Extracellular DNA and Outer Membrane Vesicles contribute to P. fluorescens SBW25 air-liquid interface biofilms.

2020 ◽  
Author(s):  
Olena Moshynets ◽  
Airat Kayumov ◽  
Olga Iungin ◽  
Svitlana Rymar ◽  
Ianina Pokholenko ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Extracellular Dna ◽  
Exogenous Dna ◽  
Rhizosphere Bacterium ◽  
Cellulose Matrix ◽  
Dnase Treatment ◽  
Biofilm Development ◽  
Biofilm Matrix

<p>Outer membrane vesicles (OMVs) and extracellular DNA (eDNA) are important for biofilm formation for many bacteria. OMVs are a perfect transport system to deliver biofilm-related components including eDNA beyond the boundaries of cells, and eDNA itself is an important structural component of biofilms as well as enabling horizontal gene transfer and local adaptation. Both OMVs and eDNA are found in the biofilms produced by the opportunistic human pathogen P. aeruginosa and the plant pathogen P. syringae, but as yet, they have not been reported in the cellulose matrix-based biofilms produced by the related model rhizosphere bacterium Pseudomonas fluorescens SBW25.</p> <p>In this work we have gone back to re-assess the complexity of SBW25 biofilms by looking for evidence of OMVs and eDNA associated with biofilm–formation. OMVs were first imaged by SEM and LC-MC analysis used to identify 51 biofilm matrix-associated proteins of which 12 were also identified in biofilm OMVs. Interestingly, only 5 proteins were identified in both biofilm matrix and OMV samples, but not in planktonic OMVs, suggesting that these may be biofilm-specific components.  </p> <p>We also observed eDNA by CLSM in both the weak and poorly-attached Viscous Mass (VM) and robust and well-attached Wrinkly Spreader (WS) air-liquid (A-L) interface biofilms produced by wild-type SBW25 and the Wrinkly Spreader mutant. The eDNA fraction could be precipitated from biofilm cell-free supernatant samples which demonstrated that WS biofilms had two-fold–higher levels than VM biofilms. DNAse treatment effected the development of both types of biofilm and reduced the strength and attachment levels when added to mature VM and WS biofilms. Testing with exogenous DNA suggests that high molecular weight (HMW) DNA is involved in both strength and attachment, perhaps by surface conditioning and interactions with the primary cellulose matrix common to both biofilms. HMW eDNA could be isolated directly from biofilm supernatants whereas two different HMW size fractions could be isolated from OMVs, presumably, from the outer OMV surface because DNAse treatment led to a substantially reduced DNA signal. This suggest that eDNA persistence and degradation in SBW25 biofilms is complex and eDNA fractions may play different roles in biofilm development, protection and adaptation.</p>

scholarly journals Pushing beyond the Envelope: the Potential Roles of OprF inPseudomonas aeruginosaBiofilm Formation and Pathogenicity

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Erin K. Cassin ◽  
Boo Shan Tseng
Keyword(s):  
Extracellular Matrix ◽  
Outer Membrane ◽  
Biofilm Formation ◽  
Matrix Protein ◽  
Cell Envelope ◽  
Outer Membrane Vesicles ◽  
Extracellular Dna ◽  
Future Research ◽  
Content Type ◽  
Biofilm Matrix

ABSTRACTThe ability ofPseudomonas aeruginosato form biofilms, which are communities of cells encased in a self-produced extracellular matrix, protects the cells from antibiotics and the host immune response. While some biofilm matrix components, such as exopolysaccharides and extracellular DNA, are relatively well characterized, the extracellular matrix proteins remain understudied. Multiple proteomic analyses of theP. aeruginosasoluble biofilm matrix and outer membrane vesicles, which are a component of the matrix, have identified OprF as an abundant matrix protein. To date, the few reports on the effects ofoprFmutations on biofilm formation are conflicting, and little is known about the potential role of OprF in the biofilm matrix. The majority of OprF studies focus on the protein as a cell-associated porin. As a component of the outer membrane, OprF assumes dual conformations and is involved in solute transport, as well as cell envelope integrity. Here, we review the current literature on OprF inP. aeruginosa, discussing how the structure and function of the cell-associated and matrix-associated protein may affect biofilm formation and pathogenesis in order to inform future research on this understudied matrix protein.

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

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.

scholarly journals Proteomic Studies of the Biofilm Matrix including Outer Membrane Vesicles of Burkholderia multivorans C1576, a Strain of Clinical Importance for Cystic Fibrosis

2020 ◽  
Vol 8 (11) ◽  
pp. 1826
Author(s):  
Lucrecia C. Terán ◽  
Marco Distefano ◽  
Barbara Bellich ◽  
Sara Petrosino ◽  
Paolo Bertoncin ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Ros Scavenging ◽  
Burkholderia Multivorans ◽  
Proteomic Approach ◽  
The Matrix ◽  
Biofilm Matrix

Biofilms are aggregates of microbial cells encased in a highly hydrated matrix made up of self-produced extracellular polymeric substances (EPS) which consist of polysaccharides, proteins, nucleic acids, and lipids. While biofilm matrix polysaccharides are unraveled, there is still poor knowledge about the identity and function of matrix-associated proteins. With this work, we performed a comprehensive proteomic approach to disclose the identity of proteins associated with the matrix of biofilm-growing Burkholderia multivorans C1576 reference strain, a cystic fibrosis clinical isolate. Transmission electron microscopy showed that B. multivorans C1576 also releases outer membrane vesicles (OMVs) in the biofilm matrix, as already demonstrated for other Gram-negative species. The proteomic analysis revealed that cytoplasmic and membrane-bound proteins are widely represented in the matrix, while OMVs are highly enriched in outer membrane proteins and siderophores. Our data suggest that cell lysis and OMVs production are the most important sources of proteins for the B. multivorans C1576 biofilm matrix. Of note, some of the identified proteins are lytic enzymes, siderophores, and proteins involved in reactive oxygen species (ROS) scavenging. These proteins might help B. multivorans C1576 in host tissue invasion and defense towards immune system assaults.

scholarly journals Carboxy-Terminal Processing Protease Controls Production of Outer Membrane Vesicles and Biofilm in Acinetobacter baumannii

2021 ◽  
Vol 9 (6) ◽  
pp. 1336
Author(s):  
Rakesh Roy ◽  
Ren-In You ◽  
Chan-Hua Chang ◽  
Chiou-Ying Yang ◽  
Nien-Tsung Lin
Keyword(s):  
Cell Wall ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Sodium Dodecyl ◽  
A549 Cells ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Extracellular Dna ◽  
Carboxy Terminal ◽  
Processing Protease

Carboxy-terminal processing protease (Ctp) is a serine protease that controls multiple cellular processes through posttranslational modification of proteins. Acinetobacter baumannii ATCC 17978 ctp mutant, namely MR14, is known to cause cell wall defects and autolysis. The objective of this study was to investigate the role of ctp mutation–driven autolysis in regulating biofilms in A. baumannii and to evaluate the vesiculation caused by cell wall defects. We found that in A. baumannii, Ctp is localized in the cytoplasmic membrane, and loss of Ctp function enhances the biofilm-forming ability of A. baumannii. Quantification of the matrix components revealed that extracellular DNA (eDNA) and proteins were the chief constituents of MR14 biofilm, and the transmission electron microscopy further indicated the presence of numerous dead cells compared with ATCC 17978. The large number of MR14 dead cells is potentially the result of compromised outer membrane integrity, as demonstrated by its high sensitivity to sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). MR14 also exhibited the hypervesiculation phenotype, producing outer-membrane vesicles (OMVs) of large mean size. The MR14 OMVs were more cytotoxic toward A549 cells than ATCC 17978 OMVs. Our overall results indicate that A. baumanniictp negatively controls pathogenic traits through autolysis and OMV biogenesis.

scholarly journals Hyperbiofilm phenotype of Pseudomonas aeruginosa defective for the PlcB and PlcN secreted phospholipases

2017 ◽  
Vol 63 (9) ◽  
pp. 780-787 ◽  
Author(s):  
Shawn Lewenza ◽  
Laetitia Charron-Mazenod ◽  
Shirin Afroj ◽  
Erik van Tilburg Bernardes
Keyword(s):  
Biofilm Formation ◽  
Membrane Vesicles ◽  
Membrane Lipid ◽  
Secretion System ◽  
Outer Membrane Vesicles ◽  
Extracellular Dna ◽  
Type Ii Secretion ◽  
Chronic Infections ◽  
Type Ii Secretion System ◽  
Biofilm Matrix

Biofilms are dense communities of bacteria enmeshed in a protective extracellular matrix composed mainly of exopolysaccharides, extracellular DNA, proteins, and outer membrane vesicles (OMVs). Given the role of biofilms in antibiotic-tolerant and chronic infections, novel strategies are needed to block, disperse, or degrade biofilms. Enzymes that degrade the biofilm matrix are a promising new therapy. We screened mutants in many of the enzymes secreted by the type II secretion system (T2SS) and determined that the T2SS, and specifically phospholipases, play a role in biofilm formation. Mutations in the xcp secretion system and in the plcB and plcN phospholipases all resulted in hyperbiofilm phenotypes. PlcB has activity against many phospholipids, including the common bacterial membrane lipid phosphatidylethanolamine, and may degrade cell membrane debris or OMVs in the biofilm matrix. Exogenous phospholipase was shown to reduce aggregation and biofilm formation, suggesting its potential role as a novel enzymatic treatment to dissolve biofilms.

scholarly journals PQS-Induced Outer Membrane Vesicles Enhance Biofilm Dispersion in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Adam C. Cooke ◽  
Catalina Florez ◽  
Elise B. Dunshee ◽  
Avery D. Lieber ◽  
Michelle L. Terry ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Antimicrobial Treatment ◽  
Outer Membrane Vesicles ◽  
Chronic Infections ◽  
Burn Wound ◽  
Enhanced Production ◽  
Biofilm Dispersion ◽  
Biofilm Development

AbstractBacterial biofilms are major contributors to chronic infections in humans. Because they are recalcitrant to conventional therapy, they present a particularly difficult treatment challenge. Identifying factors involved in biofilm development can help uncover novel targets and guide the development of anti-biofilm strategies. Pseudomonas aeruginosa causes surgical site, burn wound, and hospital acquired infections, and is also associated with aggressive biofilm formation in the lungs of cystic fibrosis patients. A potent but poorly understood contributor to P. aeruginosa virulence is the ability to produce outer membrane vesicles (OMVs). OMV trafficking has been associated with cell-to-cell communication, virulence factor delivery, and the transfer of antibiotic resistance genes. Because OMVs have almost exclusively been studied using planktonic cultures, little is known about their biogenesis and function in biofilms. Our group has shown that the Pseudomonas Quinolone Signal (PQS) induces OMV formation in P. aeruginosa, and in other species, through a biophysical mechanism that is also active in biofilms. Here, we demonstrate that PQS-induced OMV production is highly dynamic during biofilm development. Interestingly, PQS and OMV synthesis are significantly elevated during dispersion, compared to attachment and maturation stages. PQS biosynthetic and receptor mutant biofilms were significantly impaired in their ability to disperse, but this phenotype could be rescued by genetic complementation or exogenous addition of PQS. Finally, we show that purified OMVs can actively degrade extracellular protein, lipid, and DNA. We therefore propose that enhanced production of PQS-induced OMVs during biofilm dispersion facilitates cell escape by coordinating the controlled degradation of biofilm matrix components.ImportanceTreatments that manipulate biofilm dispersion hold the potential to convert chronic drug-tolerant biofilm infections from protected sessile communities into released populations that are orders-of-magnitude more susceptible to antimicrobial treatment. However, dispersed cells often exhibit increased acute virulence and dissemination phenotypes. A thorough understanding of the dispersion process is therefore critical before this promising strategy can be effectively employed. PQS has been implicated in early biofilm development, but we hypothesized that its function as an OMV inducer may contribute at multiple stages. Here, we demonstrate that PQS and OMVs are differentially produced during Pseudomonas aeruginosa biofilm development and that effective biofilm dispersion is dependent on production of PQS-induced OMVs, which likely act as delivery vehicles for matrix degrading enzymes. These findings lay the groundwork for understanding the roles of OMVs in biofilm development and suggest a model to explain the controlled matrix degradation that accompanies biofilm dispersion in many species.

scholarly journals Pseudomonas Quinolone Signal-Induced Outer Membrane Vesicles Enhance Biofilm Dispersion in Pseudomonas aeruginosa

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Adam C. Cooke ◽  
Catalina Florez ◽  
Elise B. Dunshee ◽  
Avery D. Lieber ◽  
Michelle L. Terry ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Burn Wound ◽  
Content Type ◽  
Enhanced Production ◽  
Biofilm Dispersion ◽  
Biofilm Development

ABSTRACT Bacterial biofilms are major contributors to chronic infections in humans. Because they are recalcitrant to conventional therapy, they present a particularly difficult treatment challenge. Identifying factors involved in biofilm development can help uncover novel targets and guide the development of antibiofilm strategies. Pseudomonas aeruginosa causes surgical site, burn wound, and hospital-acquired infections and is also associated with aggressive biofilm formation in the lungs of cystic fibrosis patients. A potent but poorly understood contributor to P. aeruginosa virulence is the ability to produce outer membrane vesicles (OMVs). OMV trafficking has been associated with cell-cell communication, virulence factor delivery, and transfer of antibiotic resistance genes. Because OMVs have almost exclusively been studied using planktonic cultures, little is known about their biogenesis and function in biofilms. Several groups have shown that Pseudomonas quinolone signal (PQS) induces OMV formation in P. aeruginosa. Our group described a biophysical mechanism for this and recently showed it is operative in biofilms. Here, we demonstrate that PQS-induced OMV production is highly dynamic during biofilm development. Interestingly, PQS and OMV synthesis are significantly elevated during dispersion compared to attachment and maturation stages. PQS biosynthetic and receptor mutant biofilms were significantly impaired in their ability to disperse, but this phenotype was rescued by genetic complementation or exogenous addition of PQS. Finally, we show that purified OMVs can actively degrade extracellular protein, lipid, and DNA. We therefore propose that enhanced production of PQS-induced OMVs during biofilm dispersion facilitates cell escape by coordinating the controlled degradation of biofilm matrix components. IMPORTANCE Treatments that manipulate biofilm dispersion hold the potential to convert chronic drug-tolerant biofilm infections from protected sessile communities into released populations that are orders-of-magnitude more susceptible to antimicrobial treatment. However, dispersed cells often exhibit increased acute virulence and dissemination phenotypes. A thorough understanding of the dispersion process is therefore critical before this promising strategy can be effectively employed. Pseudomonas quinolone signal (PQS) has been implicated in early biofilm development, but we hypothesized that its function as an outer membrane vesicle (OMV) inducer may contribute at multiple stages. Here, we demonstrate that PQS and OMVs are differentially produced during Pseudomonas aeruginosa biofilm development and provide evidence that effective biofilm dispersion is dependent on the production of PQS-induced OMVs, which likely act as delivery vehicles for matrix-degrading enzymes. These findings lay the groundwork for understanding OMV contributions to biofilm development and suggest a model to explain the controlled matrix degradation that accompanies biofilm dispersion in many species.

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

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 <i>Escherichia coli</i>, 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 (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) 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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