Membrane vesicle secretion and prophage induction in multidrug-resistant Stenotrophomonas maltophilia in response to ciprofloxacin stress.
Devos Simon,Van Putte Wouter,Vitse Jolien,Van Driessche Gonzalez,Stremersch Stephan,Van Den Broek Wim,Raemdonck Koen,Braeckmans Kevin,Stahlberg Henning,Kudryashev Misha,Savvides Savvas N,Devreese Bart
Environmental microbiology
Several bacterial species produce membrane vesicles (MVs) in response to antibiotic stress. However, the biogenesis and role of MVs in bacterial antibiotic resistance mechanisms have remained unclear. Here, we studied the effect of the fluoroquinolone ciprofloxacin on MV secretion by Stenotrophomonas maltophilia using a combination of electron microscopy and proteomic approaches. We found that in addition to the classical outer membrane vesicles (OMV), ciprofloxacin-stimulated cultures produced larger vesicles containing both outer and inner membranes termed outer-inner membrane vesicles (OIMV), and that such MVs are enriched with cytosolic proteins. Remarkably, OIMV were found to be decorated with filamentous structures identified as fimbriae. In addition, ciprofloxacin stress leads to the release of bacteriophages and phage tail-like particles. Prophage induction by ciprofloxacin has been linked to pathogenesis and horizontal gene transfer in several bacterial species. Together, our findings show that ciprofloxacin treatment of S. maltophilia leads to the secretion of a heterogeneous pool of MVs and the induction of prophages that are potentially involved in adverse side-effects during antibiotic treatment.
10.1111/1462-2920.13793
Outer Membrane Vesicle Production Facilitates LPS Remodeling and Outer Membrane Maintenance in Salmonella during Environmental Transitions.
Bonnington Katherine E,Kuehn Meta J
mBio
The ability of Gram-negative bacteria to carefully modulate outer membrane (OM) composition is essential to their survival. However, the asymmetric and heterogeneous structure of the Gram-negative OM poses unique challenges to the cell's successful adaption to rapid environmental transitions. Although mechanisms to recycle and degrade OM phospholipid material exist, there is no known mechanism by which to remove unfavorable lipopolysaccharide (LPS) glycoforms, except slow dilution through cell growth. As all Gram-negative bacteria constitutively shed OM vesicles (OMVs), we propose that cells may utilize OMV formation as a way to selectively remove environmentally disadvantageous LPS species. We examined the native kinetics of OM composition during physiologically relevant environmental changes in Salmonella enterica, a well-characterized model system for activation of PhoP/Q and PmrA/B two-component systems (TCSs). In response to acidic pH, toxic metals, antimicrobial peptides, and lack of divalent cations, these TCSs modify the LPS lipid A and core, lengthen the O antigen, and upregulate specific OM proteins. An environmental change to PhoP/Q- and PmrA/B-activating conditions simultaneously induced the addition of modified species of LPS to the OM, downregulation of previously dominant species of LPS, greater OMV production, and increased OMV diameter. Comparison of the relative abundance of lipid A species present in the OM and the newly budded OMVs following two sets of rapid environmental shifts revealed the retention of lipid A species with modified phosphate moieties in the OM concomitant with the selective loss of palmitoylated species via vesiculation following exposure to moderately acidic environmental conditions. IMPORTANCE:All Gram-negative bacteria alter the structural composition of LPS present in their OM in response to various environmental stimuli. We developed a system to track the native dynamics of lipid A change in Salmonella enterica serovar Typhimurium following an environmental shift to PhoP/Q- and PmrA/B-inducing conditions. We show that growth conditions influence OMV production, size, and lipid A content. We further demonstrate that the lipid A content of OMVs does not fit a stochastic model of content selection, revealing the significant retention of lipid A species containing covalent modifications that mask their 1- and 4'-phosphate moieties under host-like conditions. Furthermore, palmitoylation of the lipid A to form hepta-acylated species substantially increases the likelihood of its incorporation into OMVs. These results highlight a role for the OMV response in OM remodeling and maintenance processes in Gram-negative bacteria.
10.1128/mBio.01532-16
Outer Membrane Vesicle Induction and Isolation for Vaccine Development.
Frontiers in microbiology
Gram-negative bacteria release vesicular structures from their outer membrane, so called outer membrane vesicles (OMVs). OMVs have a variety of functions such as waste disposal, communication, and antigen or toxin delivery. These vesicles are the promising structures for vaccine development since OMVs carry many surface antigens that are identical to the bacterial surface. However, isolation is often difficult and results in low yields. Several methods to enhance OMV yield exist, but these do affect the resulting OMVs. In this review, our current knowledge about OMVs will be presented. Different methods to induce OMVs will be reviewed and their advantages and disadvantages will be discussed. The effects of the induction and isolation methods used in several immunological studies on OMVs will be compared. Finally, the challenges for OMV-based vaccine development will be examined and one example of a successful OMV-based vaccine will be presented.
10.3389/fmicb.2021.629090
Comparative Analysis of Outer Membrane Vesicle Isolation Methods With an Mutant Reveals a Hypervesiculating Phenotype With Outer-Inner Membrane Vesicle Content.
Reimer Shelby L,Beniac Daniel R,Hiebert Shannon L,Booth Timothy F,Chong Patrick M,Westmacott Garrett R,Zhanel George G,Bay Denice C
Frontiers in microbiology
Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are mediators of cell survival and pathogenesis by facilitating virulence factor dissemination and resistance to antimicrobials. Studies of OMV properties often focus on hypervesiculating mutants that have increased OMV production when compared to their corresponding wild-type (WT) strains. Currently, two conventional techniques, ultracentrifugation (UC) and ultradiafiltration (UF), are used interchangeably to isolate OMVs, however, there is concern that each technique may inadvertently alter the properties of isolated OMVs during study. To address this concern, we compared two OMV isolation methods, UC and UF, with respect to final OMV quantities, size distributions, and morphologies using a hypervesiculating K-12 Δ mutant. Nanoparticle tracking analysis (NTA) indicated that UC techniques result in lower vesicle yields compared to UF. However, UF permitted isolation of OMVs with smaller average sizes than UC, highlighting a potential OMV isolation size bias by each technique. Cryo-transmission electron microscopy (cryo-TEM) visualization of isolated OMVs revealed distinct morphological differences between WT and Δ OMVs, where Δ OMVs isolated by either UC or UF method possessed a greater proportion of OMVs with two or more membranes. Proteomic OMV analysis of WT and Δ OMVs confirmed that Δ enhances inner plasma membrane carryover in multi-lamellar OMVs. This study demonstrates that UC and UF are useful techniques for OMV isolation, where UF may be preferable due to faster isolation, higher OMV yields and enrichment of smaller sized vesicles.
10.3389/fmicb.2021.628801
Reciprocal Packaging of the Main Structural Proteins of Type 1 Fimbriae and Flagella in the Outer Membrane Vesicles of "Wild Type" Strains.
Frontiers in microbiology
Fundamental aspects of outer membrane vesicle (OMV) biogenesis and the engineering of producer strains have been major research foci for many in recent years. The focus of this study was OMV production in a variety of strains including wild type (WT) (K12 and BW25113), mutants (from the Keio collection) and proprietary [BL21 and BL21 (DE3)] strains. The present study investigated the proteome and prospective mechanism that underpinned the key finding that the dominant protein present in K-12 WT OMVs was fimbrial protein monomer (FimA) (a polymerizable protein which is the key structural monomer from which Type 1 fimbriae are made). However, mutations in genes involved in fimbriae biosynthesis (Δ, , , and ) resulted in the packaging of flagella protein monomer (FliC) (the major structural protein of flagella) into OMVs instead of FimA. Other mutations (Δ, and Δ-a transcriptional regulator of fimbriation and flagella biosynthesis) lead to the packaging of both FimA and Flagellin into the OMVs. In the majority of instances shown within this research, the production of OMVs is considered in K-12 WT strains where structural appendages including fimbriae or flagella are temporally co-expressed throughout the growth curve as shown previously in the literature. The hypothesis, proposed and supported within the present paper, is that the vesicular packaging of the major FimA is reciprocally regulated with the major FliC in K-12 OMVs but this is abrogated in a range of mutated, non-WT strains. We also demonstrate, that a protein of interest (GFP) can be targeted to OMVs in an K-12 strain by protein fusion with FimA and that this causes normal packaging to be disrupted. The findings and underlying implications for host interactions and use in biotechnology are discussed.
10.3389/fmicb.2021.557455
A Protocol for Isolation and Proteomic Characterization of Distinct Extracellular Vesicle Subtypes by Sequential Centrifugal Ultrafiltration.
Xu Rong,Simpson Richard J,Greening David W
Methods in molecular biology (Clifton, N.J.)
Scientific and clinical interest in extracellular vesicles (EVs) has increased rapidly as evidence mounts that they may constitute a new signaling paradigm. Recent studies have highlighted EVs carry preassembled complex biological information that elicit pleiotropic responses in target cells. It is well recognized that cells secrete essentially two EV subtypes that can be partially separated by differential centrifugation (DC): the larger size class (referred to as "microvesicles" or "shed microvesicles," sMVs) is heterogeneous (100-1500 nm), while the smaller size class (referred to as "exosomes") is relatively homogeneous in size (50-150 nm). A key issue hindering progress in understanding underlying mechanisms of EV subtype biogenesis and cargo selectivity has been the technical challenge of isolating homogeneous EV subpopulations suitable for molecular analysis. In this protocol we reveal a novel method for the isolation, purification, and characterization of distinct EV subtypes: exosomes and sMVs. This method, based on sequential centrifugal ultrafiltration (SCUF), affords unbiased isolation of EVs from conditioned medium from a human colon cancer cell model. For both EV subtypes, this protocol details extensive purification and characterization based on dynamic light scattering, cryoelectron microscopy, quantitation, immunoblotting, and comparative label-free proteome profiling. This analytical SCUF method developed is potentially scalable using tangential flow filtration and provides a solid foundation for future in-depth functional studies of EV subtypes from diverse cell types.
10.1007/978-1-4939-6728-5_7
Identification of small extracellular vesicle subtypes in follicular fluid: Insights into the function and miRNA profiles.
Wang Xiaomei,Meng Kai,Wang Hengqin,Wang Ying,Zhao Yunqi,Kang Jian,Zhang Yong,Quan Fusheng
Journal of cellular physiology
The study of small extracellular vesicles (sEVs) heterogeneity is one of the main problems that must be solved, and the different sEV subtypes in follicular fluid are still unclear, limiting our understanding of their function. This study first separated sEV subtypes from follicular fluid using differential ultracentrifugation combined with iodixanol density gradient flotation and then evaluated their miRNA profile and effects on the proliferation and apoptosis of granulosa cells (GCs). We also performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of potential target genes of differentially expressed miRNAs (DEMs) and KEGG analysis of potential target genes of non-DEMs. Low-density sEVs (sEV_F6) were enriched in TSG101, while high-density sEVs (sEV_F8) were enriched in CD63. The miRNA profiles of sEV_F6 and sEV_F8 were heterogeneous, and the differential signaling pathways were mainly related to the adhesion and hypoxic stress pathways, while the same signaling pathways were mainly related to cell proliferation, apoptosis, cell cycle, and autophagy pathways. In addition, the highly expressed miRNAs in both subtypes were mainly related to cell proliferation and apoptosis. Both subtypes transferred their miRNAs into GCs and promoted the proliferation ability of the GCs and inhibited their apoptosis. The results showed for the first time that there are different subtypes of sEVs in follicular fluid and that the miRNA profiles of subtypes are heterogeneous.
10.1002/jcp.30251
DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis.
Nakayama-Imaohji Haruyuki,Hirota Katsuhiko,Yamasaki Hisashi,Yoneda Saori,Nariya Hirofumi,Suzuki Motoo,Secher Thomas,Miyake Yoichiro,Oswald Eric,Hayashi Tetsuya,Kuwahara Tomomi
PloS one
Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.
10.1371/journal.pone.0148887
Regulation of outer-membrane proteins (OMPs) A and F, during -induced outer-membrane vesicle (OMV) biosynthesis.
Heliyon
BACKGROUND:Gram-negative bacteria actively secrete outer membrane vesicles into the surrounding environment and these vesicles have been shown to play various physiological and protective roles such as carrying antibiotic-degrading enzymes and acting as decoys against host defences, therefore promoting the pathogenesis of the bacterium. It has been shown that avian pathogenic species can increase vesicle biosynthesis through the acquisition of the gene but the effect this has on the cell by scavenging outer-membrane associated proteins (OmpA, OmpF) into the vesicles during vesicle release have not yet been investigated. RESULTS:Relative quantitative real-time PCR data obtained from expressing and non-expressing cells showed that during induction, showed a nearly 2-fold down regulation relative to the non-expressing cells during the entire 24 hours, while was expressed at the same level as the non-expressing cells during the first 8 hours of expression. At 24 hours post- expression, was up-regulated 4-fold. CONCLUSIONS:The regulatory effects of the newly described outer-membrane vesicle biosynthesis-related gene, , on has not previously been investigated. As -induced vesicles contain OmpA and OmpF scavenged from the bacterial outer-membrane, potential regulatory effects on the host was investigated. An increase in expression and an insignificant decrease in expression was observed during induction demonstrating that -related biosynthesis is not related to decreased expression, which is one of the potential mechanisms discussed in literature for biosynthesis. Outer-membrane vesicle biosynthesis during over-expression could potentially be accomplished through a different mechanism(s).
10.1016/j.heliyon.2019.e02014
Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis Mutant.
Takaki Kotaro,Tahara Yuhei O,Nakamichi Nao,Hasegawa Yusuke,Shintani Masaki,Ohkuma Moriya,Miyata Makoto,Futamata Hiroyuki,Tashiro Yosuke
Applied and environmental microbiology
Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of , which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming type strain JCM 1090. The Δ mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the Δ mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of Δ mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the Δ mutant to form unique vesicles. Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, JCM 1090 Δ mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications.
10.1128/AEM.01131-20
Prophage-triggered membrane vesicle formation through peptidoglycan damage in Bacillus subtilis.
Toyofuku Masanori,Cárcamo-Oyarce Gerardo,Yamamoto Tatsuya,Eisenstein Fabian,Hsiao Chien-Chi,Kurosawa Masaharu,Gademann Karl,Pilhofer Martin,Nomura Nobuhiko,Eberl Leo
Nature communications
Bacteria release membrane vesicles (MVs) that play important roles in various biological processes. However, the mechanisms of MV formation in Gram-positive bacteria are unclear, as these cells possess a single cytoplasmic membrane that is surrounded by a thick cell wall. Here we use live cell imaging and electron cryo-tomography to describe a mechanism for MV formation in Bacillus subtilis. We show that the expression of a prophage-encoded endolysin in a sub-population of cells generates holes in the peptidoglycan cell wall. Through these openings, cytoplasmic membrane material protrudes into the extracellular space and is released as MVs. Due to the loss of membrane integrity, the induced cells eventually die. The vesicle-producing cells induce MV formation in neighboring cells by the enzymatic action of the released endolysin. Our results support the idea that endolysins may be important for MV formation in bacteria, and this mechanism may potentially be useful for the production of MVs for applications in biomedicine and nanotechnology.It is unclear how Gram-positive bacteria, with a thick cell wall, can release membrane vesicles. Here, Toyofuku et al. show that a prophage-encoded endolysin can generate holes in the cell wall through which cytoplasmic membrane material protrudes and is released as vesicles.
10.1038/s41467-017-00492-w
Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms.
Turnbull Lynne,Toyofuku Masanori,Hynen Amelia L,Kurosawa Masaharu,Pessi Gabriella,Petty Nicola K,Osvath Sarah R,Cárcamo-Oyarce Gerardo,Gloag Erin S,Shimoni Raz,Omasits Ulrich,Ito Satoshi,Yap Xinhui,Monahan Leigh G,Cavaliere Rosalia,Ahrens Christian H,Charles Ian G,Nomura Nobuhiko,Eberl Leo,Whitchurch Cynthia B
Nature communications
Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.
10.1038/ncomms11220
A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria.
Roier Sandro,Zingl Franz G,Cakar Fatih,Durakovic Sanel,Kohl Paul,Eichmann Thomas O,Klug Lisa,Gadermaier Bernhard,Weinzerl Katharina,Prassl Ruth,Lass Achim,Daum Günther,Reidl Joachim,Feldman Mario F,Schild Stefan
Nature communications
Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.
10.1038/ncomms10515