scholarly journals Antibiotic action revealed by real-time imaging of the mycobacterial membrane

2022 ◽  
Author(s):  
Michael G. Wuo ◽  
Charles L Dulberger ◽  
Robert A. Brown ◽  
Alexander Sturm ◽  
Eveline Ultee ◽  
...  
Keyword(s):  
Real Time ◽  
Immune Modulation ◽  
Cell Envelope ◽  
Membrane Vesicles ◽  
Time Lapse ◽  
Outer Membrane Vesicles ◽  
Imaging Data ◽  
Minimal Cell ◽  
Mycobacterial Cell ◽  
Cellular Phenotypes

The current understanding of mycobacterial cell envelope remodeling in response to antibiotics is limited. Chemical tools that report on phenotypic changes with minimal cell wall perturbation are critical to gaining insight into this time-dependent phenomenon. Herein we describe a fluorogenic chemical probe that reports on mycobacterial cell envelope assembly in real time. We used time-lapse microscopy to reveal distinct spatial and temporal changes in the mycobacterial membrane upon treatment with frontline antibiotics. Differential antibiotic treatment elicited unique cellular phenotypes, providing a platform for monitoring cell envelope construction and remodeling responses simultaneously. Analysis of the imaging data indicates a role for antibiotic-derived outer membrane vesicles in immune modulation.

scholarly journals Lipopolysaccharide composition determines the preferred route and entry kinetics of bacterial outer membrane vesicles into host cells

2016 ◽  
Author(s):  
Eloise J O’Donoghue ◽  
Douglas F. Browning ◽  
Ewa Bielska ◽  
Luke Alderwick ◽  
Sara Jabbari ◽  
...  
Keyword(s):  
Chemical Composition ◽  
Outer Membrane ◽  
Real Time ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Gram Negative ◽  
Immune Priming ◽  
Cargo Delivery ◽  
Kinetics Of

SUMMARYOuter membrane vesicles are microvesicles shed by Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle - host cell interactions is limited by a lack of methods to study the kinetics of vesicle entry and cargo delivery to host cells in real-time. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded probe targeted to vesicles. We found that route of vesicular uptake, and thus entry kinetics and efficiency of cargo release, are determined by the chemical composition of the bacterial lipopolysaccharide. The presence of O-antigen facilitates receptor-independent entry, which enhances both rate and efficiency of cargo uptake by host cells. Collectively, our findings highlight the chemical composition of the bacterial cell wall as a major determinant of secretion-independent delivery of virulence factors during Gram-negative infections.

Immune modulation by bacterial outer membrane vesicles

2015 ◽  
Vol 15 (6) ◽  
pp. 375-387 ◽  
Author(s):  
Maria Kaparakis-Liaskos ◽  
Richard L. Ferrero
Keyword(s):  
Outer Membrane ◽  
Immune Modulation ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Bacterial Outer Membrane

scholarly journals Outer Membrane Vesicle Production by Escherichia coli Is Independent of Membrane Instability

2006 ◽  
Vol 188 (15) ◽  
pp. 5385-5392 ◽  
Author(s):  
Amanda J. McBroom ◽  
Alexandra P. Johnson ◽  
Sreekanth Vemulapalli ◽  
Meta J. Kuehn
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Physiological Basis ◽  
Gram Negative

ABSTRACT It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over- or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the σE cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.

scholarly journals The peptidoglycan-associated protein NapA plays an important role in the envelope integrity and in the pathogenesis of the lyme disease spirochete

2021 ◽  
Vol 17 (5) ◽  
pp. e1009546
Author(s):  
Marisela M. Davis ◽  
Aaron M. Brock ◽  
Tanner G. DeHart ◽  
Brittany P. Boribong ◽  
Katherine Lee ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Outer Membrane ◽  
Structural Integrity ◽  
Molecular Beacon ◽  
Cell Envelope ◽  
Protein A ◽  
Bacterial Pathogen ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Gram Negative

The bacterial pathogen responsible for causing Lyme disease, Borrelia burgdorferi, is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. In diderms, peptidoglycan (PG) is sandwiched between the inner and outer membrane of the cell envelope. In many other Gram-negative bacteria, PG is bound by protein(s), which provide both structural integrity and continuity between envelope layers. Here, we present evidence of a peptidoglycan-associated protein (PAP) in B. burgdorferi. Using an unbiased proteomics approach, we identified Neutrophil Attracting Protein A (NapA) as a PAP. Interestingly, NapA is a Dps homologue, which typically functions to bind and protect cellular DNA from damage during times of stress. While B. burgdorferi NapA is known to be involved in the oxidative stress response, it lacks the critical residues necessary for DNA binding. Biochemical and cellular studies demonstrate that NapA is localized to the B. burgdorferi periplasm and is indeed a PAP. Cryo-electron microscopy indicates that mutant bacteria, unable to produce NapA, have structural abnormalities. Defects in cell-wall integrity impact growth rate and cause the napA mutant to be more susceptible to osmotic and PG-specific stresses. NapA-linked PG is secreted in outer membrane vesicles and augments IL-17 production, relative to PG alone. Using microfluidics, we demonstrate that NapA acts as a molecular beacon—exacerbating the pathogenic properties of B. burgdorferi PG. These studies further our understanding of the B. burgdorferi cell envelope, provide critical information that underlies its pathogenesis, and highlight how a highly conserved bacterial protein can evolve mechanistically, while maintaining biological function.

Fine structure of the cell envelope layers of Flexibacter polymorphus

1975 ◽  
Vol 21 (11) ◽  
pp. 1733-1750 ◽  
Author(s):  
H. F. Ridgway ◽  
R. M. Wagner ◽  
W. T. Dawsey ◽  
R. A. Lewin
Keyword(s):  
Outer Membrane ◽  
Intermediate Layer ◽  
Cytoplasmic Membrane ◽  
Membrane Separation ◽  
Cell Envelope ◽  
Single Cells ◽  
Membrane Vesicles ◽  
Structural Data ◽  
Outer Membrane Vesicles ◽  
Thin Sections

Electron microscopy of the filamentous gliding marine bacterium Flexibacter polymorphus demonstrated that the cell envelope consists of an electron-dense intermediate layer located between two unit-type membranes: an outer membrane, presumably of lipopolysaccharide, and an inner cytoplasmic membrane. Separation of living filaments into single cells by lysozyme suggests that a peptidoglycan moiety, possibly corresponding to the intermediate layer, might be situated between the two membranes. Cell division proceeds by invagination of the cytoplasmic membrane and intermediate layer forming a transverse septum. Cells generally fail to separate after the division process, so that a common outer membrane encloses all of the cells in a single filament.There is a continuous layer of macromolecular cup-shaped elements ('goblets') attached to the outermost surface of the lipopolysaccharide membrane. Tangential thin sections, as well as negatively stained preparations of envelope fragments (produced by sonication of autolyzed cells), showed that the goblets are arranged in a close-packed hexagonal array. The presence of electron-dense structures located between the outer and inner membranes, and exhibiting the same periodicity as the goblets, suggests that some part of the goblets penetrates the outer membrane and extends across the periplasmic space to the dense intermediate layer or cytoplasmic membrane. Spontaneous autolysis in aging cultures is accompanied by the formation and release into the culture medium of large numbers of outer membrane vesicles coated with goblets. A tentative reconstruction of the envelope of F. polymorphus, based on the fine-structural data, is presented.

scholarly journals Bacterial Outer Membrane Vesicles as Antibiotic Delivery Vehicles

2021 ◽  
Vol 12 ◽  
Author(s):  
Shannon M. Collins ◽  
Angela C. Brown
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Great Promise ◽  
Gram Negative ◽  
Delivery Vehicles ◽  
Bacterial Outer Membrane ◽  
Antibiotic Delivery

Bacterial outer membrane vesicles (OMVs) are nanometer-scale, spherical vehicles released by Gram-negative bacteria into their surroundings throughout growth. These OMVs have been demonstrated to play key roles in pathogenesis by delivering certain biomolecules to host cells, including toxins and other virulence factors. In addition, this biomolecular delivery function enables OMVs to facilitate intra-bacterial communication processes, such as quorum sensing and horizontal gene transfer. The unique ability of OMVs to deliver large biomolecules across the complex Gram-negative cell envelope has inspired the use of OMVs as antibiotic delivery vehicles to overcome transport limitations. In this review, we describe the advantages, applications, and biotechnological challenges of using OMVs as antibiotic delivery vehicles, studying both natural and engineered antibiotic applications of OMVs. We argue that OMVs hold great promise as antibiotic delivery vehicles, an urgently needed application to combat the growing threat of antibiotic resistance.

scholarly journals Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity

2002 ◽  
Vol 184 (3) ◽  
pp. 754-759 ◽  
Author(s):  
Eric Cascales ◽  
Alain Bernadac ◽  
Marthe Gavioli ◽  
Jean-Claude Lazzaroni ◽  
Roland Lloubes
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Permeability Barrier ◽  
Oligomeric State ◽  
Periplasmic Proteins ◽  
Outer Membrane Permeability

ABSTRACT The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.

scholarly journals Siglec-7 Mediates Immunomodulation by Colorectal Cancer-Associated Fusobacterium nucleatum ssp. animalis

2021 ◽  
Vol 12 ◽  
Author(s):  
Dimitra Lamprinaki ◽  
Pilar Garcia-Vello ◽  
Roberta Marchetti ◽  
Charlotte Hellmich ◽  
Kelli A. McCord ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Immune Cells ◽  
Immune Modulation ◽  
Immune Cell ◽  
Membrane Vesicles ◽  
Fusobacterium Nucleatum ◽  
Human Monocyte ◽  
Outer Membrane Vesicles ◽  
Innate Immune ◽  
Inflammatory Profile

Fusobacterium nucleatum is involved in the development of colorectal cancer (CRC) through innate immune cell modulation. However, the receptors of the interaction between F. nucleatum ssp. and immune cells remain largely undetermined. Here, we showed that F. nucleatum ssp. animalis interacts with Siglecs (sialic acid–binding immunoglobulin-like lectins) expressed on innate immune cells with highest binding to Siglec-7. Binding to Siglec-7 was also observed using F. nucleatum-derived outer membrane vesicles (OMVs) and lipopolysaccharide (LPS). F. nucleatum and its derived OMVs or LPS induced a pro-inflammatory profile in human monocyte-derived dendritic cells (moDCs) and a tumour associated profile in human monocyte-derived macrophages (moMϕs). Siglec-7 silencing in moDCs or CRISPR-cas9 Siglec-7-depletion of U-937 macrophage cells altered F. nucleatum induced cytokine but not marker expression. The molecular interaction between Siglec-7 and the LPS O-antigen purified from F. nucleatum ssp. animalis was further characterised by saturation transfer difference (STD) NMR spectroscopy, revealing novel ligands for Siglec-7. Together, these data support a new role for Siglec-7 in mediating immune modulation by F. nucleatum strains and their OMVs through recognition of LPS on the bacterial cell surface. This opens a new dimension in our understanding of how F. nucleatum promotes CRC progression through the generation of a pro-inflammatory environment and provides a molecular lead for the development of novel cancer therapeutic approaches targeting F. nucleatum-Siglec-7 interaction.

scholarly journals PSORTdb 4.0: expanded and redesigned bacterial and archaeal protein subcellular localization database incorporating new secondary localizations

2020 ◽  
Vol 49 (D1) ◽  
pp. D803-D808
Author(s):  
Wing Yin Venus Lau ◽  
Gemma R Hoad ◽  
Vivian Jin ◽  
Geoffrey L Winsor ◽  
Ashmeet Madyan ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Protein Function ◽  
Drug Targets ◽  
Cell Envelope ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Protein Subcellular Localization ◽  
Atypical Cell ◽  
Important Species ◽  
Archaeal Protein

Abstract Protein subcellular localization (SCL) is important for understanding protein function, genome annotation, and aids identification of potential cell surface diagnostic markers, drug targets, or vaccine components. PSORTdb comprises ePSORTdb, a manually curated database of experimentally verified protein SCLs, and cPSORTdb, a pre-computed database of PSORTb-predicted SCLs for NCBI’s RefSeq deduced bacterial and archaeal proteomes. We now report PSORTdb 4.0 (http://db.psort.org/). It features a website refresh, in particular a more user-friendly database search. It also addresses the need to uniquely identify proteins from NCBI genomes now that GI numbers have been retired. It further expands both ePSORTdb and cPSORTdb, including additional data about novel secondary localizations, such as proteins found in bacterial outer membrane vesicles. Protein predictions in cPSORTdb have increased along with the number of available microbial genomes, from approximately 13 million when PSORTdb 3.0 was released, to over 66 million currently. Now, analyses of both complete and draft genomes are included. This expanded database will be of wide use to researchers developing SCL predictors or studying diverse microbes, including medically, agriculturally and industrially important species that have both classic or atypical cell envelope structures or vesicles.

scholarly journals TolA, TolB, and TolR Are Critical Cell Envelope Proteins of Salmonella Enterica Serovar Choleraesuis Affecting Cell Morphology, OMVs Production, and Virulence

2021 ◽  
Author(s):  
Quan Li ◽  
Zheng Li ◽  
Xia Fei ◽  
Yichen Tian ◽  
Guodong Zhou ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Membrane Vesicles ◽  
High Sensitivity ◽  
Sodium Deoxycholate ◽  
Envelope Proteins ◽  
Outer Membrane Vesicles ◽  
Infection Model

Abstract The Tol-Pal system of Gram-negative bacteria is necessary for maintaining outer membrane integrity. It is a multiprotein complex of five envelope proteins, TolQ, TolR, TolA, TolB, and Pal. These proteins were first investigated in E. coli, and subsequently been identified in many other bacterial genera. However, the function of the Tol-Pal system in Salmonella Choleraesuis pathogenesis is still unclear. Here, we reported the role of three of these proteins in the phenotype and biology of S. Choleraesuis. We found that mutations in tolA, tolB, and tolR caused severe damage to the cell wall, which was supported by observing the microstructure of spherical forms, long chains, flagella defects, and membrane blebbing. We confirmed that all the mutants significantly decreased S. Choleraesuis survival when exposed to sodium deoxycholate and exhibited a high sensitivity to vancomycin, which may be explained by the disruption of envelope integrity. In addition, tolA, tolB, and tolR mutants displayed attenuated virulence in a mouse infection model. This could be interpreted as a series of defective phenotypes in the mutants, such as severe defects in envelope integrity, growth, and motility. Further investigation showed that all the genes participate in outer membrane vesicles (OMVs) biogenesis. Interestingly, immunization with OMVs from ΔtolB efficiently enhanced murine viability in contrast to OMVs from the wild-type S. Choleraesuis, suggesting its potential use in vaccination strategies. Collectively, this study provides an insight into the biological role of the S. Choleraesuis Tol-Pal system.

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