scholarly journals Penetration of Enveloped Double-Stranded RNA Bacteriophages φ13 and φ6 into Pseudomonas syringae Cells

2005 ◽  
Vol 79 (8) ◽  
pp. 5017-5026 ◽  
Author(s):  
Rimantas Daugelavičius ◽  
Virginija Cvirkaitė ◽  
Aušra Gaidelytė ◽  
Elena Bakienė ◽  
Rasa Gabrėnaitė-Verkhovskaya ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Outer Membrane ◽  
Pseudomonas Syringae ◽  
Cell Envelope ◽  
Active Role ◽  
Lytic Enzyme ◽  
Virus Infections ◽  
Virus Binding ◽  
Double Stranded Rna

ABSTRACT Bacteriophages φ6 and φ13 are related enveloped double-stranded RNA viruses that infect gram-negative Pseudomonas syringae cells. φ6 uses a pilus as a receptor, and φ13 attaches to the host lipopolysaccharide. We compared the entry-related events of these two viruses, including receptor binding, envelope fusion, peptidoglycan penetration, and passage through the plasma membrane. The infection-related events are dependent on the multiplicity of infection in the case of φ13 but not with φ6. A temporal increase of host outer membrane permeability to lipophilic ions was observed from 1.5 to 4 min postinfection in both virus infections. This enhanced permeability period coincided with the fast dilution of octadecyl rhodamine B-labeled virus-associated lipid molecules. This result is in agreement with membrane fusion, and the presence of temporal virus-derived membrane patches on the outer membrane. Similar to φ6, φ13 contains a thermosensitive lytic enzyme involved in peptidoglycan penetration. The phage entry also caused a limited depolarization of the plasma membrane. Inhibition of host respiration considerably decreased the efficiency of irreversible virus binding and membrane fusion. An active role of cell energy metabolism in restoring the infection-induced defects in the cell envelope was also observed.

scholarly journals Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4+ cells from virus binding.

1990 ◽  
Vol 172 (4) ◽  
pp. 1233-1242 ◽  
Author(s):  
D Healey ◽  
L Dianda ◽  
J P Moore ◽  
J S McDougal ◽  
M J Moore ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Monoclonal Antibodies ◽  
Membrane Fusion ◽  
Syncytium Formation ◽  
Active Role ◽  
Virus Infectivity ◽  
Virus Binding ◽  
Virus Envelope ◽  
Immunodeficiency Virus ◽  
Glycoprotein Gp120

Human immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and syncytium formation. Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain of CD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and syncytium formation by a number of HIV isolates are blocked. These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion.

scholarly journals Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC

2016 ◽  
Vol 198 (23) ◽  
pp. 3176-3185 ◽  
Author(s):  
Abigail T. Ntreh ◽  
Jon W. Weeks ◽  
Logan M. Nickels ◽  
Helen I. Zgurskaya
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Fusion ◽  
Outer Membrane ◽  
Fusion Proteins ◽  
Efflux Pump ◽  
Cell Envelope ◽  
Functional Integration ◽  
Content Type ◽  
Membrane Fusion Proteins ◽  
Stimulation Of

ABSTRACTTriABC-OpmH is an efflux pump fromPseudomonas aeruginosawith an unusual substrate specificity and protein composition. When overexpressed, this pump confers a high level of resistance to the biocide triclosan and the detergent SDS, which are commonly used in combinations for antimicrobial treatments. This activity requires an RND transporter (TriC), an outer membrane channel (OpmH), and two periplasmic membrane fusion proteins (TriA and TriB) with nonequivalent functions. In the active complex, TriA is responsible for the recruitment of OpmH, while TriB is responsible for stimulation of the transporter TriC. Here, we used the functional and structural differences between the two membrane fusion proteins to link their functional roles to specific interactions with OpmH. Our results provide evidence that the TriB-dependent stimulation of the TriC transporter is coupled to opening of the OpmH aperture through binding to the interprotomer groove of OpmH.IMPORTANCEMultidrug efflux transporters are important contributors to intrinsic and acquired antibiotic resistance in clinics. In Gram-negative bacteria, these transporters have a characteristic tripartite architecture spanning the entire two-membrane cell envelope. How such complexes are assembled and how the reactions separated in two different membranes are coupled to provide efficient efflux of various compounds across the cell envelope remain unclear. This study addressed these questions, and the results suggest a mechanism for functional integration of drug efflux by the inner membrane transporter and opening of the channel for transport across the outer membrane.

Isolation and characterization of the plasma membrane and the outer membrane of Deinococcus radiodurans strain Sark

1981 ◽  
Vol 27 (7) ◽  
pp. 729-734 ◽  
Author(s):  
B. G. Thompson ◽  
R. G. E. Murray
Keyword(s):  
Plasma Membrane ◽  
Outer Membrane ◽  
Deinococcus Radiodurans ◽  
Cell Envelope ◽  
Growth Cycle ◽  
Outer Cell ◽  
Batch Cultures ◽  
Lysobacter Enzymogenes ◽  
Isolation And Characterization ◽  
Wall Profile

Deinococcus radiodurans strain Sark, although gram-positive, has a complex cell wall profile that includes an outer membrane-like structure. The outer cell envelope layers formed blebs throughout the growth cycle, which were shed as large vesicles (0.5–3.5 μm in diameter) from approximately 5% of the cell population. Instability of the compartmentalized layer immediately beneath the outer membrane was the cause of the vesicle production. This instability was accentuated by treatment with 10% NaCl, which released the outer membrane from all cells without disrupting the peptidoglycan layer, and provided an outer membrane fraction uncontaminated by plasma membrane. Cells so treated formed protoplasts after sequential treatment with 6 M urea, trypsin, and the supernatant from batch cultures of Lysobacter enzymogenes 495. The plasma membrane was isolated from lysed protoplasts. The absence or presence of catalase activity, and differences in lipid composition, were used to differentiate between plasma membrane and outer membrane.

scholarly journals High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex function

2021 ◽  
Vol 118 (32) ◽  
pp. e2100369118
Author(s):  
Muralidhar Tata ◽  
Santosh Kumar ◽  
Sarah R. Lach ◽  
Shreya Saha ◽  
Elizabeth M. Hart ◽  
...  
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
Cell Envelope ◽  
Complex Function ◽  
Membrane Integrity ◽  
Active Role ◽  
Central Component ◽  
Loss Of Function ◽  
Bam Complex ◽  
Suppressor Screen

The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the β-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF’s partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494L and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.

scholarly journals Molecular basis for the maintenance of lipid asymmetry in the outer membrane ofEscherichia coli

2017 ◽  
Author(s):  
Jiang Yeow ◽  
Kang Wei Tan ◽  
Daniel A. Holdbrook ◽  
Zhi-Soon Chong ◽  
Jan K. Marzinek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Molecular Basis ◽  
Cell Envelope ◽  
Active Role ◽  
Molecular Architecture ◽  
Hairpin Loop ◽  
Lipid Asymmetry ◽  
Gram Negative ◽  
Outer Leaflet

AbstractA distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides (LPS) and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults. InEscherichia coli, the OmpC-MlaA complex is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How it performs this function is unknown. Here, we define the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. We establish that MlaA sits entirely within the bilayer in complex with OmpC and provides a hydrophilic channel possibly for PL translocation across the OM. Furthermore, we show that flexibility in a hairpin loop adjacent to the channel modulates MlaA activity. Finally, we demonstrate that OmpC plays an active role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antimicrobial drug development.

scholarly journals Stress-Based High-Throughput Screening Assays to Identify Inhibitors of Cell Envelope Biogenesis

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 808
Author(s):  
Maurice Steenhuis ◽  
Corinne M. ten Hagen-Jongman ◽  
Peter van Ulsen ◽  
Joen Luirink
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
Stress Responses ◽  
High Throughput Screening ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Compound Screening ◽  
Outer Membrane Biogenesis

The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.

scholarly journals Insulin Stimulates Membrane Fusion and GLUT4 Accumulation in Clathrin Coats on Adipocyte Plasma Membranes

2007 ◽  
Vol 27 (9) ◽  
pp. 3456-3469 ◽  
Author(s):  
Shaohui Huang ◽  
Larry M. Lifshitz ◽  
Christine Jones ◽  
Karl D. Bellve ◽  
Clive Standley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Insulin Signaling ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Tirf Microscopy ◽  
Adipocyte Plasma Membranes ◽  
Kinetics Of

ABSTRACT Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by ∼4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.

scholarly journals Ugo1 and Mdm30 act sequentially during Fzo1-mediated mitochondrial outer membrane fusion

2011 ◽  
Vol 124 (7) ◽  
pp. 1126-1135 ◽  
Author(s):  
F. Anton ◽  
J. M. Fres ◽  
A. Schauss ◽  
B. Pinson ◽  
G. J. K. Praefcke ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane
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