are especially useful to decipher the mechanisms by which sRNA will adopt the proper conformation thanks to the action of proteins such as Hfq or ProQ in the regulation of the expression of their target mRNAs.
Assuntos
Pequeno RNA não Traduzido , Pequeno RNA não Traduzido/genética , Proteínas/metabolismo , RNA Mensageiro/metabolismo , DNA , Dicroísmo Circular , Fator Proteico 1 do HospedeiroRESUMO
Microcin E492 (MccE492) is an antimicrobial peptide and proposed virulence factor produced by some Klebsiella pneumoniae strains, which, under certain conditions, form amyloid fibers, leading to the loss of its antibacterial activity. Although this protein has been characterized as a model functional amyloid, the secondary structure transitions behind its formation, and the possible effect of molecules that inhibit this process, have not been investigated. In this study, we examined the ability of the green tea flavonoid epigallocatechin gallate (EGCG) to interfere with MccE492 amyloid formation. Aggregation kinetics followed by thioflavin T binding were used to monitor amyloid formation in the presence or absence of EGCG. Additionally, synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) were used to study the secondary structure, thermal stability, and morphology of microcin E492 fibers. Our results showed that EGCG significantly inhibited the formation of the MccE492 amyloid, resulting in mainly amorphous aggregates and small oligomers. However, these aggregates retained part of the ß-sheet SRCD signal and a high resistance to heat denaturation, suggesting that the aggregation process is sequestered or deviated at some stage but not completely prevented. Thus, EGCG is an interesting inhibitor of the amyloid formation of MccE492 and other bacterial amyloids.
Assuntos
Catequina , Polifenóis , Polifenóis/farmacologia , Chá , Amiloide/química , Proteínas Amiloidogênicas , Catequina/farmacologia , Catequina/químicaRESUMO
The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs. Moreover, we provide evidence that Hfq interacts with and is inserted into OMV membranes, suggesting a role for this protein in the release of sRNA outside the vesicle. These findings provide clues to the mechanism of host-bacteria interactions which may not be defined solely by protein-protein and protein-outer membrane contacts, but also by the exchange of RNAs, and in particular sRNAs.
Assuntos
Proteínas de Escherichia coli , Pequeno RNA não Traduzido , Escherichia coli/genética , Escherichia coli/metabolismo , Dicroísmo Circular , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Pequeno RNA não Traduzido/genética , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/genética , Regulação Bacteriana da Expressão GênicaRESUMO
Due to their two-cell membranes, Gram-negative bacteria are particularly resistant to antibiotics. Recent investigations aimed at exploring new target proteins involved in Gram-negative bacteria adaptation helped to identify environmental changes encountered during infection. One of the most promising approaches in finding novel targets for antibacterial drugs consists of blocking noncoding RNA-based regulation using the protein cofactor, Hfq. Although Hfq is important in many bacterial pathogens, its involvement in antibiotics response is still unclear. Indeed, Hfq may mediate drug resistance by regulating the major efflux system in Escherichia coli, but it could also play a role in the influx of antibiotics. Here, using an imaging approach, we addressed this problem quantitatively at the single-cell level. More precisely, we analyzed how Hfq affects the dynamic influx and efflux of ciprofloxacin, an antibiotic from the group of fluoroquinolones that is used to treat bacterial infections. Our results indicated that the absence of either whole Hfq or its C-terminal domain resulted in a more effective accumulation of ciprofloxacin, irrespective of the presence of the functional AcrAB-TolC efflux pump. However, overproduction of the MicF small regulatory RNA, which reduces the efficiency of expression of the ompF gene (coding for a porin involved in antibiotics influx) in a Hfq-dependent manner, resulted in impaired accumulation of ciprofloxacin. These results led us to propose potential mechanisms of action of Hfq in the regulation of fluoroquinolone fluxes across the E. coli envelope.
RESUMO
The bacterial chromosomic DNA is packed within a membrane-less structure, the nucleoid, due to the association of DNA with proteins called Nucleoid Associated Proteins (NAPs). Among these NAPs, Hfq is one of the most intriguing as it plays both direct and indirect roles on DNA structure. Indeed, Hfq is best known to mediate post-transcriptional regulation by using small noncoding RNA (sRNA). Although Hfq presence in the nucleoid has been demonstrated for years, its precise role is still unclear. Recently, it has been shown in vitro that Hfq forms amyloid-like structures through its C-terminal region, hence belonging to the bridging family of NAPs. Here, using cryo soft X-ray tomography imaging of native unlabeled cells and using a semi-automatic analysis and segmentation procedure, we show that Hfq significantly remodels the Escherichia coli nucleoid. More specifically, Hfq influences nucleoid density especially during the stationary growth phase when it is more abundant. Our results indicate that Hfq could regulate nucleoid compaction directly via its interaction with DNA, but also at the post-transcriptional level via its interaction with RNAs. Taken together, our findings reveal a new role for this protein in nucleoid remodeling in vivo, that may serve in response to stress conditions and in adapting to changing environments.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Tomografia por Raios X , DNA , Proteínas de Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genéticaRESUMO
Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNAs. Previously, we showed that the Hfq C-terminal region forms an amyloid-like structure and that these fibrils interact with membranes. The immediate consequence of this interaction is a disruption of the membrane, but the effect on Hfq structure was unknown. To investigate details of the mechanism of interaction, the present work uses different in vitro biophysical approaches. We show that the Hfq C-terminal region influences membrane integrity and, conversely, that the membrane specifically affects the amyloid assembly. The reported effect of this bacterial master regulator on membrane integrity is discussed in light of the possible consequence on small regulatory RNA-based regulation.
Assuntos
Proteínas de Escherichia coli , RNA Bacteriano , Proteínas Amiloidogênicas/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/metabolismoRESUMO
Amyloid inhibitors, such as the green tea compound epigallocatechin gallate EGCG, apomorphine or curlicide, have antibacterial properties. Conversely, antibiotics such as tetracycline derivatives or rifampicin also affect eukaryotic amyloids formation and may be used to treat neurodegenerative diseases. This opens the possibility for existing drugs to be repurposed in view of new therapy, targeting amyloid-like proteins from eukaryotes to prokaryotes and conversely. Here we present how to evaluate the effect of these amyloid-forming inhibitors on bacterial amyloid self-assemblies in vitro and on bacterial survival. The different approaches possible are presented.
Assuntos
Amiloidose , Catequina , Amiloide/metabolismo , Proteínas Amiloidogênicas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/metabolismo , Catequina/farmacologia , HumanosRESUMO
Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and orientated circular dichroism (OCD) are complementary spectroscopies widely used for the analysis of protein samples such as the amyloids commonly renowned as neurodegenerative agents. Determining the secondary structure content of proteins, such as aggregated ß-sheets inside the amyloids and in various environments, including membranes and lipids, has made these techniques very valuable and complemental to high-resolution techniques such as nuclear magnetic resonance (NMR), X-ray crystallography, and cryo-electron microscopy. FTIR and CD are extremely sensitive to structural changes of proteins due to environmental changes. Furthermore, FTIR provides information on lipid modifications upon protein binding, whereas synchrotron radiation CD (SRCD) and OCD are sensitive to the subtle structural changes occurring in ß-sheet-rich proteins and their orientation or alignment with lipid bilayers. FTIR and CD techniques allow the identification of parallel and antiparallel ß-sheet content and are therefore complementary. In this chapter, we present FTIR and CD/OCD applications to study the interactions of bacterial amyloids with membranes and lipids. Moreover, we show how to decipher the spectroscopic signals to obtain information on the molecular structure of amyloids and their interaction with lipids, addressing potential amyloid insertion into membranes and the lipid bilayer adjustments observed.
Assuntos
Amiloide , Proteínas Amiloidogênicas , Amiloide/química , Dicroísmo Circular , Microscopia Crioeletrônica , Bicamadas Lipídicas/química , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
Bacterial chromosomal DNA is packed within a non-membranous structure, the nucleoid, thanks to nucleoid associated proteins (NAPs). The role of bacterial amyloid has recently emerged among these NAPs, particularly with the nucleoid-associated protein Hfq that plays a direct role in DNA compaction. In this chapter, we present a 3D imaging technique, cryo-soft X-ray tomography (cryo-SXT) to obtain a detailed 3D visualization of subcellular bacterial structures, especially the nucleoid. Cryo-SXT imaging of native unlabeled cells enables observation of the nucleoid in 3D with a high resolution, allowing to evidence in vivo the role of amyloids on DNA compaction. The precise experimental methods to obtain 3D tomograms will be presented.
Assuntos
Organelas , Tomografia por Raios X , Proteínas Amiloidogênicas , Proteínas de Bactérias , DNA , DNA Bacteriano , Imageamento Tridimensional/métodos , Organelas/ultraestrutura , Tomografia por Raios X/métodosRESUMO
Hfq is a bacterial RNA chaperone which promotes the pairing of small noncoding RNAs to target mRNAs, allowing post-transcriptional regulation. This RNA annealing activity has been attributed for years to the N-terminal region of the protein that forms a toroidal structure with a typical Sm-fold. Nevertheless, many Hfqs, including that of Escherichia coli, have a C-terminal region with unclear functions. Here we use a biophysical approach, Synchrotron Radiation Circular Dichroism (SRCD), to probe the interaction of the E. coli Hfq C-terminal amyloid region with RNA and its effect on RNA annealing. This C-terminal region of Hfq, which has been described to be dispensable for sRNA:mRNA annealing, has an unexpected and significant effect on this activity. The functional consequences of this novel property of the amyloid region of Hfq in relation to physiological stress are discussed.
RESUMO
Hfq is a bacterial regulator with key roles in gene expression. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, thanks to its binding to small regulatory noncoding RNAs. This property is of primary importance for bacterial adaptation and survival in hosts. Small RNAs and Hfq are, for instance, involved in the response to antibiotics. Previous work has shown that the E. coli Hfq C-terminal region (Hfq-CTR) self-assembles into an amyloid structure. It was also demonstrated that the green tea compound EpiGallo Catechin Gallate (EGCG) binds to Hfq-CTR amyloid fibrils and remodels them into nonamyloid structures. Thus, compounds that target the amyloid region of Hfq may be used as antibacterial agents. Here, we show that another compound that inhibits amyloid formation, apomorphine, may also serve as a new antibacterial. Our results provide an alternative in order to repurpose apomorphine, commonly used in the treatment of Parkinson's disease, as an antibiotic to block bacterial adaptation to treat infections.
RESUMO
Nucleic acid amyloid proteins interactions have been observed in the past few years. These interactions often promote protein aggregation. Nevertheless, molecular basis and physiological consequences of these interactions are still poorly understood. Additionally, it is unknown whether the nucleic acid promotes the formation of self-assembly due to direct interactions or indirectly via sequences surrounding the amyloid region. Here we focus our attention on a bacterial amyloid, Hfq. This protein is a pleiotropic bacterial regulator that mediates many aspects of nucleic acids metabolism. The protein notably mediates mRNA stability and translation efficiency by using stress-related small non coding regulatory RNA. In addition, Hfq, thanks to its amyloid C-terminal region, binds and compacts DNA. A combination of experimental methodologies, including synchrotron radiation circular dichroism (SRCD), gel shift assay and infrared (FTIR) spectroscopy have been used to probe the interaction of Hfq C-terminal region with DNA. We clearly identify important amino acids in this region involved in DNA binding and polymerization properties. This allows to understand better how this bacterial amyloid interacts with DNA. Possible functional consequence to answer to stresses are discussed.
RESUMO
Hfq is a pleiotropic regulator that has key roles in the control of genetic expression. The protein noticeably regulates translation efficiency and RNA decay in Gram-negative bacteria, due to the Hfq-mediated interaction between small regulatory noncoding RNA and mRNA. This property is of primary importance for bacterial adaptation and virulence. We have previously shown that the Hfq E. coli protein, and more precisely its C-terminal region (CTR), self-assembles into an amyloid-like structure. In the present work, we demonstrate that epigallocatechin gallate (EGCG), a major green tea polyphenol compound, targets the Hfq amyloid region and can be used as a potential antibacterial agent. We analysed the effect of this compound on Hfq amyloid fibril stability and show that EGCG both disrupts Hfq-CTR fibrils and inhibits their formation. We show that, even if EGCG affects other bacterial amyloids, it also specifically targets Hfq-CTR in vivo. Our results provide an alternative approach for the utilisation of EGCG that may be used synergistically with conventional antibiotics to block bacterial adaptation and treat infections.
RESUMO
Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNA. Besides these RNA-related functions, Hfq has also been described as one of the nucleoid associated proteins shaping the bacterial chromosome. Therefore, Hfq appears as a versatile nucleic acid-binding protein, which functions are probably even more numerous than those initially suggested. For instance, E. coli Hfq, and more precisely its C-terminal region (CTR), has been shown to induce DNA compaction into a condensed form. In this paper, we establish that DNA induces Hfq-CTR amyloidogenesis, resulting in a change of DNA local conformation. Furthermore, we clarify the effect of Hfq on DNA topology. Our results evidence that, even if the protein has a strong propensity to compact DNA thanks to its amyloid region, it does not affect overall DNA topology. We confirm however that hfq gene disruption influences plasmid supercoiling in vivo, indicating that the effect on DNA topology in former reports was indirect. Most likely, this effect is related to small regulatory sRNA-Hfq-based regulation of another protein that influences DNA supercoiling, possibly a nucleoid associated protein such as H-NS or Dps. Finally, we hypothesise that this indirect effect on DNA topology explains, at least partially, the previously reported effect of Hfq on plasmid replication efficiency.