Bacterial lipopolysaccharide induces settlement and metamorphosis in a marine larva

AbstractBacterially induced metamorphosis has been observed in marine invertebrate larvae from nearly every major marine phylum. Despite the widespread nature of this phenomenon the mechanism of this process remains poorly understood. The serpulid polychaete Hydroides elegans is a well-established model system for understanding bacteria-mediated larval development. A broad range of bacterial biofilm species elicit larval metamorphosis in this species via at least two mechanisms, including outer membrane vesicles and phage-tail bacteriocins. Here, we investigated the interaction between larvae of H. elegans and the inductive bacterium Cellulophaga lytica, which produces an abundance of OMVs but not phage-tail bacteriocins. We asked whether the OMVs of C. lytica induce larval settlement due to cell membrane components or through delivery of specific cargo. Employing a biochemical structure-function approach, and with a strong ecological focus, the cells and outer membrane vesicles produced by C. lytica were interrogated to determine the structure of the inductive molecule. Here we report that lipopolysaccharide is the inductive molecule produced by C. lytica that induces larvae of H. elegans to metamorphose. The widespread prevalence of LPS and its associated taxonomic and structural variability suggest that it could be a broadly employed cue to bacterially induced larval settlement of marine invertebrates.Significance StatementWhenever new surfaces are created in the sea, they are quickly populated by dense communities of invertebrate animals, whose establishment and maintenance requires site-specific settlement of larvae from the plankton. Although it is recognized that larvae selectively settle in sites where they can metamorphose and thrive and that the bacteria residing in biofilms on these surfaces are important suppliers of cues, the nature of the cues used to identify the ‘right places’ has remained enigmatic. In this paper, we reveal that lipopolysaccharide (LPS) molecules from a marine Gram-negative bacterium are the cuing molecules for a tropical marine worm and demonstrate the likelihood that LPS provides the variation necessary to be the settlement cue for the majority of bottom-living invertebrate animals.

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Pseudomonas aeruginosa leucine aminopeptidase influences early biofilm composition and structure via vesicle-associated anti-biofilm activity

10.1101/784918 ◽

2019 ◽

Author(s):

Caitlin N. Esoda ◽

Meta J. Kuehn

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Biofilm Formation ◽

Leucine Aminopeptidase ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Bacterial Biofilm ◽

Biofilm Architecture ◽

Biofilm Development

AbstractPseudomonas aeruginosa, known as one of the leading causes of disease in cystic fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds and chronic wounds. We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known to contribute to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content, and that OMVs from PaAP expressing strains but not PaAP alone or in combination with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally found that OMVs from PaAP expressing strains could cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. Finally, we showed that the OMVs could also mediate the detachment of biofilms formed by both non-self P. aeruginosa strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture.ImportanceBiofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug- resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In order to treat these infections more successfully, the mechanisms of bacterial biofilm development must be elucidated. While both bacterially-secreted aminopeptidase and outer membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the contributions of each of these factors to the steps in biofilm generation have not been well studied. This work provides new insight as to how these bacterial components mediate the formation of a robust, drug-resistant extracellular matrix and implicates outer membrane vesicles as active components of biofilm architecture, expanding our overall understanding of P. aeruginosa biofilm biology.

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Outer Membrane Vesicles Released From Aeromonas Strains Are Involved in the Biofilm Formation

Frontiers in Microbiology ◽

10.3389/fmicb.2020.613650 ◽

2021 ◽

Vol 11 ◽

Author(s):

Soshi Seike ◽

Hidetomo Kobayashi ◽

Mitsunobu Ueda ◽

Eizo Takahashi ◽

Keinosuke Okamoto ◽

...

Keyword(s):

Amino Acid ◽

Outer Membrane ◽

Biofilm Formation ◽

Bacterial Species ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Amino Acid Sequences ◽

Bacterial Biofilm ◽

Gram Negative Bacteria ◽

Gram Negative

Aeromonas spp. are Gram-negative rod-shaped bacteria ubiquitously distributed in diverse water sources. Several Aeromonas spp. are known as human and fish pathogens. Recently, attention has been focused on the relationship between bacterial biofilm formation and pathogenicity or drug resistance. However, there have been few reports on biofilm formation by Aeromonas. This study is the first to examine the in vitro formation and components of the biofilm of several Aeromonas clinical and environmental strains. A biofilm formation assay using 1% crystal violet on a polystyrene plate revealed that most Aeromonas strains used in this study formed biofilms but one strain did not. Analysis of the basic components contained in the biofilms formed by Aeromonas strains confirmed that they contained polysaccharides containing GlcNAc, extracellular nucleic acids, and proteins, as previously reported for the biofilms of other bacterial species. Among these components, we focused on several proteins fractionated by SDS-PAGE and determined their amino acid sequences. The results showed that some proteins existing in the Aeromonas biofilms have amino acid sequences homologous to functional proteins present in the outer membrane of Gram-negative bacteria. This result suggests that outer membrane components may affect the biofilm formation of Aeromonas strains. It is known that Gram-negative bacteria often release extracellular membrane vesicles from the outer membrane, so we think that the outer membrane-derived proteins found in the Aeromonas biofilms may be derived from such membrane vesicles. To examine this idea, we next investigated the ability of Aeromonas strains to form outer membrane vesicles (OMVs). Electron microscopic analysis revealed that most Aeromonas strains released OMVs outside the cells. Finally, we purified OMVs from several Aeromonas strains and examined their effect on the biofilm formation. We found that the addition of OMVs dose-dependently promoted biofilm formation, except for one strain that did not form biofilms. These results suggest that the OMVs released from the bacterial cells are closely related to the biofilm formation of Aeromonas strains.

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Pseudomonas aeruginosa Leucine Aminopeptidase Influences Early Biofilm Composition and Structure via Vesicle-Associated Antibiofilm Activity

mBio ◽

10.1128/mbio.02548-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 4

Author(s):

Caitlin N. Esoda ◽

Meta J. Kuehn

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Biofilm Formation ◽

Leucine Aminopeptidase ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Bacterial Biofilm ◽

Content Type ◽

Biofilm Architecture

ABSTRACT Pseudomonas aeruginosa, known as one of the leading causes of disease in cystic fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds, and chronic wounds. We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known to contribute to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content, and that OMVs from PaAP-expressing strains but not PaAP alone or in combination with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally found that OMVs from PaAP-expressing strains could cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. Finally, we showed that the OMVs could also mediate the detachment of biofilms formed by both nonself P. aeruginosa strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture. IMPORTANCE Biofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In order to treat these infections more successfully, the mechanisms of bacterial biofilm development must be elucidated. While both bacterially secreted aminopeptidase and outer membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the contributions of each of these factors to the steps in biofilm generation have not been well studied. This work provides new insight into how these bacterial components mediate the formation of a robust, drug-resistant extracellular matrix and implicates outer membrane vesicles as active components of biofilm architecture, expanding our overall understanding of P. aeruginosa biofilm biology.

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The Effect of Helicobacter Pylori Outer Membrane Vesicles On Vacuolation in Gastric and Colonic Epithelial Cells

Endoscopy ◽

10.1055/s-2006-956848 ◽

2006 ◽

Vol 38 (11) ◽

Author(s):

EM Mullaney ◽

AM Terres ◽

DP Kelleher

Keyword(s):

Helicobacter Pylori ◽

Epithelial Cells ◽

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Colonic Epithelial Cells

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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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