scholarly journals Bacteriophage infection produces membrane vesicles in Escherichia coli via both explosive cell lysis and membrane blebbing

2020 ◽  
Author(s):  
Pappu K. Mandal ◽  
Giulia Ballerin ◽  
Laura M. Nolan ◽  
Nicola K. Petty ◽  
Cynthia B. Whitchurch
Keyword(s):  
Outer Membrane ◽  
Bacteriophage T4 ◽  
Super Resolution ◽  
Membrane Vesicles ◽  
Cell Lysis ◽  
Outer Membrane Vesicles ◽  
Lytic Bacteriophage ◽  
Bacteriophage Infection ◽  
Domains Of Life ◽  
Membrane Bound

AbstractMembrane Vesicles (MVs) are membrane-bound spherical nanostructures that prevail in all three domains of life. In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane and are often referred to as outer membrane vesicles (OMVs). We have recently described another mechanism of MV biogenesis in Pseudomonas aeruginosa that involves explosive cell lysis events which shatters cellular membranes into fragments that rapidly anneal into MVs. Interestingly, MVs are often observed within preparations of lytic bacteriophage, however the source of these MVs and their association with bacteriophage infection has not been explored. In this study we aimed to determine if MV biogenesis is associated with lytic bacteriophage infection. Live super-resolution microscopy demonstrated that explosive cell lysis of E. coli cells infected with either bacteriophage T4 or T7, produced MVs derived from shattered membrane fragments. Infection by either bacteriophage was also associated with the formation of membrane blebs on intact bacteria. TEM revealed multiple classes of MVs within phage lysates, consistent with multiple mechanisms of MV biogenesis. These findings suggest that bacteriophage infection may be a major contributor to the abundance of bacterial MVs in nature.

scholarly journals Bacteriophage infection of Escherichia coli leads to the formation of membrane vesicles via both explosive cell lysis and membrane blebbing

Microbiology ◽  
2021 ◽  
Vol 167 (4) ◽  
Author(s):  
Pappu K. Mandal ◽  
Giulia Ballerin ◽  
Laura M. Nolan ◽  
Nicola K. Petty ◽  
Cynthia B. Whitchurch
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Type Species ◽  
Bacteriophage T4 ◽  
Membrane Vesicles ◽  
Cell Lysis ◽  
Lytic Bacteriophage ◽  
Content Type ◽  
Link Type ◽  
Bacteriophage Infection

Membrane vesicles (MVs) are membrane-bound spherical nanostructures that prevail in all three domains of life. In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane and are often referred to as outer membrane vesicles (OMVs). We have recently described another mechanism of MV formation in Pseudomonas aeruginosa that involves explosive cell-lysis events, which shatters cellular membranes into fragments that rapidly anneal into MVs. Interestingly, MVs are often observed within preparations of lytic bacteriophage, however the source of these MVs and their association with bacteriophage infection has not been explored. In this study we aimed to determine if MV formation is associated with lytic bacteriophage infection. Live super-resolution microscopy demonstrated that explosive cell lysis of Escherichia coli cells infected with either bacteriophage T4 or T7, resulted in the formation of MVs derived from shattered membrane fragments. Infection by either bacteriophage was also associated with the formation of membrane blebs on intact bacteria. TEM revealed multiple classes of MVs within phage lysates, consistent with multiple mechanisms of MV formation. These findings suggest that bacteriophage infection may be a major contributor to the abundance of bacterial MVs in nature.

scholarly journals Outer membrane vesicles engineered to express membrane-bound antigen program dendritic cells for cross-presentation to CD8+ T cells

2019 ◽  
Vol 91 ◽  
pp. 248-257 ◽  
Author(s):  
Sjoerd T.T. Schetters ◽  
Wouter S.P. Jong ◽  
Sophie K. Horrevorts ◽  
Laura J.W. Kruijssen ◽  
Steef Engels ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Outer Membrane ◽  
Cd8 T Cells ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Cross Presentation ◽  
Membrane Bound

scholarly journals Biogenesis of Outer Membrane Vesicles Concentrates the Unsaturated Fatty Acid of Phosphatidylinositol in Capnocytophaga ochracea

2021 ◽  
Vol 12 ◽  
Author(s):  
Divya Naradasu ◽  
Waheed Miran ◽  
Shruti Sharma ◽  
Satoshi Takenawa ◽  
Takamitsu Soma ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Outer Membrane ◽  
Unsaturated Fatty Acids ◽  
Protein Profile ◽  
Membrane Vesicles ◽  
Cell Lysis ◽  
Outer Membrane Vesicles ◽  
Growth Phases ◽  
Oral Biofilms ◽  
Lipidome Analysis

Bacterial outer membrane vesicles (OMVs) are spherical lipid bilayer nanostructures released by bacteria that facilitate oral biofilm formation via cellular aggregation and intercellular communication. Recent studies have revealed that Capnocytophaga ochracea is one of the dominant members of oral biofilms; however, their potential for OMV production has yet to be investigated. This study demonstrated the biogenesis of OMVs in C. ochracea associated with the concentration of unsaturated fatty acids of phosphatidylinositol (PI) and characterized the size and protein profile of OMVs produced at growth phases. Transmission electron microscopy showed isolated spherical structures from cells stained with heavy metals, indicating the production of OMVs with a size ranging from 25 to 100 nm. Lipidome analysis revealed the presence of phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, and PI as the main lipids. Some unsaturated fatty acids of PI were present specifically in OMV and little in the outer membrane, suggesting that OMVs are generated from a specific region of the membrane through blebbing rather than a random process such as cell lysis. Furthermore, the lack of similar PI accumulation in the OMV of Porphyromonas gingivalis suggests that C. ochracea has a different biogenesis mechanism. The blebbing mechanism was further supported by higher OMV production occurring at the exponential phase in comparison to the stationary phase, where cell lysis is more likely to occur. Further, comparative protein profile of OMVs isolated under different growth phases may indicate that the OMV cargo does not largely vary with growth phases. The present study provides a basis for further understanding the roles of C. ochracea OMVs in oral biofilms as well as systemic diseases that C. ochracea involves.

scholarly journals Salmonella Outer Membrane Vesicles contain tRNA Fragments (tRFs) that Inhibit Bacteriophage P22 infection

2021 ◽  
Author(s):  
Dominika Houserova ◽  
Yulong Huang ◽  
Kasukurthi Kasukurthi ◽  
Brianna Watters ◽  
Fiza Khan ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Dependent Manner ◽  
Antiviral Defense ◽  
Bacteriophage P22 ◽  
Bacteriophage Infection ◽  
Serovar Typhimurium ◽  
Dose Dependent

Abstract Salmonella Outer Membrane Vesicles (OMVs) were recently shown to inhibit P22 bacteriophage infection. Interestingly, we identify 31 recurrent tRFs abundantly expressed by Salmonella enterica serovar Typhimurium and find these tRFs are highly complementary to known Salmonella enterica-infecting bacteriophage (17 averaging 97.4% complementarity over 22.9 nt) and specifically enriched in S. Typhimurium OMVs. Most notably, tRNA-Thr-CGT-1-1, 44-73, bears 100% complementary over its entire 30 nt length to 29 distinct Salmonella enterica-infecting bacteriophage including P22. Importantly, we find inhibiting this tRF in secreted OMVs improves P22 infectivity in a dose dependent manner whereas raising OMV tRF levels conversely inhibits P22. Furthermore, we find P22 pre-incubation with OMVs isolated from naïve S. Typhimurium, rescues the ability of S. Typhimurium depleted of tRNA-Thr-CGT-1-1, 44-73 tRF to defend against P22. Collectively, these experiments confirm tRFs secreted in S. Typhimurium OMVs are directly involved with and required for the ability of OMVs to defend against bacteriophage predation. As we find the majority of OMV tRFs are highly complementary to an array of known Salmonella enterica-infecting bacteriophage, we suggest OMV tRFs may primarily function as a broadly acting, previously uncharacterized ancient antiviral defense.

scholarly journals Salmonella Outer Membrane Vesicles contain tRNA Fragments (tRFs) that Inhibit Bacteriophage P22 infection

2021 ◽  
Author(s):  
Dominika Houserova ◽  
Yulong Huang ◽  
Mohan V. Kasukurthi ◽  
Brianna C. Watters ◽  
Fiza F. Khan ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Dependent Manner ◽  
Rna Seq ◽  
Bacteriophage P22 ◽  
Bacteriophage Infection ◽  
Serovar Typhimurium ◽  
Dose Dependent

Salmonella Outer Membrane Vesicles (OMVs) were recently shown to inhibit P22 bacteriophage infection. Furthermore, despite there being several published reports now independently describing (1) the marked prevalence of tRFs within secreted vesicle transcriptomes and (2) roles for specific tRFs in facilitating/inhibiting viral replication, there have been no examinations of the effects of vesicle-secreted tRFs on viral infection reported to date. Notably, while specific tRFs have been reported in a number of bacteria, the tRFs expressed by salmonellae have not been previously characterized. As such, we recently screened small RNA-seq datasets for the presence of recurrent, specifically excised tRFs and identified 31 recurrent, relatively abundant tRFs expressed by Salmonella enterica serovar Typhimurium (SL1344). Furthermore, we find S. Typhimurium OMVs contain significant levels of tRFs highly complementary to known Salmonella enterica-infecting bacteriophage with 17 of 31 tRFs bearing marked complementarity to at least one known Salmonella enterica-infecting phage (averaging 97.4% complementarity over 22.9 nt). Most notably, tRNA-Thr-CGT-1-1, 44-73, bears 100% sequence complementary over its entire 30 nt length to 29 distinct, annotated Salmonella enterica-infecting bacteriophage including P22. Importantly, we find inhibiting this tRF in secreted OMVs improves P22 infectivity in a dose dependent manner whereas raising OMV tRF levels conversely inhibits P22 infectivity. Furthermore, we find P22 phage pre-incubation with OMVs isolated from naive, control SL1344 S. Typhimurium, successfully rescues the ability of S. Typhimurium transformed with a specific tRNA-Thr-CGT-1-1, 44-73 tRF inhibitor to defend against P22. Collectively, these experiments confirm tRFs secreted in S. Typhimurium OMVs are directly involved with and required for the ability of OMVs to defend against bacteriophage predation. As we find the majority of OMV tRFs are highly complementary to an array of known Salmonella enterica-infecting bacteriophage, we suggest OMV tRFs may primarily function as a broadly acting, previously uncharacterized innate antiviral defense.

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.

Sign in / Sign up

Export Citation Format

Share Document