RESUMEN
Outer membrane vesicles (OMVs) are nanostructures derived from the outer membrane of Gram-negative bacteria. We previously demonstrated that vaccination with endotoxin-free OMVs isolated from an Acinetobacter baumannii strain lacking lipooligosaccharide (LOS) biosynthesis, due to a mutation in lpxD, provides full protection in a murine sepsis model. The present study characterizes the protein content of highly-purified OMVs isolated from LOS-replete and LOS-deficient strains. Four purification methods were evaluated to obtain highly purified OMV preparations: ultracentrifugation, size exclusion chromatography (SEC), ultracentrifugation followed by SEC, and Optiprep™. OMVs from each method were characterized using nanoparticle tracking analysis and electron microscopy. OMVs from LOS-deficient and LOS-replete strains purified using the Optiprep™ method were subjected to LC-MS/MS analysis to determine protein content. Significant differences in protein composition between OMVs from LOS-deficient and LOS-replete strains were found. Computational analyses using Bepipred 3.0 and SEMA 2.0 indicated that the lack of LOS led to the overexpression of immunogenic proteins found in LOS-containing OMVs and the presence of immune-stimulating proteins absent in LOS-replete OMVs. These findings have important implications for developing OMV-based vaccines against A. baumannii, using both LOS-containing and LOS-free OMVs preparations.
Asunto(s)
Acinetobacter baumannii , Proteínas de la Membrana Bacteriana Externa , Lipopolisacáridos , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/genética , Lipopolisacáridos/biosíntesis , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Infecciones por Acinetobacter/microbiología , Animales , Ratones , Espectrometría de Masas en Tándem , Membrana Externa Bacteriana/metabolismoRESUMEN
Helicobacter pylori has been recognized not only as a causative agent of a spectrum of gastroduodenal diseases including chronic gastritis, peptic ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer, but also as the culprit in several extra-gastric diseases. However, the association of H. pylori infection with extra-gastric diseases remains elusive, prompting a reevaluation of the role of H. pylori-derived outer membrane vesicles (OMVs). Like other gram-negative bacteria, H. pylori constitutively sheds biologically active OMVs for long-distance delivery of bacterial virulence factors in a concentrated and protected form, averting the need of direct bacterial contact with distant host cells to induce extra-gastric diseases associated with this gastric pathogen. Additionally, H. pylori-derived OMVs contribute to bacterial survival and chronic gastric pathogenesis. Moreover, the immunogenic activity, non-replicable nature, and anti-bacterial adhesion effect of H. pylori OMVs make them a desirable vaccine candidate against infection. The immunogenic potency and safety concerns of the OMV contents are challenges in the development of H. pylori OMV-based vaccines. In this review, we discuss recent advances regarding H. pylori OMVs, focusing on new insights into their biogenesis mechanisms and biological functions.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Humanos , Animales , Factores de Virulencia/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismoRESUMEN
The discovery of Replication Competent Circular DNA molecules in mammalian cells and tissues is being linked to debilitating diseases, such as multiple sclerosis (MS), bovine spongiform encephalopathy (BSE), and colorectal cancer (CRC). These circular DNA molecules, otherwise known as bovine meat and milk factors (BMMFs) and Slow Progressive Hidden INfections of variable (X) latency (SPHINX), bear significant (80%) sequence similarity with the plasmids of Acinetobacter baumannii strains. Nanostructures, such as bacterial outer membrane vesicles (OMVs) serve as vehicles for transporting biomolecular cargo and have the potential to facilitate interkingdom lateral mobility of DNA. Strengthening the proposed hypothesis, this study demonstrates that OMVs derived from A. baumannii DS002 carrying four plasmids and genome (pTS236) of phage, AbDs1, successfully reached different parts of the body, including the central nervous system, following the injection of fluorescein isothiocyanate (FITC)-labeled OMVs into experimental mice. Out of the four OMV-associated plasmids, three (pTS4586, pTS9900, and pTS134338) were identified within the lumen, and the fourth one (pTS11291) was found on the surface of OMVs. In addition to the indigenous plasmids, the phage-encoded protein, Orf96, anchored on the surface of the OMVs by establishing a strong interaction with the OMV-associated porin, OmpA. Intriguingly, a subset of labeled OMVs, when incubated with Neuro2A cells, translocated across the membrane and reached to the cytoplasmic space of the cells. Collectively, the experimental evidence presented herein underscores the promising potential of OMVs as vehicles for delivering molecular cargo containing plasmids and phage genomes to diverse mammalian tissues and cells. IMPORTANCE: Several independent studies have demonstrated the existence of replication competent circular DNA molecules of bacterial and viral origin in mammalian cells and tissues. However, studies about their origin and lateral mobility to mammalian cells are scarce. Our work describes the existence of circular DNA, similar to that of DNA molecules identified in mammalian cells, OMVs derived from soil isolate of A. baumannii DS002. Furthermore, the work also provides visual evidence that demonstrates the passage of labeled OMVs to different organs of experimental mice within hours after intravenously administering OMVs into experimental mice. Some of the labeled OMVs have even crossed the membrane of Neuro2A, suggesting the existence of interkingdom horizontal mobility between bacteria and mammals.
Asunto(s)
Acinetobacter baumannii , ADN Circular , Transferencia de Gen Horizontal , Plásmidos , Animales , Ratones , Plásmidos/genética , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Bovinos , ADN Circular/genética , ADN Circular/metabolismo , Membrana Externa Bacteriana/metabolismo , Femenino , Bacteriófagos/genética , Bacteriófagos/fisiología , Carne/microbiología , Leche/microbiología , Infecciones por Acinetobacter/microbiología , Vesículas Extracelulares/metabolismo , Ratones Endogámicos BALB C , ADN Bacteriano/genéticaRESUMEN
The persistent emergence of multidrug-resistant bacterial pathogens is leading to a decline in the therapeutic efficacy of antibiotics, with Pseudomonas aeruginosa (P. aeruginosa) emerging as a notable threat. We investigated the antibiotic resistance and quorum sensing (QS) system of P. aeruginosa, with a particular focused on outer membrane vesicles (OMVs) and polymyxin B as the last line of antibiotic defense. Our findings indicate that OMVs increase the resistance of P. aeruginosa to polymyxin B. The overall gene transcription levels within P. aeruginosa also reveal that OMVs can reduce the efficacy of polymyxin B. However, both OMVs and sublethal concentrations of polymyxin B suppressed the transcription levels of genes associated with the QS system. Furthermore, OMVs and polymyxin B acted in concert on the QS system of P. aeruginosa to produce a more potent inhibitory effect. This suppression was evidenced by a decrease in the secretion of virulence factors, impaired bacterial motility, and a notable decline in the ability to form biofilms. These results reveal that OMVs enhance the resistance of P. aeruginosa to polymyxin B, yet they collaborate with polymyxin B to inhibit the QS system. Our research contribute to a deeper understanding of the resistance mechanisms of P. aeruginosa in the environment, and provide new insights into the reduction of bacterial infections caused by P. aeruginosa through the QS system.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Polimixina B , Pseudomonas aeruginosa , Percepción de Quorum , Pseudomonas aeruginosa/efectos de los fármacos , Percepción de Quorum/efectos de los fármacos , Antibacterianos/farmacología , Polimixina B/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Biopelículas/efectos de los fármacos , Membrana Externa Bacteriana/efectos de los fármacos , Membrana Externa Bacteriana/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
BACKGROUND: Klebsiella pneumoniae is the most commonly encountered pathogen in clinical practice. Widespread use of broad-spectrum antibiotics has led to the current global dissemination of carbapenem-resistant K. pneumoniae, which poses a significant threat to antibacterial treatment efficacy and public health. Outer membrane vesicles (OMVs) have been identified as carriers capable of facilitating the transfer of virulence and resistance genes. However, the role of OMVs in carbapenem-resistant K. pneumoniae under external pressures such as antibiotic and phage treatments remains unclear. METHODS: To isolate and purify OMVs under the pressure of phages and tigecycline, we subjected K. pneumoniae 0692 harboring plasmid-mediated blaNDM-1 and blaKPC-2 genes to density gradient separation. The double-layer plate method was used to isolate MJ1, which efficiently lysed K. pneumoniae 0692 cells. Transmission electron microscopy (TEM) was used to characterize the isolated phages and extract OMV groups for relevant morphological identification. Determination of protein content of each OMV group was conducted through bicinchoninic acid assay (BCA) and proteomic analysis. RESULTS: K. pneumoniae 0692 released OMVs in response to different environmental stimuli, which were characterized through TEM as having the typical structure and particle size of OMVs. Phage or tigecycline treatment alone resulted in a slight increase in the mean protein concentration of OMVs secreted by K. pneumoniae 0692 compared to that in the untreated group. However, when phage treatment was combined with tigecycline, there was a significant reduction in the average protein concentration of OMVs compared to tigecycline treatment alone. Proteomics showed that OMVs encapsulated numerous functional proteins and that under different external stresses of phages and tigecycline, the proteins carried by K. pneumoniae 0692-derived OMVs were significantly upregulated or downregulated compared with those in the untreated group. CONCLUSIONS: This study confirmed the ability of OMVs to carry abundant proteins and highlighted the important role of OMV-associated proteins in bacterial responses to phages and tigecycline, representing an important advancement in microbial resistance research.
Asunto(s)
Antibacterianos , Bacteriófagos , Carbapenémicos , Klebsiella pneumoniae , Proteómica , Tigeciclina , Tigeciclina/farmacología , Klebsiella pneumoniae/virología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Bacteriófagos/genética , Bacteriófagos/fisiología , Antibacterianos/farmacología , Carbapenémicos/farmacología , Infecciones por Klebsiella/microbiología , Humanos , Vesículas Extracelulares/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/efectos de los fármacos , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Plásmidos/genética , Pruebas de Sensibilidad Microbiana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have key roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogen Salmonella Typhimurium increases OMV production inside the acidic vacuoles of host cells by changing expression of its outer membrane proteins and modifying the composition of lipid A. However, the molecular mechanisms that translate pH changes into OMV production are not completely understood. Here, we show that the outer membrane protein PagC promotes OMV production through pH-dependent interactions between its extracellular loops and surrounding lipopolysaccharide (LPS). Structural comparisons and mutational studies indicate that a pH-responsive amino acid motif in PagC extracellular loops, containing PagC-specific histidine residues, is crucial for OMV formation. Molecular dynamics simulations suggest that protonation of histidine residues leads to changes in the structure and flexibility of PagC extracellular loops and their interactions with the surrounding LPS, altering membrane curvature. Consistent with that hypothesis, mimicking acidic pH by mutating those histidine residues to lysine increases OMV production. Thus, our findings reveal a mechanism for sensing and responding to environmental pH and for control of membrane dynamics by outer membrane proteins.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Lipopolisacáridos , Simulación de Dinámica Molecular , Salmonella typhimurium , Concentración de Iones de Hidrógeno , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/genética , Lipopolisacáridos/metabolismo , Membrana Externa Bacteriana/metabolismo , Secuencias de Aminoácidos , Histidina/metabolismoRESUMEN
Bacteroides spp. are prominent gut commensals that are believed to modulate the intestinal environment, in part, by producing outer membrane vesicles (OMVs). Bacteroides OMVs have been ascribed many functions in vitro, but the genetic underpinnings behind OMV biogenesis and regulation are unclear. Understanding the mechanism of OMV biogenesis is required to determine the importance of Bacteroides OMVs in vivo. Here, we describe our methodology for screening Bacteroides thetaiotaomicron VPI-5482 to identify genes required for OMV biogenesis and regulation in a high-throughput format. This protocol is easily adaptable and can potentially be employed to further our knowledge of OMV biogenesis in other bacteria.
Asunto(s)
Bacteroides thetaiotaomicron , Bacteroides thetaiotaomicron/genética , Bacteroides thetaiotaomicron/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismoRESUMEN
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 molecular pathogenesis of Gram-negative bacteria remains a complex and incompletely understood phenomenon. Various factors are believed to contribute to the pathogenicity of these bacteria. One key mechanism utilized by Gram-negative bacteria is the production of outer membrane vesicles (OMVs), which are small spherical particles derived from the bacterial outer membrane. These OMVs are crucial in delivering virulence factors to the host, facilitating host-pathogen interactions. Within these OMVs, small regulatory RNAs (sRNAs) have been identified as important players in modulating the host immune response. One of the main challenges in studying OMVs and their cargo of sRNAs is the difficulty in isolating and purifying sufficient quantities of OMVs, as well as accurately predicting genuine sRNAs computationally. In this chapter, we present protocols aimed at overcoming these obstacles.
Asunto(s)
Membrana Externa Bacteriana , Biología Computacional , ARN Pequeño no Traducido , Biología Computacional/métodos , ARN Pequeño no Traducido/genética , Membrana Externa Bacteriana/metabolismo , ARN Bacteriano/genética , Bacterias Gramnegativas/genéticaRESUMEN
Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs.
Asunto(s)
Antígenos Bacterianos , Biotinilación , Vesículas Extracelulares , Vesículas Extracelulares/inmunología , Vesículas Extracelulares/metabolismo , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Vacunas Bacterianas/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Desarrollo de Vacunas , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/inmunologíaRESUMEN
Outer membrane vesicles (OMVs) are small, spherical, nanoscale proteoliposomes released from Gram-negative bacteria that play an important role in cellular defense, pathogenesis, and signaling, among other functions. The functionality of OMVs can be enhanced by engineering developed for biomedical and biochemical applications. Here, we describe methods for directed packaging of enzymes into bacterial OMVs of E. coli using engineered molecular systems, such as localizing proteins to the inner or outer surface of the vesicle. Additionally, we detail some modification strategies for OMVs such as lyophilization and surfactant conjugation that enable the protection of activity of the packaged enzyme when exposed to non-physiological conditions such as elevated temperature, organic solvents, and repeated freeze/thaw that otherwise lead to a substantial loss in the activity of the free enzyme.
Asunto(s)
Escherichia coli , Proteolípidos , Escherichia coli/metabolismo , Escherichia coli/genética , Proteolípidos/metabolismo , Membrana Externa Bacteriana/metabolismo , Liofilización/métodos , Proteínas de la Membrana Bacteriana Externa/metabolismo , Enzimas/metabolismo , Enzimas/químicaRESUMEN
This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo. The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD600=0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 â. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs (P=0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.
Asunto(s)
Membrana Externa Bacteriana , Carcinoma Hepatocelular , Glipicanos , Neoplasias Hepáticas , Anticuerpos de Cadena Única , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/química , Animales , Ratones , Humanos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/inmunología , Células Hep G2 , Glipicanos/inmunología , Glipicanos/metabolismo , Glipicanos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Sistemas de Liberación de Medicamentos , Ratones DesnudosRESUMEN
Autonomous nanorobots represent an advanced tool for precision therapy to improve therapeutic efficacy. However, current nanorobotic designs primarily rely on inorganic materials with compromised biocompatibility and limited biological functions. Here, we introduce enzyme-powered bacterial outer membrane vesicle (OMV) nanorobots. The immobilized urease on the OMV membrane catalyzes the decomposition of bioavailable urea, generating effective propulsion for nanorobots. This OMV nanorobot preserves the unique features of OMVs, including intrinsic biocompatibility, immunogenicity, versatile surface bioengineering for desired biofunctionalities, capability of cargo loading and protection. We present OMV-based nanorobots designed for effective tumor therapy by leveraging the membrane properties of OMVs. These involve surface bioengineering of robotic body with cell-penetrating peptide for tumor targeting and penetration, which is further enhanced by active propulsion of nanorobots. Additionally, OMV nanorobots can effectively safeguard the loaded gene silencing tool, small interfering RNA (siRNA), from enzymatic degradation. Through systematic in vitro and in vivo studies using a rodent model, we demonstrate that these OMV nanorobots substantially enhanced siRNA delivery and immune stimulation, resulting in the utmost effectiveness in tumor suppression when juxtaposed with static groups, particularly evident in the orthotopic bladder tumor model. This OMV nanorobot opens an inspiring avenue to design advanced medical robots with expanded versatility and adaptability, broadening their operation scope in practical biomedical domains.
Asunto(s)
Membrana Externa Bacteriana , Animales , Humanos , Membrana Externa Bacteriana/metabolismo , Ratones , Robótica/métodos , Ureasa/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismoRESUMEN
The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic ß-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Periplasma/metabolismo , Dominios Proteicos , Pliegue de ProteínaRESUMEN
Bacteria can repurpose their own bacteriophage viruses (phage) to kill competing bacteria. Phage-derived elements are frequently strain specific in their killing activity, although there is limited evidence that this specificity drives bacterial population dynamics. Here, we identified intact phage and their derived elements in a metapopulation of wild plant-associated Pseudomonas genomes. We discovered that the most abundant viral cluster encodes a phage remnant resembling a phage tail called a tailocin, which bacteria have co-opted to kill bacterial competitors. Each pathogenic Pseudomonas strain carries one of a few distinct tailocin variants that target the variable polysaccharides in the outer membrane of co-occurring pathogenic Pseudomonas strains. Analysis of herbarium samples from the past 170 years revealed that the same tailocin and bacterial receptor variants have persisted in Pseudomonas populations. These results suggest that tailocin genetic diversity can be mined to develop targeted "tailocin cocktails" for microbial control.
Asunto(s)
Bacteriocinas , Fagos Pseudomonas , Pseudomonas , Proteínas de la Cola de los Virus , Antibiosis , Membrana Externa Bacteriana/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Variación Genética , Genoma Bacteriano , Polisacáridos Bacterianos/metabolismo , Pseudomonas/metabolismo , Pseudomonas/virología , Fagos Pseudomonas/genética , Fagos Pseudomonas/metabolismo , Proteínas de la Cola de los Virus/metabolismo , Proteínas de la Cola de los Virus/genética , Terapia de Fagos/métodosRESUMEN
Antigen 43 (Ag43) proteins, found on the outer membrane of Escherichia coli, are ß-sheets that fold into a unique cylindrical structure known as a ß-barrel. There are several known structural similarities between bacterial Ag43 autotransporters and physical components; however, the factors that stabilize the barrel and the mechanism for Ag43 passenger domainmediated translocation across the pore of the ß-barrel remain unclear. In this study, we analyzed Ag43ß-enhanced green fluorescent protein chimeric variants to provide new insights into the autotransporter Ag43ß-barrel assembly, focusing on the impact of the α-helical linker domain. Among the chimeric variants, Ag43ß700 showed the highest surface display, which was confirmed through extracellular protease digestion, flow cytometry, and an evaluation of outer membrane vesicles (OMVs). The Ag43ß700 module offered reliable information on stable barrel folding and chimera expression at the exterior of the OMVs. [BMB Reports 2024; 57(8): 369-374].
Asunto(s)
Membrana Externa Bacteriana , Escherichia coli , Proteínas Fluorescentes Verdes , Escherichia coli/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Adhesinas de Escherichia coli/metabolismo , Adhesinas de Escherichia coli/química , Adhesinas de Escherichia coli/genética , Pliegue de Proteína , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/químicaRESUMEN
Escherichia coli Nissle 1917 (EcN 1917) exhibits distinct tumor-targeting activity, and early studies demonstrated that outer membrane vesicles (OMVs) mediate bacteria-host interactions. To decipher the molecular mechanism underlying the interaction between EcN 1917 and host cells via OMV-mediated communication, we investigated the phenotypic changes in Caco-2 cells perturbed by EcN 1917-derived OMVs and constructed proteomic maps of the EcN 1917-derived OMV components and OMV-perturbed host cells. Our findings revealed that the size of the EcN 1917-derived OMV proteome increased 4-fold. Treatment with EcN 1917-derived OMVs altered the proteomic and phosphoproteomic profiles of host cells. Importantly, for the first time, we found that treatment with EcN 1917-derived OMVs inhibited cancer cell migration by suppressing the expression of ANXA9. In addition, phosphoproteomic data suggested that the ErbB pathway may be involved in OMV-mediated cell migration. Taken together, our study provides valuable data for further investigations of OMV-mediated bacteria-host interactions and offers great insights into the underlying mechanism of probiotic-assisted colorectal cancer therapy.
Asunto(s)
Movimiento Celular , Escherichia coli , Proteoma , Proteómica , Humanos , Células CACO-2 , Proteómica/métodos , Escherichia coli/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Membrana Externa Bacteriana/metabolismoRESUMEN
Extended-spectrumß-lactam producing Escherichia coli (ESBL-EC) readily colonizes live poultry and serves as a major source of contamination in retail chicken meat, posing significant threats to public health. This study aims to investigate the impact of inappropriate antibiotic use on the dissemination and exacerbation of antibiotic resistance in ESBL-EC and explore the underlying molecular mechanisms. Through experimental analysis, we propose a hypothesis that inappropriate antibiotic use may exacerbate resistance by affecting vesicle formation and protein secretion. Experimental results demonstrate that under the influence of amoxicillin, the concentration of proteins secreted in outer membrane vehicles (OMVs) by ESBL-EC significantly increases, along with a significant upregulation in the expression of the CTX-M-55-type Extended-spectrum beta-lactamase (CTX-M-55). Proteomic analysis and differential gene knockout experiments identified the key protein YdcZ, associated with OMVs formation and protein transportation in ESBL-EC under amoxicillin treatment. Further investigations reveal direct interactions between YdcZ and other proteins (YdiH and BssR). Upon ydcz gene knockout, a significant decrease in protein concentration within OMVs is observed, accompanied by a noticeable reduction in protection against sensitive bacteria. These findings suggest a critical role of YdcZ in regulating the process of protein transportation to OMVs in ESBL-EC under the influence of amoxicillin. In summary, our research uncovers the significant role of inappropriate antibiotic use in promoting the secretion of OMVs by ESBL-EC, aiding the survival of antibiotic-sensitive bacteria in the vicinity of infection sites. These findings provide new insights into the mechanisms underlying antibiotic-induced bacterial resistance dissemination and offer novel avenues for exploring prevention and control strategies against bacterial resistance propagation.
Asunto(s)
Amoxicilina , Antibacterianos , Proteínas de Escherichia coli , Escherichia coli , Transporte de Proteínas , beta-Lactamasas , Antibacterianos/farmacología , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , beta-Lactamasas/metabolismo , beta-Lactamasas/genética , Amoxicilina/farmacología , Animales , Pruebas de Sensibilidad Microbiana , Proteómica , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Pollos/microbiología , Farmacorresistencia Bacteriana , Membrana Externa Bacteriana/efectos de los fármacos , Membrana Externa Bacteriana/metabolismo , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/tratamiento farmacológicoRESUMEN
Radiotherapy (RT), including external beam radiation therapy (EBRT) and radionuclide therapy (RNT), realizes physical killing of local tumors and activates systemic anti-tumor immunity. However, these effects need to be further strengthened and the difference between EBRT and RNT should be discovered. Herein, bacterial outer membrane (OM) was biomineralized with manganese oxide (MnO2) to obtain OM@MnO2-PEG nanoparticles for enhanced radio-immunotherapy via amplifying EBRT/RNT-induced immunogenic cell death (ICD) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) activation. OM@MnO2-PEG can react with H2O2 and then gradually produce O2, Mn2+ and OM fragments in the tumor microenvironment. The relieved tumor hypoxia improves the radio-sensitivity of tumor cells, resulting in enhanced ICD and DNA damage. Mn2+ together with the DNA fragments in the cytoplasm activate the cGAS-STING pathway, further exhibiting a positive role in various aspects of innate immunity and adaptive immunity. Besides, OM fragments promote tumor antigen presentation and anti-tumor macrophages polarization. More importantly, our study reveals that OM@MnO2-PEG-mediated RNT triggers much stronger cGAS-STING pathway-involved immunotherapy than that of EBRT, owing to the duration difference of RT. Therefore, this study develops a powerful sensitizer of radio-immunotherapy and uncovers some differences between EBRT and RNT in the activation of cGAS-STING pathway-related anti-tumor immunity.
Asunto(s)
Membrana Externa Bacteriana , Inmunoterapia , Compuestos de Manganeso , Proteínas de la Membrana , Nucleotidiltransferasas , Óxidos , Nucleotidiltransferasas/metabolismo , Compuestos de Manganeso/química , Proteínas de la Membrana/metabolismo , Ratones , Inmunoterapia/métodos , Óxidos/química , Animales , Membrana Externa Bacteriana/metabolismo , Microambiente Tumoral , Línea Celular Tumoral , Transducción de Señal , Humanos , Radioterapia/métodos , Nanopartículas/química , Biomineralización , Muerte Celular Inmunogénica/efectos de los fármacos , Neoplasias/terapia , Peróxido de Hidrógeno/metabolismo , Inmunidad InnataRESUMEN
Polymyxins are gram-negative antibiotics that target lipid A, the conserved membrane anchor of lipopolysaccharide in the outer membrane. Despite their clinical importance, the molecular mechanisms underpinning polymyxin activity remain unresolved. Here, we use surface plasmon resonance to kinetically interrogate interactions between polymyxins and lipid A and derive a phenomenological model. Our analyses suggest a lipid A-catalyzed, three-state mechanism for polymyxins: transient binding, membrane insertion, and super-stoichiometric cluster accumulation with a long residence time. Accumulation also occurs for brevicidine, another lipid A-targeting antibacterial molecule. Lipid A modifications that impart polymyxin resistance and a non-bactericidal polymyxin derivative exhibit binding that does not evolve into long-lived species. We propose that transient binding to lipid A permeabilizes the outer membrane and cluster accumulation enables the bactericidal activity of polymyxins. These findings could establish a blueprint for discovery of lipid A-targeting antibiotics and provide a generalizable approach to study interactions with the gram-negative outer membrane.