RESUMO
Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.
Assuntos
Anticorpos Antibacterianos , Anticorpos Neutralizantes , Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Anticorpos Antibacterianos/farmacologia , Microscopia Crioeletrônica , Imunoglobulinas/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Infecções por Pseudomonas/tratamento farmacológicoRESUMO
The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.
Assuntos
ATPases Associadas a Diversas Atividades Celulares , Proteínas de Bactérias , DNA Helicases , DNA Cruciforme , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , DNA Cruciforme/química , DNA Cruciforme/metabolismo , DNA Cruciforme/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Recombinação Homóloga , Hidrólise , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Nucleotídeos , Conformação Proteica , RotaçãoRESUMO
Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year1. Specialized protein transport systems-known as type VII secretion systems (T7SSs)-are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope2,3. Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP5 protease. A trimer of MycP5 caps a central periplasmic dome-like chamber that is formed by three EccB5 dimers, with the proteolytic sites of MycP5 facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP5 show disruption of the EccB5 periplasmic assembly and increased flexibility, which highlights the importance of MycP5 for complex integrity. Beneath the EccB5-MycP5 chamber, dimers of the EccC5 ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC5 domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen.
Assuntos
Microscopia Crioeletrônica , Mycobacterium tuberculosis , Sistemas de Secreção Tipo VII/metabolismo , Sistemas de Secreção Tipo VII/ultraestrutura , Citosol/química , Citosol/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/ultraestrutura , Periplasma/química , Periplasma/metabolismo , Domínios Proteicos , Multimerização Proteica , Estabilidade Proteica , Tuberculose/virologia , Sistemas de Secreção Tipo VII/químicaRESUMO
Particle localization (picking) in digital tomograms is a laborious and time-intensive step in cryogenic electron tomography (cryoET) analysis often requiring considerable user involvement, thus becoming a bottleneck for automated cryoET subtomogram averaging (STA) pipelines. In this paper, we introduce a deep learning framework called PickYOLO to tackle this problem. PickYOLO is a super-fast, universal particle detector based on the deep-learning real-time object recognition system YOLO (You Only Look Once), and tested on single particles, filamentous structures, and membrane-embedded particles. After training with the centre coordinates of a few hundred representative particles, the network automatically detects additional particles with high yield and reliability at a rate of 0.24-3.75 s per tomogram. PickYOLO can automatically detect number of particles comparable to those manually selected by experienced microscopists. This makes PickYOLO a valuable tool to substantially reduce the time and manual effort needed to analyse cryoET data for STA, greatly aiding in high-resolution cryoET structure determination.
Assuntos
Aprendizado Profundo , Elétrons , Reprodutibilidade dos Testes , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodosRESUMO
The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin-binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca2+ in nonmuscle cells. Here we report the mechanism of Ca2+-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca2+-free and Ca2+-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca2+, binding of which can only be regulated in the presence of physiological concentrations of Mg2+ Ca2+ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin-binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery.
Assuntos
Actinina/metabolismo , Cálcio/farmacologia , Entamoeba histolytica/metabolismo , Actinina/química , Actinina/genética , Domínio Catalítico , Entamoeba histolytica/genética , Regulação da Expressão Gênica , Modelos Moleculares , Conformação Proteica , Domínios ProteicosRESUMO
Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo-EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, without observing toxic effects.
Assuntos
Acinetobacter baumannii , Fenilpropionatos , Antibacterianos/farmacologia , Antibacterianos/química , Microscopia Crioeletrônica , Fenilpropionatos/farmacologia , Bactérias Gram-Negativas , Testes de Sensibilidade MicrobianaRESUMO
Type III protein-secretion machines are essential for the interactions of many pathogenic or symbiotic bacterial species with their respective eukaryotic hosts. The core component of these machines is the injectisome, a multiprotein complex that mediates the selection of substrates, their passage through the bacterial envelope, and ultimately their delivery into eukaryotic target cells. The injectisome is composed of a large cytoplasmic complex or sorting platform, a multiring base embedded in the bacterial envelope, and a needle-like filament that protrudes several nanometers from the bacterial surface and is capped at its distal end by the tip complex. A characteristic feature of these machines is that their activity is stimulated by contact with target host cells. The sensing of target cells, thought to be mediated by the distal tip of the needle filament, generates an activating signal that must be transduced to the secretion machine by the needle filament. Here, through a multidisciplinary approach, including solid-state NMR (SSNMR) and cryo electron microscopy (cryo-EM) analyses, we have identified critical residues of the needle filament protein of a Salmonella Typhimurium type III secretion system that are involved in the regulation of the activity of the secretion machine. We found that mutations in the needle filament protein result in various specific phenotypes associated with different steps in the type III secretion process. More specifically, these studies reveal an important role for a polymorphic helix of the needle filament protein and the residues that line the lumen of its central channel in the control of type III secretion.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Complexos Multiproteicos/metabolismo , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/química , Sistemas de Secreção Bacterianos/genética , Microscopia Crioeletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Mutação , Conformação Proteica , Transporte Proteico/genética , Salmonella typhimurium/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/ultraestruturaRESUMO
Viral inhibitors, such as pleconaril and vapendavir, target conserved regions in the capsids of rhinoviruses (RVs) and enteroviruses (EVs) by binding to a hydrophobic pocket in viral capsid protein 1 (VP1). In resistant RVs and EVs, bulky residues in this pocket prevent their binding. However, recently developed pyrazolopyrimidines inhibit pleconaril-resistant RVs and EVs, and computational modeling has suggested that they also bind to the hydrophobic pocket in VP1. We studied the mechanism of inhibition of pleconaril-resistant RVs using RV-B5 (1 of the 7 naturally pleconaril-resistant rhinoviruses) and OBR-5-340, a bioavailable pyrazolopyrimidine with proven in vivo activity, and determined the 3D-structure of the protein-ligand complex to 3.6 Å with cryoelectron microscopy. Our data indicate that, similar to other capsid binders, OBR-5-340 induces thermostability and inhibits viral adsorption and uncoating. However, we found that OBR-5-340 attaches closer to the entrance of the pocket than most other capsid binders, whose viral complexes have been studied so far, showing only marginal overlaps of the attachment sites. Comparing the experimentally determined 3D structure with the control, RV-B5 incubated with solvent only and determined to 3.2 Å, revealed no gross conformational changes upon OBR-5-340 binding. The pocket of the naturally OBR-5-340-resistant RV-A89 likewise incubated with OBR-5-340 and solved to 2.9 Å was empty. Pyrazolopyrimidines have a rigid molecular scaffold and may thus be less affected by a loss of entropy upon binding. They interact with less-conserved regions than known capsid binders. Overall, pyrazolopyrimidines could be more suitable for the development of new, broadly active inhibitors.
Assuntos
Antivirais/metabolismo , Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Farmacorresistência Viral , Oxidiazóis/farmacologia , Rhinovirus/metabolismo , Proteínas Virais/química , Antivirais/farmacologia , Sítios de Ligação , Capsídeo/efeitos dos fármacos , Capsídeo/ultraestrutura , Células HeLa , Humanos , Modelos Moleculares , Estrutura Molecular , Oxazóis , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/metabolismo , Infecções por Picornaviridae/virologia , Ligação Proteica , Conformação Proteica , Rhinovirus/efeitos dos fármacos , Rhinovirus/ultraestrutura , Relação Estrutura-Atividade , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The type III secretion system (T3SS) is an essential virulence factor of many pathogenic bacterial species including Salmonella, Yersinia, Shigella and enteropathogenic Escherichia coli (EPEC). It is an intricate molecular machine that spans the bacterial membranes and injects effector proteins into target host cells, enabling bacterial infection. The T3SS needle complex comprises of proteinaceous rings supporting a needle filament which extends out into the extracellular environment. It serves as the central conduit for translocating effector proteins. Multiple laboratories have dedicated a remarkable effort to decipher the structure and function of the needle complex. A combination of structural biology techniques such as cryo-electron microscopy (cryoEM), X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and computer modelling have been utilized to study different structural components at progressively higher resolutions. This chapter will provide an overview of the structural details of the T3SS needle complex, shedding light on this essential component of this fascinating bacterial system.
Assuntos
Sistemas de Secreção Tipo III/química , Proteínas de Bactérias , Simulação por Computador , Microscopia Crioeletrônica , Cristalografia por Raios X , Ressonância Magnética Nuclear Biomolecular , Sistemas de Secreção Tipo III/ultraestrutura , Fatores de VirulênciaRESUMO
One of the most exciting developments in the field of bacterial pathogenesis in recent years is the discovery that many pathogens utilize complex nanomachines to deliver bacterially encoded effector proteins into target eukaryotic cells. These effector proteins modulate a variety of cellular functions for the pathogen's benefit. One of these protein-delivery machines is the type III secretion system (T3SS). T3SSs are widespread in nature and are encoded not only by bacteria pathogenic to vertebrates or plants but also by bacteria that are symbiotic to plants or insects. A central component of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins across the bacterial envelope. Working in conjunction with several cytoplasmic components, the needle complex engages specific substrates in sequential order, moves them across the bacterial envelope, and ultimately delivers them into eukaryotic cells. The central role of T3SSs in pathogenesis makes them great targets for novel antimicrobial strategies.
Assuntos
Bactérias/química , Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Animais , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Humanos , Transporte ProteicoRESUMO
We describe a general approach for refining protein structure models on the basis of cryo-electron microscopy maps with near-atomic resolution. The method integrates Monte Carlo sampling with local density-guided optimization, Rosetta all-atom refinement and real-space B-factor fitting. In tests on experimental maps of three different systems with 4.5-Å resolution or better, the method consistently produced models with atomic-level accuracy largely independently of starting-model quality, and it outperformed the molecular dynamics-based MDFF method. Cross-validated model quality statistics correlated with model accuracy over the three test systems.
Assuntos
Físico-Química/métodos , Microscopia Crioeletrônica , Modelos Moleculares , Proteínas/química , Método de Monte CarloRESUMO
Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called injectisome. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central "cup" substructure of a Salmonella Typhimurium secretion system. However, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we present evidence that a SpaP pentamer forms a 15 Å wide pore and provide a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further refine the current view of export apparatus assembly, consolidate transmembrane topology models for SpaP and SpaR, and present intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, indicating how export apparatus and needle filament are connected to create a continuous conduit for substrate translocation.
Assuntos
Salmonella typhimurium/metabolismo , Salmonella typhimurium/ultraestrutura , Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo III/ultraestrutura , Cromatografia em Gel , Processamento de Imagem Assistida por Computador , Immunoblotting , Espectrometria de Massas , Microscopia EletrônicaRESUMO
Key cellular processes are frequently accompanied by protein-facilitated shape changes in the plasma membrane. N-BAR-domain protein modules generate curvature by means of complex interactions with the membrane surface. The way they assemble and the mechanism by which they operate are largely dependent on their binding density. Although the mechanism at lower densities has recently begun to emerge, how membrane scaffolds form at high densities remains unclear. By combining electron microscopy and multiscale simulations, we show that N-BAR proteins at high densities can transform a lipid vesicle into a 3D tubular network. We show that this process is a consequence of excess adhesive energy combined with the local stiffening of the membrane, which occurs in a narrow range of mechanical properties of both the membrane and the protein. We show that lipid diffusion is significantly reduced by protein binding at this density regime and even more in areas of high Gaussian curvature, indicating a potential effect on molecular transport in cells. Finally, we reveal that the breaking of the bilayer topology is accompanied by the nematic arrangement of the protein on the surface, a structural motif that likely drives the formation of reticular structures in living cells.
Assuntos
Lipossomos/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Difusão , Bicamadas Lipídicas/química , Lipossomos/ultraestrutura , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Type III protein secretion systems are unique bacterial nanomachines with the capacity to deliver bacterial effector proteins into eukaryotic cells. These systems are critical to the biology of many pathogenic or symbiotic bacteria for insects, plants, animals, and humans. Essential components of these systems are multiprotein envelope-associated organelles known as the needle complex and a group of membrane proteins that compose the so-called export apparatus. Here, we show that components of the export apparatus associate intimately with the needle complex, forming a structure that can be visualized by cryo-electron microscopy. We also show that formation of the needle complex base is initiated at the export apparatus and that, in the absence of export apparatus components, there is a significant reduction in the levels of needle complex base assembly. Our results show a substantial coordination in the assembly of the two central elements of type III secretion machines.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Complexos Multiproteicos/metabolismo , Salmonella typhimurium/fisiologia , Via Secretória/fisiologia , Western Blotting , Microscopia Crioeletrônica , Processamento de Imagem Assistida por Computador , Imunoprecipitação , Complexos Multiproteicos/ultraestrutura , Salmonella typhimurium/metabolismo , Salmonella typhimurium/ultraestruturaRESUMO
Holliday junctions are key intermediate DNA structures during genetic recombination. One of the first Holliday junction-processing protein complexes to be discovered was the well conserved RuvAB branch migration complex present in bacteria that mediates an ATP-dependent movement of the Holliday junction (branch migration). Although the RuvAB complex served as a paradigm for the processing of the Holliday junction, due to technical limitations the detailed structure and underlying mechanism of the RuvAB branch migration complex has until now remained unclear. Recently, structures of a reconstituted RuvAB complex actively-processing a Holliday junction were resolved using time-resolved cryo-electron microscopy. These structures showed distinct conformational states at different stages of the migration process. These structures made it possible to propose an integrated model for RuvAB Holliday junction branch migration. Furthermore, they revealed unexpected insights into the highly coordinated and regulated mechanisms of the nucleotide cycle powering substrate translocation in the hexameric AAA+ RuvB ATPase. Here, we review these latest advances and describe areas for future research.
Assuntos
DNA Cruciforme , Movimento , Microscopia Crioeletrônica , ATPases Associadas a Diversas Atividades Celulares , NucleotídeosRESUMO
Building accurate protein models into moderate resolution (3-5 Å) cryoelectron microscopy (cryo-EM) maps is challenging and error prone. We have developed MEDIC (Model Error Detection in Cryo-EM), a robust statistical model that identifies local backbone errors in protein structures built into cryo-EM maps by combining local fit-to-density with deep-learning-derived structural information. MEDIC is validated on a set of 28 structures that were subsequently solved to higher resolutions, where we identify the differences between low- and high-resolution structures with 68% precision and 60% recall. We additionally use this model to fix over 100 errors in 12 deposited structures and to identify errors in 4 refined AlphaFold predictions with 80% precision and 60% recall. As modelers more frequently use deep learning predictions as a starting point for refinement and rebuilding, MEDIC's ability to handle errors in structures derived from hand-building and machine learning methods makes it a powerful tool for structural biologists.
Assuntos
Aprendizado de Máquina , Proteínas , Conformação Proteica , Microscopia Crioeletrônica/métodos , Modelos Moleculares , Proteínas/químicaRESUMO
Cryogenic electron microscopy (cryo-EM) data represent density maps of macromolecular systems at atomic or near-atomic resolution. However, building and refining 3D atomic models by using data from cryo-EM maps is not straightforward and requires significant hands-on experience and manual intervention. We recently developed StarMap, an easy-to-use interface between the popular structural display program ChimeraX and Rosetta, a powerful molecular modeling engine. StarMap offers a general approach for refining structural models of biological macromolecules into cryo-EM density maps by combining Monte Carlo sampling with local density-guided optimization, Rosetta-based all-atom refinement and real-space B-factor calculations in a straightforward workflow. StarMap includes options for structural symmetry, local refinements and independent model validation. The overall quality of the refinement and the structure resolution is then assessed via analytical outputs, such as magnification calibration (pixel size calibration) and Fourier shell correlations. Z-scores reported by StarMap provide an easily interpretable indicator of the goodness of fit for each residue and can be plotted to evaluate structural models and improve local residue refinements, as well as to identify flexible regions and potentially functional sites in large macromolecular complexes. The protocol requires general computer skills, without the need for coding expertise, because most parts of the workflow can be operated by clicking tabs within the ChimeraX graphical user interface. Time requirements for the model refinement depend on the size and quality of the input data; however, this step can typically be completed within 1 d. The analytical parts of the workflow are completed within minutes.
Assuntos
Estrutura Molecular , Fluxo de Trabalho , Microscopia Crioeletrônica/métodos , Modelos Moleculares , Conformação Proteica , Substâncias MacromolecularesRESUMO
Secreted bacterial type III secretion system (T3SS) proteins are essential for successful infection by many human pathogens. Both T3SS translocator SipC and effector SipA are critical for Salmonella infection by subversion of the host cell cytoskeleton, but the precise molecular interplay between them remains unknown. Here, using cryo-electron microscopy, we show that SipA binds along the F-actin grooves with a unique binding pattern. SipA stabilizes F-actin through charged interface residues and appears to prevent inorganic phosphate release through closure of the "back door" of adenosine 5'-triphosphate pocket. We also show that SipC enhances the binding of SipA to F-actin, thus demonstrating that a sequential presence of T3SS proteins in host cells is associated with a sequence of infection events-starting with actin nucleation, filament growth, and stabilization. Together, our data explain the coordinated interplay of a precisely tuned and highly effective mechanism during Salmonella infection and provide a blueprint for interfering with Salmonella effectors acting on actin.
Assuntos
Actinas , Infecções por Salmonella , Humanos , Actinas/metabolismo , Microscopia Crioeletrônica , Proteínas de Bactérias/metabolismo , Citoesqueleto de Actina/metabolismoRESUMO
Biosynthetic engineering of bicyclic darobactins, selectively sealing the lateral gate of the outer membrane protein BamA, leads to active analogues, which are up to 128-fold more potent against Gram-negative pathogens compared to native counterparts. Because of their excellent antibacterial activity, darobactins represent one of the most promising new antibiotic classes of the past decades. Here, we present a series of structure-driven biosynthetic modifications of our current frontrunner, darobactin 22 (D22), to investigate modifications at the understudied positions 2, 4, and 5 for their impact on bioactivity. Novel darobactins were found to be highly active against critical pathogens from the WHO priority list. Antibacterial activity data were corroborated by dissociation constants with BamA. The most active derivatives D22 and D69 were subjected to ADMET profiling, showing promising features. We further evaluated D22 and D69 for bioactivity against multidrug-resistant clinical isolates and found them to have strong activity.
Assuntos
Antibacterianos , Fenilpropionatos , Antibacterianos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
The correct organization of single subunits of multi-protein machines in a three dimensional context is critical for their functionality. Type III secretion systems (T3SS) are molecular machines with the capacity to deliver bacterial effector proteins into host cells and are fundamental for the biology of many pathogenic or symbiotic bacteria. A central component of T3SSs is the needle complex, a multiprotein structure that mediates the passage of effector proteins through the bacterial envelope. We have used cryo electron microscopy combined with bacterial genetics, site-specific labeling, mutational analysis, chemical derivatization and high-resolution mass spectrometry to generate an experimentally validated topographic map of a Salmonella typhimurium T3SS needle complex. This study provides insights into the organization of this evolutionary highly conserved nanomachinery and is the basis for further functional analysis.