Application of cryo-electron microscopy for investigation of Bax-induced pores in apoptosis

2017 ◽  
Vol 6 (1) ◽  
pp. 47-55
Author(s):  
Tomomi Kuwana
Keyword(s):  
Electron Microscopy ◽  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Experimental System ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Cryo Electron Microscopy

AbstractMitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis, the molecular mechanisms of which have been a subject of intensive study. This process is important for therapeutic intervention in various diseases, such as cancer. Pro-apoptotic Bax and Bak are functionally redundant and structurally homologous. When activated at the mitochondrial outer membrane, they cause the membrane to permeabilize and release apoptogenic proteins from the intermembrane space. To unravel the molecular mechanisms of this unique and important event, we systematically reduced the experimental system. Simple outer membrane vesicles and liposomes recapitulated many features of MOMP. Although conventional transmission electron microscopy could not detect any membrane changes during MOMP in these vesicles, cryo-electron microscopy successfully revealed Bax-induced delicate pores, owing to its ability to preserve native, hydrated membrane structure. The data are consistent with the idea that Bax is unfolded and embedded in the bilayer and deforms the membrane to form a large pore. Together with the biochemical and structure data in the literature, we now have more comprehensive models of the key function of Bax. We hope that new tools, such as lipid nanodiscs, will give us an atomic-level resolution and finally solve Bax structure in the membrane, where it functions.

scholarly journals Protein Import Channel of the Outer Mitochondrial Membrane: a Highly Stable Tom40-Tom22 Core Structure Differentially Interacts with Preproteins, Small Tom Proteins, and Import Receptors

2001 ◽  
Vol 21 (7) ◽  
pp. 2337-2348 ◽  
Author(s):  
Chris Meisinger ◽  
Michael T. Ryan ◽  
Kerstin Hill ◽  
Kirstin Model ◽  
Joo Hyun Lim ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Outer Membrane ◽  
Stable Complex ◽  
Intermembrane Space ◽  
Import Receptors ◽  
Conductance States ◽  
Coupled Channel

ABSTRACT The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a ∼400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.

scholarly journals Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins

2020 ◽  
Vol 21 (15) ◽  
pp. 5496
Author(s):  
Claire Siebert ◽  
Corinne Mercier ◽  
Donald K. Martin ◽  
Patricia Renesto ◽  
Beatrice Schaack
Keyword(s):  
Outer Membrane ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Iron Transport ◽  
Molecular Mechanisms ◽  
Protein A ◽  
Membrane Vesicles ◽  
Disodium Salt ◽  
Outer Membrane Vesicles ◽  
Novel Therapeutic Strategies

Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections.

scholarly journals Isolation and Characterization of Outer Membrane Vesicles of Pectobacterium brasiliense 1692

2021 ◽  
Vol 9 (9) ◽  
pp. 1918
Author(s):  
Silindile Maphosa ◽  
Lucy Novungayo Moleleki
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Outer Membrane ◽  
Critical Role ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Broad Host Range ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Animal Pathogens

Pectobacterium brasiliense (Pbr) 1692 is an aggressive phytopathogen affecting a broad host range of crops and ornamental plants, including potatoes. Previous research on animal pathogens, and a few plant pathogens, revealed that Outer Membrane Vesicles (OMVs) are part of Gram-negative bacteria’s (GNB) adaptive toolkit. For this reason, OMV production and subsequent release from bacteria is a conserved process. Therefore, we hypothesized that OMVs might transport proteins that play a critical role in causing soft rot disease and in the survival and fitness of Pbr1692. Here, we show that the potato pathogen, Pbr1692, releases OMVs of various morphologies in Luria Bertani media at 31 °C. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) confirmed the production of OMVs by Pbr1692 cells. Transmission Electron Microscopy showed that these exist as chain-, single-, and double-membrane morphologies. Mass spectrometry followed by Gene Ontology, Clusters of Orthologous Groups, Virulence Factor, CAZymes, Antibiotic Resistance Ontology, and Bastion6 T6SE annotations identified 129 OMV-associated proteins with diverse annotated roles, including antibiotic stress response, virulence, and competition. Pbr1692 OMVs contributed to virulence in potato tubers and elicited a hypersensitive response in Nicotiana benthamiana leaves. Furthermore, Pbr1692 OMVs demonstrated antibacterial activity against Dickeya dadantii.

Outer Membrane Vesicles of Bordetella pertussis encapsulated into sodium alginate nanoparticles as novel vaccine delivery system

2021 ◽  
Vol 27 ◽  
Author(s):  
Abbas Rami ◽  
Fatemeh Kazemi-Lomedasht ◽  
Ali Mirjalili ◽  
Mojtaba Noofeli ◽  
Fereshteh Shahcheraghi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Outer Membrane ◽  
Sodium Alginate ◽  
Delivery System ◽  
Outer Membrane Proteins ◽  
Vaccine Delivery ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Vaccine Delivery System

Background: Outer membrane vesicles (OMVs) release from Gram-negative bacteria and are interesting alternatives that can replace those vaccines that contain naturally incorporated bacterial surface antigens, lipopolysaccharides (LPS) and outer membrane proteins (OMPs). Nanoparticles can be used to encapsulate vesicles for slow release and prevent macromolecular degradation. Objective: Therefore, encapsulation of OMVs of B. pertussis into sodium alginate nanoparticles was the main aim of the current study. Method: The OMVs of B. pertussis extracted and characterized by particle sizer, electron microscopy, SDSPAGE and Western blot assays. The extracted OMVs were encapsulated in sodium alginate nanoparticles (OMV-NP) using unique gelation process and injected into BALB/c mice. Immunogenicity indices such as different classes of antibodies and interleukins related to different T cell lines were evaluated in immunized mice by ELISA. The respiratory challenge was evaluated in the groups of mice. The existence of pertussis toxin (PTX), filamentous haemagglutinin (FHA) and PRN (pertactin) in B. pertussis OMVs was verified using SDS-PAGE and Western blot analysis. Results: TEM electron microscopy showed the size of these OMVs to be around 20-80 nm. The OMVs with appropriate quality were encapsulated into sodium alginate nanoparticles (OMV-NP), and the final size was about 500 nm after encapsulation. Immunity indices were significantly higher in the OMV-NP receiving group. In challenge tests, the OMV-NP vaccine was able to show the highest rate of lung clearance compared to the control groups (OMV and wPV) at the lowest injection dose. Conclusion: The results indicate the potential of OMV-NP as a novel vaccine delivery system.

scholarly journals LPS Remodeling Triggers Formation of Outer Membrane Vesicles inSalmonella

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Wael Elhenawy ◽  
Michael Bording-Jorgensen ◽  
Ezequiel Valguarnera ◽  
M. Florencia Haurat ◽  
Eytan Wine ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Lipid A ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mass Spectrometry Analysis ◽  
Membrane Remodeling ◽  
Macrophage Infection ◽  
Spectrometry Analysis

ABSTRACTOuter membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employingSalmonella entericaserovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in apagLmutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane.IMPORTANCEThe role of lipid remodeling in vesiculation is well documented in eukaryotes. Similarly, bacteria produce membrane-derived vesicles; however, the molecular mechanisms underlying their production are yet to be determined. In this work, we investigated the role of outer membrane remodeling in OMV biogenesis inS. Typhimurium. We showed that the expression of the lipid A deacylase PagL results in overvesiculation with deacylated lipid A accumulation exclusively in OMV. AnS. Typhimurium ΔpagLstrain showed a significant reduction in intracellular OMV secretion relative to the wild-type strain. Our results suggest a novel mechanism for OMV biogenesis that involves outer membrane remodeling through lipid A modification. Understanding how OMV are produced by bacteria is important to advance our understanding of the host-pathogen interactions.

scholarly journals Comparison of the degradative fate of monoamine oxidase in endogenous and transplanted mitochondrial outer membrane in rat hepatocytes. Implications for the cytomorphological basis of protein catabolism

1984 ◽  
Vol 219 (1) ◽  
pp. 61-72 ◽  
Author(s):  
P J Evans ◽  
R J Mayer
Keyword(s):  
Monoamine Oxidase ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Fluorescein Isothiocyanate ◽  
Rat Hepatocytes ◽  
Similar Rate ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Outer Membrane ◽  
Protein Catabolism

The degradative fate of monoamine oxidase in endogenous and transplanted mitochondrial outer membrane has been compared in rat hepatocyte monolayers. Monoamine oxidase was specifically irreversibly radiolabelled by the suicide inhibitor [3H]pargyline. Hepatocyte monolayers were cultured in conditions in which rates of protein catabolism like those in vivo are maintained [Evans & Mayer (1983) Biochem. J. 216, 151-161]. Incubation of hepatocyte monolayers for 17 h with [3H]pargyline specifically radiolabels mitochondrial monoamine oxidase, as shown by Percoll-gradient fractionation of broken hepatocytes. Monoamine oxidase is degraded at a similar rate to that observed in liver in vivo (t1/2 approx. 63 h). The effects of leupeptin, methylamine and colchicine on the degradation of endogenous radiolabelled enzyme has been studied over prolonged culture periods. Culture of hepatocytes for periods of up to 80 h with inhibitors was not cytotoxic, as demonstrated by measurements of several intrinsic biochemical parameters. Leupeptin, methylamine and colchicine inhibit the degradation of endogenous monoamine oxidase by 60, 38 and 18% respectively. Monoamine oxidase in mitochondrial-outer-membrane vesicles introduced into hepatocytes by poly(ethylene glycol)-mediated vesicle-cell transplantation is degraded at a similar rate (t1/2 55 h) to the endogenous mitochondrial enzyme. Whereas leupeptin inhibits the degradation of endogenous and transplanted enzyme to a similar extent, methylamine and colchicine inhibit the degradation of transplanted enzyme to a much greater extent (85 and 56% respectively). Fluorescence microscopy (with fluorescein isothiocyanate-conjugated mitochondrial outer membrane) shows that transplanted mitochondrial outer membrane undergoes internalization and translocation to a sided perinuclear site, as observed previously with whole mitochondria [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The effects of the inhibitors on the distribution of transplanted membrane material in the cell and inhibition of proteolysis show the importance of cytomorphology for intracellular protein catabolism.

scholarly journals Electroporation of outer membrane vesicles derived from Pseudomonas aeruginosa with gold nanoparticles

2019 ◽  
Vol 1 (12) ◽  
Author(s):  
Zeineb Ayed ◽  
Luana Cuvillier ◽  
Garima Dobhal ◽  
Renee V. Goreham
Keyword(s):  
Electron Microscopy ◽  
Drug Delivery ◽  
Gold Nanoparticles ◽  
Outer Membrane ◽  
Drug Delivery System ◽  
Delivery System ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Novel Drug Delivery System ◽  
Novel Drug

Abstract Since their discovery, extracellular vesicles have gained considerable scientific interest as a novel drug delivery system. In particular, outer membrane vesicles (OMVs) play a critical role in bacteria–bacteria communication and bacteria–host interactions by trafficking cell signalling biochemicals (i.e. DNA, RNA, proteins). Although previous studies have focused on the use of OMVs as vaccines, little work has been done on loading them with functional nanomaterials for drug delivery. We have developed a novel drug delivery system by loading OMVs with gold nanoparticles (AuNPs). AuNPs are versatile nanoparticles that have been extensively used in disease therapeutics. The particles were loaded into the vesicles via electroporation, which uses an electric pulse to create a short-lived electric field. The resulting capacitance on the membrane generates pores in the lipid bilayer of the OMVs allowing AuNPs (or any nanoparticle under 10 nm) inside the vesicles. Closure of the pores of the lipid membrane of the OMVs entraps the nanoparticles as cargo. Transmission electron microscopy was used to confirm the loading of AuNPs inside the OMVs and dynamic light scattering (DLS) and cryogenic scanning electron microscopy (cryo-SEM) verified the size and integrity of the OMVs. This is the first report to load nanoparticles into OMVs, demonstrating a potential method for drug delivery. Graphic abstract

scholarly journals Outer Membrane Vesicle Biosynthesis in Salmonella : Is There More to Gram-Negative Bacteria?

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Joachim Reidl
Keyword(s):  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Serovar Typhimurium ◽  
Reported Study

ABSTRACT Recent research has focused on the biological role of outer membrane vesicles (OMVs), which are derived from the outer membranes (OMs) of Gram-negative bacteria, and their potential exploitation as therapeutics. OMVs have been characterized in many ways and functions. Until recently, research focused on hypothetical and empirical models that addressed the molecular mechanisms of OMV biogenesis, such as vesicles bulging from the OM in various ways. The recently reported study by Elhenawy et al. (mBio 7:e00940-16, 2016, http://dx.doi.org/10.1128/mBio.00940-16 ) provided further insights into OMV biogenesis of Salmonella enterica serovar Typhimurium. That study showed that deacylation of lipopolysaccharides (LPS) influences the level of OMV production and, furthermore, determines a sorting of high versus low acylated LPS in OMs and OMVs, respectively. Interestingly, deacylation may inversely correlate with other LPS modifications, suggesting some synergy toward optimized host resistance via best OM compositions for S . Typhimurium.

scholarly journals Pertussis Vaccine Candidate Based on Outer Membrane Vesicles Derived From Biofilm Culture

2021 ◽  
Vol 12 ◽  
Author(s):  
Francisco Carriquiriborde ◽  
Pablo Martin Aispuro ◽  
Nicolás Ambrosis ◽  
Eugenia Zurita ◽  
Daniela Bottero ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Immune Response ◽  
Outer Membrane ◽  
Pertussis Vaccine ◽  
Bacterial Colonization ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Protective Capacity ◽  
Scanning Electron

Outer membrane vesicles (OMV) derived from Bordetella pertussis—the etiologic agent of the resurgent disease called pertussis—are safe and effective in preventing bacterial colonization in the lungs of immunized mice. Vaccine formulations containing those OMV are capable of inducing a mixed Th1/Th2/Th17 profile, but even more interestingly, they may induce a tissue-resident memory immune response. This immune response is recommended for the new generation of pertussis-vaccines that must be developed to overcome the weaknesses of current commercial acellular vaccines (second-generation of pertussis vaccine). The third-generation of pertussis vaccine should also deal with infections caused by bacteria that currently circulate in the population and are phenotypically and genotypically different [in particular those deficient in the expression of pertactin antigen, PRN(-)] from those that circulated in the past. Here we evaluated the protective capacity of OMV derived from bacteria grown in biofilm, since it was observed that, by difference with older culture collection vaccine strains, circulating clinical B. pertussis isolates possess higher capacity for this lifestyle. Therefore, we performed studies with a clinical isolate with good biofilm-forming capacity. Biofilm lifestyle was confirmed by both scanning electron microscopy and proteomics. While scanning electron microscopy revealed typical biofilm structures in these cultures, BipA, fimbria, and other adhesins described as typical of the biofilm lifestyle were overexpressed in the biofilm culture in comparison with planktonic culture. OMV derived from biofilm (OMVbiof) or planktonic lifestyle (OMVplank) were used to formulate vaccines to compare their immunogenicity and protective capacities against infection with PRN(+) or PRN(-) B. pertussis clinical isolates. Using the mouse protection model, we detected that OMVbiof-vaccine was more immunogenic than OMVplank-vaccine in terms of both specific antibody titers and quality, since OMVbiof-vaccine induced antibodies with higher avidity. Moreover, when OMV were administered at suboptimal quantity for protection, OMVbiof-vaccine exhibited a significantly adequate and higher protective capacity against PRN(+) or PRN(-) than OMVplank-vaccine. Our findings indicate that the vaccine based on B. pertussis biofilm-derived OMV induces high protection also against pertactin-deficient strains, with a robust immune response.

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