RESUMEN
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.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Dicroismo Circular , Sincrotrones , Dicroismo Circular/métodos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química , Estructura Secundaria de Proteína , Membrana Dobles de Lípidos/metabolismo , Membrana Dobles de Lípidos/químicaRESUMEN
The cell envelope of Gram-negative bacteria is composed of an outer membrane (OM) and an inner membrane (IM) and a peptidoglycan cell wall (CW) between them. Combined with Braun's lipoprotein (Lpp), which connects the OM and the CW, and numerous membrane proteins that exist in both OM and IM, the cell envelope creates a mechanically stable environment that resists various physical and chemical perturbations to the cell, including turgor pressure caused by the solute concentration difference between the cytoplasm of the cell and the extracellular environment. Previous computational studies have explored how individual components (OM, IM, and CW) can resist turgor pressure although combinations of them have been less well studied. To that end, we constructed multiple OM-CW systems, including the Lpp connections with the CW under increasing degrees of strain. The results show that the OM can effectively resist the tension imposed by the CW, shrinking by only 3-5% in area even when the CW is stretched to 2.5× its relaxed area. The area expansion modulus of the system increases with increasing CW strain, although the OM remains a significant contributor to the envelope's mechanical stability. Additionally, we find that when the protein TolC is embedded in the OM, its stiffness increases.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Pared Celular , Peptidoglicano , Pared Celular/química , Pared Celular/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Simulación de Dinámica MolecularRESUMEN
Recent advances in molecular dynamics (MD) simulations have led to rapid improvement in our understanding of the molecular details of the outer membranes (OMs) of Gram-negative bacteria. In this review, we highlight the latest discoveries from MD simulations of OMs, shedding light on the dynamic nature of these bacteria's first line of defense. With the focus on cutting-edge approaches, we explore the OM's sensitivity to structural features, including divalent cations and membrane composition, which have emerged as crucial determinants of antimicrobial passage. Additionally, studies have provided novel insights into outer-membrane proteins (OMPs), revealing their intricate roles in substrate translocation and their distinct interactions with lipopolysaccharides (LPS) in the OM. Finally, we explore the challenging process of ß-barrel membrane protein insertion, showcasing recent findings that have enhanced our grasp of this fundamental biological phenomenon.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Bacterias Gramnegativas , Simulación de Dinámica Molecular , Bacterias Gramnegativas/metabolismo , Bacterias Gramnegativas/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química , Lipopolisacáridos/química , Lipopolisacáridos/metabolismoRESUMEN
Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from Cellulophaga pacifica KMM 3664T isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that C. pacifica KMM 3664T LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial Salmonella typhimurium LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry.
Asunto(s)
Lípido A , Lípido A/química , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química , Animales , Lipopolisacáridos/química , RatonesRESUMEN
The cell envelope of the poly-extremophile bacterium Deinococcus radiodurans is renowned for its highly organized structure and unique functional characteristics. In this bacterium, a precise regularity characterizes not just the S-layer, but it also extends to the underlying cell envelope layers, resulting in a dense and tightly arranged configuration. This regularity is attributed to a minimum of three protein complexes located at the outer membrane level. Together, they constitute a recurring structural unit that extends across the cell envelope, effectively tiling the entirety of the cell body. Nevertheless, a comprehensive grasp of the vacant spaces within each layer and their functional roles remains limited. In this study, we delve into these aspects by integrating the state of the art with structural calculations. This approach provides crucial evidence supporting an evolutive pressure intricately linked to surface phenomena depending on the environmental conditions.
Asunto(s)
Membrana Celular , Deinococcus , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Membrana Celular/química , Pared Celular/química , Pared Celular/metabolismo , Deinococcus/metabolismo , Deinococcus/químicaRESUMEN
Gram-negative bacteria are surrounded by two membranes. A special feature of the outer membrane is its asymmetry. It contains lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet1-3. The proper assembly of LPS in the outer membrane is required for cell viability and provides Gram-negative bacteria intrinsic resistance to many classes of antibiotics. LPS biosynthesis is completed in the inner membrane, so the LPS must be extracted, moved across the aqueous periplasm that separates the two membranes and translocated through the outer membrane where it assembles on the cell surface4. LPS transport and assembly requires seven conserved and essential LPS transport components5 (LptA-G). This system has been proposed to form a continuous protein bridge that provides a path for LPS to reach the cell surface6,7, but this model has not been validated in living cells. Here, using single-molecule tracking, we show that Lpt protein dynamics are consistent with the bridge model. Half of the inner membrane Lpt proteins exist in a bridge state, and bridges persist for 5-10 s, showing that their organization is highly dynamic. LPS facilitates Lpt bridge formation, suggesting a mechanism by which the production of LPS can be directly coupled to its transport. Finally, the bridge decay kinetics suggest that there may be two different types of bridges, whose stability differs according to the presence (long-lived) or absence (short-lived) of LPS. Together, our data support a model in which LPS is both a substrate and a structural component of dynamic Lpt bridges that promote outer membrane assembly.
Asunto(s)
Membrana Externa Bacteriana , Proteínas Portadoras , Bacterias Gramnegativas , Lipopolisacáridos , Proteínas de la Membrana , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Escherichia coli/química , Escherichia coli/citología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Bacterias Gramnegativas/química , Bacterias Gramnegativas/citología , Bacterias Gramnegativas/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismoRESUMEN
The Maintenance of outer membrane (OM) Lipid Asymmetry system mediates retrograde phospholipid transport from the OM to the inner membrane (IM) in Gram-negative bacteria. However, the interactions between the various subunits of the IM and OM complexes are not well understood. In a recent study in 2023 by MacRae et al. in the Journal of Biological Chemistry, the authors examine components in the Maintenance of OM Lipid Asymmetry complex, define the interaction interfaces between members of the pathway, and propose a molecular model of the lipid transfer process from the OM to the IM that will help elucidate intricacies of lipid transport.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Membrana Externa Bacteriana , Bacterias Gramnegativas , Metabolismo de los Lípidos , Lípidos de la Membrana , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Bacterias Gramnegativas/citología , Bacterias Gramnegativas/metabolismo , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismoRESUMEN
Gram-negative bacteria surround their cytoplasmic membrane with a peptidoglycan (PG) cell wall and an outer membrane (OM) with an outer leaflet composed of lipopolysaccharide (LPS)1. This complex envelope presents a formidable barrier to drug entry and is a major determinant of the intrinsic antibiotic resistance of these organisms2. The biogenesis pathways that build the surface are also targets of many of our most effective antibacterial therapies3. Understanding the molecular mechanisms underlying the assembly of the Gram-negative envelope therefore promises to aid the development of new treatments effective against the growing problem of drug-resistant infections. Although the individual pathways for PG and OM synthesis and assembly are well characterized, almost nothing is known about how the biogenesis of these essential surface layers is coordinated. Here we report the discovery of a regulatory interaction between the committed enzymes for the PG and LPS synthesis pathways in the Gram-negative pathogen Pseudomonas aeruginosa. We show that the PG synthesis enzyme MurA interacts directly and specifically with the LPS synthesis enzyme LpxC. Moreover, MurA was shown to stimulate LpxC activity in cells and in a purified system. Our results support a model in which the assembly of the PG and OM layers in many proteobacterial species is coordinated by linking the activities of the committed enzymes in their respective synthesis pathways.
Asunto(s)
Membrana Externa Bacteriana , Pared Celular , Pseudomonas aeruginosa , Pared Celular/metabolismo , Lipopolisacáridos/metabolismo , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Pseudomonas aeruginosa/citología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/metabolismo , Peptidoglicano/biosíntesis , Peptidoglicano/metabolismoRESUMEN
Inducing immunogenic cell death (ICD) is a critical strategy for enhancing cancer immunotherapy. However, inefficient and risky ICD inducers along with a tumor hypoxia microenvironment seriously limit the immunotherapy efficacy. Non-specific delivery is also responsible for this inefficiency. In this work, we report a drug-free bacteria-derived outer membrane vesicle (OMV)-functionalized Fe3O4-MnO2 (FMO) nanoplatform that realized neutrophil-mediated targeted delivery and photothermally enhanced cancer immunotherapy. In this system, modification of OMVs derived from Escherichia coli enhanced the accumulation of FMO NPs at the tumor tissue through neutrophil-mediated targeted delivery. The FMO NPs underwent reactive decomposition in the tumor site, generating manganese and iron ions that induced ICD and O2 that regulated the tumor hypoxia environment. Moreover, OMVs are rich in pathogen-associated pattern molecules that can overcome the tumor immunosuppressive microenvironment and effectively activate immune cells, thereby enhancing specific immune responses. Photothermal therapy (PTT) caused by MnO2 and Fe3O4 can not only indirectly stimulate systemic immunity by directly destroying tumor cells but also promote the enrichment of neutrophil-equipped nanoparticles by enhancing the inflammatory response at the tumor site. Finally, the proposed multi-modal treatment system with targeted delivery capability realized effective tumor immunotherapy to prevent tumor growth and recurrence.
Asunto(s)
Bioingeniería , Inmunoterapia , Nanopartículas Multifuncionales , Neoplasias , Humanos , Línea Celular Tumoral , Inmunoterapia/métodos , Nanopartículas Multifuncionales/uso terapéutico , Neoplasias/terapia , Microambiente Tumoral/inmunología , Vesículas Transportadoras/química , Vesículas Transportadoras/inmunología , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/inmunología , Escherichia coliRESUMEN
The outer membrane of Gram-negative bacteria plays a critical role in protecting the cell against external stressors, including antibiotics, and therefore is a prime target for antimicrobial discovery. To facilitate the discovery efforts, a precise knowledge of the outer membrane proteome, and possible variations during pathogenesis, is important. Characterization of the bacterial outer membrane remain challenging, however, and low throughput, due to the high hydrophobicity and relatively low abundance of this cell compartment. Here we adapt our peptidisc-based method to selectively isolate the outer membrane proteome before analysis by mass spectrometry. Using a dual detergent membrane solubilization approach, followed by protein purification in peptidiscs, we capture over 70 outer membrane proteins, including 26 integral ß-barrels and 26 lipoproteins. Many of these proteins are present at high peptide intensities, indicative of a high abundance in the library sample. We further show that the isolated outer membrane proteome can be employed in downstream ligand-binding assays. This peptidisc library made of outer membrane proteins may therefore be useful to systematically survey other bacterial outer membrane proteomes, but also as a nanoparticle format able to support the discovery of next-generation antimicrobials. Data are available via ProteomeXchange identifier PXD036749.
Asunto(s)
Detergentes , Proteoma , Proteoma/metabolismo , Detergentes/química , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana Bacteriana Externa/químicaRESUMEN
Deinococcus radiodurans is a phylogenetically deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet (UV) radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of D. radiodurans has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in D. radiodurans and related bacteria. Here, we report a electron cryomicroscopy (cryo-EM) structure of the abundant D. radiodurans OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded ß-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Furthermore, using protein structure prediction and sequence-based bioinformatic analysis, we show that SlpA-like putative OM-PG connector proteins are widespread in phylogenetically deep-branching Gram-negative bacteria. Finally, combining our atomic structures with fluorescence and electron microscopy of cell envelopes of wild-type and mutant bacterial strains, we report a model for the cell surface of D. radiodurans. Our results will have important implications for understanding the cell surface organization and hyperstability of D. radiodurans and related bacteria and the evolutionary transition between Gram-negative and Gram-positive bacteria.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas , Pared Celular , Deinococcus , Membrana Externa Bacteriana/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/química , Pared Celular/química , Microscopía por Crioelectrón , Deinococcus/química , Deinococcus/clasificación , Peptidoglicano/química , Filogenia , Dominios ProteicosRESUMEN
The type IX secretion system (T9SS) transports cargo proteins through the outer membrane of Bacteroidetes and attaches them to the cell surface for functions including pathogenesis, gliding motility, and degradation of carbon sources. The T9SS comprises at least 20 different proteins and includes several modules: the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, the outer membrane Sov translocon, and the cell attachment complex. However, the spatial organization of these modules is unknown. We have characterized the protein interactome of the Sov translocon in Porphyromonas gingivalis and identified Sov-PorV-PorA as well as Sov-PorW-PorN-PorK to be novel networks. PorW also interacted with PGN_1783 (PorD), which was required for maximum secretion efficiency. The identification of PorW as the missing link completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore. IMPORTANCE The T9SS is a newly identified protein secretion system of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum used by pathogens associated with diseases of humans, fish, and poultry for the secretion and cell surface attachment of virulence factors. The T9SS comprises three known modules: (i) the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, (ii) the outer membrane Sov translocon, and (iii) the cell surface attachment complex. The spatial organization and interaction of these modules have been a mystery. Here, we describe the protein interactome of the Sov translocon in the human pathogen Porphyromonas gingivalis and have identified PorW as the missing link which bridges PorN with Sov and so completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing, for the first time, a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Porphyromonas gingivalis/metabolismo , Secuencia de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/química , Sistemas de Secreción Bacterianos/genética , Porphyromonas gingivalis/química , Porphyromonas gingivalis/genética , Unión Proteica , Transporte de Proteínas , Alineación de SecuenciaRESUMEN
Approximately one-third of the human colonic microbiome is formed by bacteria from the genus Bacteroides. These bacteria produce a large amount of uniformly sized outer membrane vesicles (OMVs), which are equipped with hydrolytic enzymes that play a role in the degradation of diet- and host-derived glycans. In this work, we characterize the lipid composition of membranes and OMVs from Bacteroides thetaiotaomicron VPI-5482. Liquid chromatography-mass spectrometry (LC-MS) analysis indicated that OMVs carry sphingolipids, glycerophospholipids, and serine-dipeptide lipids. Sphingolipid species represent more than 50% of the total lipid content of OMVs. The most abundant sphingolipids in OMVs are ethanolamine phosphoceramide (EPC) and inositol phosphoceramide (IPC). Bioinformatics analysis allowed the identification of the BT1522-1526 operon putatively involved in IPC synthesis. Mutagenesis studies revealed that BT1522-1526 is essential for the synthesis of phosphatidylinositol (PI) and IPC, confirming the role of this operon in the biosynthesis of IPC. BT1522-1526 mutant strains lacking IPC produced OMVs that were indistinguishable from the wild-type strain, indicating that IPC sphingolipid species are not involved in OMV biogenesis. Given the known role of sphingolipids in immunomodulation, we suggest that OMVs may act as long-distance vehicles for the delivery of sphingolipids in the human gut. IMPORTANCE Sphingolipids are essential membrane lipid components found in eukaryotes that are also involved in cell signaling processes. Although rare in bacteria, sphingolipids are produced by members of the phylum Bacteroidetes, human gut commensals. Here, we determined that OMVs carry sphingolipids and other lipids of known signaling function. Our results demonstrate that the BT1522-1526 operon is required for IPC biosynthesis in B. thetaiotaomicron.
Asunto(s)
Bacteroides thetaiotaomicron/metabolismo , Ceramidas/biosíntesis , Inositol/metabolismo , Vesículas Transportadoras/metabolismo , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteroides thetaiotaomicron/genética , Vías Biosintéticas , Ceramidas/química , Glicerofosfolípidos/química , Glicerofosfolípidos/metabolismo , Lipidómica , Espectrometría de Masas , Operón , Esfingolípidos/química , Esfingolípidos/metabolismo , Vesículas Transportadoras/química , Vesículas Transportadoras/genéticaRESUMEN
Gram-negative bacteria resist external stresses due to cell envelope rigidity, which is provided by two membranes and a peptidoglycan layer. The outer membrane (OM) surface contains lipopolysaccharide (LPS; contains O-antigen) or lipooligosaccharide (LOS). LPS/LOS are essential in most Gram-negative bacteria and may contribute to cellular rigidity. Acinetobacter baumannii is a useful tool for testing these hypotheses as it can survive without LOS. Previously, our group found that strains with naturally high levels of penicillin binding protein 1A (PBP1A) could not become LOS deficient unless the gene encoding it was deleted, highlighting the relevance of peptidoglycan biosynthesis and suggesting that high PBP1A levels were toxic during LOS deficiency. Transposon sequencing and follow-up analysis found that axial peptidoglycan synthesis by the elongasome and a peptidoglycan recycling enzyme, ElsL, were vital in LOS-deficient cells. The toxicity of high PBP1A levels during LOS deficiency was clarified to be due to a negative impact on elongasome function. Our data suggest that during LOS deficiency, the strength of the peptidoglycan specifically imparted by elongasome synthesis becomes essential, supporting that the OM and peptidoglycan contribute to cell rigidity. IMPORTANCE Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength. Recently, the most outer membrane of the cell was also proposed to contribute to rigidity due to properties of a unique molecule called lipopolysaccharide (LPS). LPS is located on the cell surface in the outer membrane and is typically required for growth. By using Acinetobacter baumannii, a Gram-negative bacterium that can grow without LPS, we found that key features of the peptidoglycan structure also become essential. This finding supports that both the outer membrane and peptidoglycan contribute to cell rigidity.
Asunto(s)
Acinetobacter baumannii/crecimiento & desarrollo , Acinetobacter baumannii/metabolismo , Membrana Externa Bacteriana/metabolismo , Lipopolisacáridos/biosíntesis , Peptidoglicano/biosíntesis , Acinetobacter baumannii/química , Acinetobacter baumannii/genética , Membrana Externa Bacteriana/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lipopolisacáridos/química , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/química , Periplasma/química , Periplasma/genética , Periplasma/metabolismoRESUMEN
Altered intestinal microbial composition promotes intestinal barrier dysfunction and triggers the initiation and recurrence of inflammatory bowel disease (IBD). Current treatments for IBD are focused on control of inflammation rather than on maintaining intestinal epithelial barrier function. Here, we show that the internalization of Gram-negative bacterial outer membrane vesicles (OMVs) in human intestinal epithelial cells promotes recruitment of caspase-5 and PIKfyve to early endosomal membranes via sorting nexin 10 (SNX10), resulting in LPS release from OMVs into the cytosol. Caspase-5 activated by cytosolic LPS leads to Lyn phosphorylation, which in turn promotes nuclear translocalization of Snail/Slug, downregulation of E-cadherin expression, and intestinal barrier dysfunction. SNX10 deletion or treatment with DC-SX029, a novel SNX10 inhibitor, rescues OMV-induced intestinal barrier dysfunction and ameliorates colitis in mice by blocking cytosolic LPS release, caspase-5 activation, and downstream signaling. Our results show that targeting SNX10 may be a new therapeutic approach for restoring intestinal epithelial barrier function and promising strategy for IBD treatment.
Asunto(s)
Membrana Externa Bacteriana/química , Caspasas/metabolismo , Colitis/patología , Lipopolisacáridos/metabolismo , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismo , Animales , Células CACO-2 , Colitis/inducido químicamente , Colitis/genética , Citosol/metabolismo , Modelos Animales de Enfermedad , Endosomas/metabolismo , Endosomas/trasplante , Femenino , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/efectos adversos , Masculino , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Transducción de Señal/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
The Gram-negative cell envelope is a complex structure delineating the cell from its environment. Recently, we found that enterobacterial common antigen (ECA) plays a role maintaining the outer membrane (OM) permeability barrier, which excludes toxic molecules including many antibiotics. ECA is a conserved carbohydrate found throughout Enterobacterales (e.g., Salmonella, Klebsiella, and Yersinia). There are two OM forms of ECA (phosphoglyceride-linked ECAPG and lipopolysaccharide-linked ECALPS) and one periplasmic form of ECA (cyclic ECACYC). ECAPG, found in the outer leaflet of the OM, consists of a linear ECA oligomer attached to phosphoglyceride through a phosphodiester linkage. The process through which ECAPG is produced from polymerized ECA is unknown. Therefore, we set out to identify genes interacting genetically with ECAPG biosynthesis in Escherichia coli K-12 using the competition between ECA and peptidoglycan biosynthesis. Through transposon-directed insertion sequencing, we identified an interaction between elyC and ECAPG biosynthesis. ElyC is an inner membrane protein previously shown to alter peptidoglycan biosynthesis rates. We found ΔelyC was lethal specifically in strains producing ECAPG without other ECA forms, suggesting ECAPG biosynthesis impairment or dysregulation. Further characterization suggested ElyC inhibits ECAPG synthesis in a posttranscriptional manner. Moreover, the full impact of ElyC on ECA levels requires the presence of ECACYC. Our data demonstrate ECACYC can regulate ECAPG synthesis in strains wild type for elyC. Overall, our data demonstrate ElyC and ECACYC act in a novel pathway that regulates the production of ECAPG, supporting a model in which ElyC provides feedback regulation of ECAPG production based on the periplasmic levels of ECACYC. IMPORTANCE Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to Enterobacterales, including Klebsiella pneumoniae, Salmonella enterica, and Yersinia pestis. As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery. Here, we elucidated, in E. coli K-12, a new pathway for the regulation of biosynthesis of one cell surface form of ECA, ECAPG. In this pathway, an inner membrane protein, ElyC, and the periplasmic form of ECA, ECACYC, genetically interact to inhibit the synthesis of ECAPG, potentially through feedback regulation based on ECACYC levels. This is the first insight into the pathway responsible for synthesis of ECAPG and represents a potential target for weakening the OM permeability barrier. Furthermore, this pathway provides a tool for experimental manipulation of ECAPG levels.
Asunto(s)
Antígenos Bacterianos/biosíntesis , Escherichia coli/metabolismo , Glicerofosfolípidos/biosíntesis , Antígenos Bacterianos/química , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Vías Biosintéticas , Escherichia coli/genética , Glicerofosfolípidos/químicaRESUMEN
Human α-defensin 5 (HD5) is one of cationic antimicrobial peptides which plays a crucial role in an innate immune system in human body. HD5 shows the killing activity against a broad spectrum of pathogenic bacteria by making a pore in a bacterial membrane and penetrating into a cytosol. Nonetheless, its pore-forming mechanisms remain unclear. Thus, in this work, the constant-velocity steered molecular dynamics (SMD) simulation was used to simulate the permeation of a dimeric HD5 into a gram-negative lipopolysaccharide (LPS) membrane model. Arginine-rich HD5 is found to strongly interact with a LPS surface. Upon arrival, arginines on HD5 interact with lipid A head groups (a top part of LPS) and then drag these charged moieties down into a hydrophobic core resulting in the formation of water-filled pore. Although all arginines are found to interact with a membrane, Arg13 and Arg32 appear to play a dominant role in the HD5 adsorption on a gram-negative membrane. Furthermore, one chain of a dimeric HD5 is required for HD5 adhesion. The interactions of arginine-lipid A head groups play a major role in adhering a cationic HD5 on a membrane surface and retarding a HD5 passage in the meantime.
Asunto(s)
Membrana Externa Bacteriana/química , alfa-Defensinas/química , Arginina/química , Membrana Externa Bacteriana/metabolismo , Bacterias Gramnegativas/química , Humanos , Enlace de Hidrógeno , Lipopolisacáridos/química , Simulación de Dinámica Molecular , Multimerización de Proteína , alfa-Defensinas/metabolismoRESUMEN
Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.
Asunto(s)
Bacteriocinas/química , Colicinas/química , Escherichia coli/metabolismo , Porinas/química , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Sitios de Unión , Colicinas/genética , Colicinas/metabolismo , Microscopía por Crioelectrón , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Porinas/genética , Porinas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , TermodinámicaRESUMEN
Bacteroidetocins are a family of antibacterial peptide toxins that are produced by and target members of the phylum Bacteroidetes. To date, 19 bacteroidetocins have been identified, and four have been tested and shown to kill diverse Bacteroidales species (M. J. Coyne, N. Béchon, L. M. Matano, V. L. McEneany, et al., Nat Commun 10:3460, 2019, https://doi.org/10.1038/s41467-019-11494-1). Here, we identify the target and likely mechanism of action of the bacteroidetocins. We selected seven spontaneous mutants of four different genera, all resistant to bacteroidetocin A (Bd-A) and found that all contained mutations in a single gene, bamA. Construction of three of these bamA mutants in the wild-type (WT) strains confirmed they confer resistance to Bd-A as well as to other bacteroidetocins. We identified an aspartate residue of BamA at the beginning of exterior loop 3 (eL3) that, when altered, renders strains resistant to Bd-A. Analysis of a panel of diverse Bacteroidales strains showed a correlation between the presence of this aspartate residue and Bd-A sensitivity. Fluorescence microscopy and transmission electron microscopy (TEM) analysis of Bd-A-treated cells showed cellular morphological changes consistent with a BamA defect. Transcriptomic analysis of Bd-A-treated cells revealed gene expression changes indicative of cell envelope stress. Studies in mice revealed that bacteroidetocin-resistant mutants are outcompeted by their WT strain in vivo. Analyses of longitudinal human gut isolates showed that bamA mutations leading to bacteroidetocin resistance do not become fixed in the human gut, even in bacteroidetocin-producing strains and nonproducing coresident strains. Together, these data lend further support to the applicability of the bacteroidetocins as therapeutic peptides in the treatment of maladies involving Bacteroidales species. IMPORTANCE The bacteroidetocins are a newly discovered class of bacteriocins specific to Bacteroidetes with a spectrum of targets extending from symbiotic gut Bacteroides, Parabacteroides, and Prevotella species to pathogenic oral and vaginal Prevotella species. We previously showed that one such bacteroidetocin, Bd-A, is active at nanomolar concentrations, is water soluble, and is bactericidal, all desirable features in a therapeutic antibacterial peptide. Here, we identify the target of several of the bacteroidetocins as the essential outer membrane protein BamA. Although mutations in bamA can be selected in bacteria grown in vitro, we show both in a mouse model and in human gut ecosystems that bamA mutants leading to Bd-A resistance are fitness attenuated and are not selected. These features further support the potential usefulness of the bacteroidetocins as therapeutics for maladies associated with pathogenic Prevotella species, such as recurrent bacterial vaginosis, for which there are few effective treatments.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteriocinas/farmacología , Bacteroidetes/efectos de los fármacos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/efectos de los fármacos , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Bacteroidetes/química , Bacteroidetes/genética , Bacteroidetes/fisiología , Farmacorresistencia Bacteriana , Femenino , Microbioma Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/microbiología , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Ratones , Alineación de Secuencia , SimbiosisRESUMEN
BACKGROUND: Biofilms are microbial communities surrounded by a self-produced extracellular matrix which protects them from environmental stress. Bacteria within biofilms are 10- to 1000-fold more resistant to antibiotics, making it challenging but imperative to develop new therapeutics that can disperse biofilms and eradicate infection. Gram-negative bacteria produce outer membrane vesicles (OMV) that play critical roles in communication, genetic exchange, cargo delivery, and pathogenesis. We have previously shown that OMVs derived from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. RESULTS: Here, we examine the antibiofilm activity of Burkholderia thailandensis OMVs against the oral biofilm-forming pathogen Streptococcus mutans. We demonstrate that OMV treatment reduces biofilm biomass, biofilm integrity, and bacterial cell viability. Both heat-labile and heat-stable components, including 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline and long-chain rhamnolipid, contribute to the antibiofilm activity of OMVs. When OMVs are co-administered with gentamicin, the efficacy of the antibiotic against S. mutans biofilms is enhanced. CONCLUSION: These studies indicate that bacterial-derived OMVs are highly effective biological nanoparticles that can inhibit and potentially eradicate biofilms.