Purification of membrane vesicles from Gram-positive bacteria using flow cytometry, after iodixanol density-gradient ultracentrifugation

2020 ◽  
Author(s):  
Tadahiro Nasukawa ◽  
Ryosuke Sugimoto ◽  
Jumpei Uchiyama ◽  
Iyo Takemura-Uchiyama ◽  
Hironobu Murakami ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Density Gradient ◽  
Membrane Vesicles ◽  
Density Gradient Ultracentrifugation ◽  
Gram Positive ◽  
Gram Positive Bacteria

scholarly journals Extracellular Vesicle Biogenesis and Functions in Gram-Positive Bacteria

2020 ◽  
Vol 88 (12) ◽  
Author(s):  
Paul Briaud ◽  
Ronan K. Carroll
Keyword(s):  
Lipid Bilayers ◽  
Membrane Vesicles ◽  
Cell Interaction ◽  
Extracellular Vesicle ◽  
Wall Structure ◽  
Future Research ◽  
Gram Positive ◽  
Content Type ◽  
Bacterial Physiology ◽  
Gram Positive Bacteria

ABSTRACT Extracellular vesicles (EVs) are membrane-derived lipid bilayers secreted by bacteria and eukaryotic cells. Bacterial membrane vesicles were discovered over 60 years ago and have been extensively studied in Gram-negative bacteria. During their production, EVs are loaded with proteins, nucleic acids, and various compounds that are subsequently released into the environment. Depending on the packaged cargo, EVs have a broad spectrum of action and are involved in pathogenesis, antibiotic resistance, nutrient uptake, and nucleic acid transfer. Due to differences in cell wall structure, EVs in Gram-positive bacteria have been disregarded for decades, and our understanding of their biogenesis and host cell interaction is incomplete. Recently, studies on bacteria such as Staphylococcus aureus, Streptococcus spp., Bacillus subtilis, and Mycobacterium spp. have demonstrated EV production in Gram-positive bacteria and shown the great importance EVs have in Gram-positive bacterial physiology and disease progression. Here, we review the latest findings on the biogenesis and functions of EVs from Gram-positive bacteria and identify key areas for future research.

Gram-positive bacteria produce membrane vesicles: Proteomics-based characterization of Staphylococcus aureus-derived membrane vesicles

PROTEOMICS ◽  
2009 ◽  
Vol 9 (24) ◽  
pp. 5425-5436 ◽  
Author(s):  
Eun-Young Lee ◽  
Do-Young Choi ◽  
Dae-Kyum Kim ◽  
Jung-Wook Kim ◽  
Jung Ok Park ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Membrane Vesicles ◽  
Gram Positive ◽  
Gram Positive Bacteria

Bactericidal effect of gentamicin-induced membrane vesicles derived fromPseudomonas aeruginosaPAO1 on gram-positive bacteria

2002 ◽  
Vol 48 (9) ◽  
pp. 810-820 ◽  
Author(s):  
Kelly L MacDonald ◽  
Terry J Beveridge
Keyword(s):  
Electron Microscopy ◽  
Therapeutic Potential ◽  
Hydrophobic Interaction Chromatography ◽  
Membrane Vesicles ◽  
Bactericidal Effect ◽  
Gram Positive ◽  
Thin Sections ◽  
Gram Negative ◽  
Induced Membrane ◽  
Gram Positive Bacteria

Previous studies have shown that gentamicin-induced membrane vesicles (g-MVs) from Pseudomonas aeruginosa PAO1 possess both the antibiotic (gentamicin) and a potent peptidoglycan hydrolase (PGase; autolysin) that is effective in killing gram-negative pathogens. This present study evaluated the therapeutic potential of g-MVs against four gram-positive bacteria. Bactericidal assays and electron microscopy of thin sections revealed that Bacillus subtilis 168 and Staphylococcus aureus D2C were susceptible to killing mediated by g-MVs, Listeria monocytogenes ATCC 19113 was slightly susceptible, whereas Enterococcus hirae ATCC 9790 was unaffected. g-MVs were generally more effective against the bacteria than was soluble gentamicin, suggesting they could have more killing power than natural membrane vesicles containing no antibiotic. Electron microscopy and hydrophobic interaction chromatography showed that more membrane vesicles (MVs) initially attached to B. subtilis (hydrophilic) than to predominantly hydrophobic E. hirae, L. monocytogenes, and S. aureus. Zymograms containing murein sacculi as an enzyme substrate illustrated that all organisms except E. hirae were sensitive to the 26-kDa autolysin to varying degrees. Peptidoglycan O-acetylation did not influence susceptibility to MV-mediated lysis. Though not universally effective, the g-MV delivery system remains a promising therapeutic alternative for specific gram-positive infections.Key words: gram-negative membrane vesicles, gentamicin, autolysin.

scholarly journals Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Nozomu Obana ◽  
Ryoma Nakao ◽  
Kyoko Nagayama ◽  
Kouji Nakamura ◽  
Hidenobu Senpuku ◽  
...  
Keyword(s):  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Host Interaction ◽  
Sensor Kinases ◽  
Toll Like Receptor 2 ◽  
Membrane Spanning ◽  
Aspartate Residue

ABSTRACT Recently, many Gram-positive bacteria as well as Gram-negative bacteria have been reported to produce membrane vesicles (MVs), but little is known regarding the regulators involved in MV formation. We found that a Gram-positive anaerobic pathogen, Clostridium perfringens, produces MVs predominantly containing membrane proteins and cell wall components. These MVs stimulated proinflammatory cytokine production in mouse macrophage-like cells. We suggested that MVs induced interleukin-6 production through the Toll-like receptor 2 (TLR2) signaling pathway. Thus, the MV could have a role in the bacterium-host interaction and bacterial infection pathogenesis. Moreover, we found that the sporulation master regulator gene spo0A was required for vesiculogenesis. A conserved, phosphorylated aspartate residue of Spo0A was indispensable for MV production, suggesting that the phosphorylation of Spo0A triggers MV production. Multiple orphan sensor kinases necessary for sporulation were also required to maximize MV production. These findings imply that C. perfringens actively produces immunoactive MVs in response to the environment changing, as recognized by membrane-spanning sensor kinases and by modulating the phosphorylation level of Spo0A.

scholarly journals A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria

2003 ◽  
Vol 69 (5) ◽  
pp. 2857-2863 ◽  
Author(s):  
Claus Holm ◽  
Lene Jespersen
Keyword(s):  
Flow Cytometry ◽  
Bacterial Species ◽  
Staining Technique ◽  
Gram Positive ◽  
Bulk Tank Milk ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Gram Staining ◽  
Flow Cytometric ◽  
Bulk Tank

ABSTRACT A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50°C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at −18°C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5°C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.

scholarly journals Extracellular vesicles: An emerging platform in gram-positive bacteria

2020 ◽  
Vol 7 (12) ◽  
pp. 312-322
Author(s):  
Swagata Bose ◽  
Shifu Aggarwal ◽  
Durg Vijai Singh ◽  
Narottam Acharya
Keyword(s):  
Extracellular Vesicles ◽  
Intercellular Communication ◽  
Pathogenic Bacteria ◽  
Membrane Vesicles ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Functional Aspects ◽  
As Stress

Extracellular vesicles (EV), also known as membrane vesicles, are produced as an end product of secretion by both pathogenic and non-pathogenic bacteria. Several reports suggest that archaea, gram-negative bacteria, and eukaryotic cells secrete membrane vesicles as a means for cell-free intercellular communication. EVs influence intercellular communication by transferring a myriad of biomolecules including genetic information. Also, EVs have been implicated in many phenomena such as stress response, intercellular competition, lateral gene transfer, and pathogenicity. However, the cellular process of secreting EVs in gram-positive bacteria is less studied. A notion with the thick cell-walled microbes such as gram-positive bacteria is that the EV release is impossible among them. The role of gram-positive EVs in health and diseases is being studied gradually. Being nano-sized, the EVs from gram-positive bacteria carry a diversity of cargo compounds that have a role in bacterial competition, survival, invasion, host immune evasion, and infection. In this review, we summarise the current understanding of the EVs produced by gram-positive bacteria. Also, we discuss the functional aspects of these components while comparing them with gram-negative bacteria.

Sign in / Sign up

Export Citation Format

Share Document