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

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.

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Lipopolysaccharide structure impacts the entry kinetics of bacterial outer membrane vesicles into host cells

PLoS Pathogens ◽

10.1371/journal.ppat.1006760 ◽

2017 ◽

Vol 13 (11) ◽

pp. e1006760 ◽

Cited By ~ 23

Author(s):

Eloise J. O’Donoghue ◽

Natalie Sirisaengtaksin ◽

Douglas F. Browning ◽

Ewa Bielska ◽

Mohammed Hadis ◽

...

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Bacterial Outer Membrane ◽

Kinetics Of

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

Journal of Bacteriology ◽

10.1128/jb.00498-06 ◽

2006 ◽

Vol 188 (15) ◽

pp. 5385-5392 ◽

Cited By ~ 191

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.

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Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria

Microbiology ◽

10.1099/mic.0.079400-0 ◽

2014 ◽

Vol 160 (10) ◽

pp. 2109-2121 ◽

Cited By ~ 83

Author(s):

Heramb M. Kulkarni ◽

Medicharla V. Jagannadham

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Future Research ◽

Gram Negative Bacteria ◽

Biological Functions ◽

Gram Negative ◽

Bacterial Quorum ◽

Membrane Components

Outer membrane vesicles (OMVs) released from Gram-negative bacteria consist of lipids, proteins, lipopolysaccharides and other molecules. OMVs are associated with several biological functions such as horizontal gene transfer, intracellular and intercellular communication, transfer of contents to host cells, and eliciting an immune response in host cells. Although hypotheses have been made concerning the mechanism of biogenesis of these vesicles, research on OMV formation is far from complete. The roles of outer membrane components, bacterial quorum sensing molecules and some specific proteins in OMV biogenesis have been studied. This review discusses the different models that have been proposed for OMV biogenesis, along with details of the biological functions of OMVs and the likely scope of future research.

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Bacterial Outer Membrane Vesicles as Antibiotic Delivery Vehicles

Frontiers in Immunology ◽

10.3389/fimmu.2021.733064 ◽

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.

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Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00031-09 ◽

2010 ◽

Vol 74 (1) ◽

pp. 81-94 ◽

Cited By ~ 474

Author(s):

Terri N. Ellis ◽

Meta J. Kuehn

Keyword(s):

Outer Membrane ◽

Virulence Factors ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Gram Negative ◽

Stress Response Pathway ◽

Degradative Enzymes ◽

Bacterial Virulence Factor ◽

Molecular Patterns

SUMMARY Outer membrane (OM) vesicles are ubiquitously produced by Gram-negative bacteria during all stages of bacterial growth. OM vesicles are naturally secreted by both pathogenic and nonpathogenic bacteria. Strong experimental evidence exists to categorize OM vesicle production as a type of Gram-negative bacterial virulence factor. A growing body of data demonstrates an association of active virulence factors and toxins with vesicles, suggesting that they play a role in pathogenesis. One of the most popular and best-studied pathogenic functions for membrane vesicles is to serve as natural vehicles for the intercellular transport of virulence factors and other materials directly into host cells. The production of OM vesicles has been identified as an independent bacterial stress response pathway that is activated when bacteria encounter environmental stress, such as what might be experienced during the colonization of host tissues. Their detection in infected human tissues reinforces this theory. Various other virulence factors are also associated with OM vesicles, including adhesins and degradative enzymes. As a result, OM vesicles are heavily laden with pathogen-associated molecular patterns (PAMPs), virulence factors, and other OM components that can impact the course of infection by having toxigenic effects or by the activation of the innate immune response. However, infected hosts can also benefit from OM vesicle production by stimulating their ability to mount an effective defense. Vesicles display antigens and can elicit potent inflammatory and immune responses. In sum, OM vesicles are likely to play a significant role in the virulence of Gram-negative bacterial pathogens.

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Outer Membrane Vesicles of Vibrio cholerae Protect and Deliver Active Cholera Toxin to Host Cells via Porin-Dependent Uptake

mBio ◽

10.1128/mbio.00534-21 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Franz G. Zingl ◽

Himadri B. Thapa ◽

Martina Scharf ◽

Paul Kohl ◽

Anna M. Müller ◽

...

Keyword(s):

Outer Membrane ◽

Cholera Toxin ◽

Vibrio Cholerae ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Intestinal Cells ◽

Gram Negative ◽

Content Type ◽

Outer Membrane Porins

ABSTRACT Outer membrane vesicles (OMVs) are an emerging research field due to their multifactorial composition and involvement in interspecies and intraspecies communication. Recent studies indicate that vesicle release by Gram-negative bacterial pathogens is increased during in vivo colonization, as exemplified by the facultative human pathogen Vibrio cholerae upon oral ingestion by the host. In this study, we investigate the fate of OMVs produced by the Gram-negative facultative pathogen V. cholerae. We show that vesicles produced by the clinically relevant El Tor biotype are readily taken up by human intestinal cell lines. We identify outer membrane porins of V. cholerae, i.e., OmpU and OmpT, as the required surface effectors on OMVs for cellular uptake, and we pinpoint the uptake mechanism as caveolin-mediated endocytosis. Furthermore, we show that OMVs derived from V. cholerae grown under virulence-inducing conditions act as potent vehicles for delivery of bioactive cholera toxin to intestinal epithelial cells. In contrast to free cholera toxin secreted via the type II secretion system, OMV-associated cholera toxin is protected from degradation by intestinal proteases. Taken together, these data show that OMV-associated cholera toxin can sustain longer periods in the intestinal tract and preserve toxin effects, as indicated by a prolonged increase of cAMP levels in the intestinal tissue. IMPORTANCE Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing. We aim to broaden our understanding of Vibrio cholerae infections, virulence, and toxicity by drawing attention to the involvement of OMVs in these core processes. Upon host entry, V. cholerae increases secretion of OMVs, which can carry the main virulence factor, cholera toxin, to distant host intestinal cells. We show that specific outer membrane porins on the vesicle surface mediate endocytosis of the vesicles into intestinal cells. With protection by the vesicles, cholera toxin activity endures even in the presence of intestinal proteases. It is tempting to hypothesize that the extended half-life of vesicle-associated cholera toxin allows it to target host cells distant from the primary colonization sites.

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Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

10.26434/chemrxiv.8282204.v1 ◽

2019 ◽

Author(s):

Jiajun Wang ◽

Rémi Terrasse ◽

Jayesh Arun Bafna ◽

Lorraine Benier ◽

Mathias Winterhalter

Keyword(s):

Outer Membrane ◽

Lipid Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Multi Drug Resistance ◽

Gram Negative Bacteria ◽

Membrane Channels ◽

Gram Negative ◽

Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from Escherichia coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (kon) and lower dissociation (koff) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

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Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

International Journal of Molecular Sciences ◽

10.3390/ijms22094823 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4823

Author(s):

María Fernanda González ◽

Paula Díaz ◽

Alejandra Sandoval-Bórquez ◽

Daniela Herrera ◽

Andrew F. G. Quest

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Outer Membrane ◽

Extracellular Vesicles ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Gastric Epithelium ◽

Infected Cells ◽

H Pylori

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.

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