scholarly journals MreB Drives De Novo Rod Morphogenesis in Caulobacter crescentus via Remodeling of the Cell Wall

2009 ◽  
Vol 192 (6) ◽  
pp. 1671-1684 ◽  
Author(s):  
Constantin N. Takacs ◽  
Sebastian Poggio ◽  
Godefroid Charbon ◽  
Mathieu Pucheault ◽  
Waldemar Vollmer ◽  
...  
Keyword(s):  
High Performance ◽  
Culture Medium ◽  
De Novo ◽  
Caulobacter Crescentus ◽  
Morphological Changes ◽  
Bacterial Species ◽  
Membrane Vesicles ◽  
Structural Analog ◽  
Outer Membrane Vesicles ◽  
Insight Into

ABSTRACT MreB, the bacterial actin-like cytoskeleton, is required for the rod morphology of many bacterial species. Disruption of MreB function results in loss of rod morphology and cell rounding. Here, we show that the widely used MreB inhibitor A22 causes MreB-independent growth inhibition that varies with the drug concentration, culture medium conditions, and bacterial species tested. MP265, an A22 structural analog, is less toxic than A22 for growth yet equally efficient for disrupting the MreB cytoskeleton. The action of A22 and MP265 is enhanced by basic pH of the culture medium. Using this knowledge and the rapid reversibility of drug action, we examined the restoration of rod shape in lemon-shaped Caulobacter crescentus cells pretreated with MP265 or A22 under nontoxic conditions. We found that reversible restoration of MreB function after drug removal causes extensive morphological changes including a remarkable cell thinning accompanied with elongation, cell branching, and shedding of outer membrane vesicles. We also thoroughly characterized the composition of C. crescentus peptidoglycan by high-performance liquid chromatography and mass spectrometry and showed that MreB disruption and recovery of rod shape following restoration of MreB function are accompanied by considerable changes in composition. Our results provide insight into MreB function in peptidoglycan remodeling and rod shape morphogenesis and suggest that MreB promotes the transglycosylase activity of penicillin-binding proteins.

scholarly journals Surface Exposure and Packing of Lipoproteins into Outer Membrane Vesicles Are Coupled Processes inBacteroides

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Ezequiel Valguarnera ◽  
Nichollas E. Scott ◽  
Philippe Azimzadeh ◽  
Mario F. Feldman
Keyword(s):  
Gut Microbiota ◽  
Public Goods ◽  
Outer Membrane ◽  
Bacterial Species ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Alpha Amylase ◽  
Coupled Processes ◽  
Human Gut ◽  
Human Gut Microbiota

ABSTRACTOuter membrane vesicles (OMVs) are spherical structures derived from the outer membranes (OMs) of Gram-negative bacteria.Bacteroidesspp. are prominent components of the human gut microbiota, and OMVs produced by these species are proposed to play key roles in gut homeostasis. OMV biogenesis inBacteroidesis a poorly understood process. Here, we revisited the protein composition ofBacteroides thetaiotaomicronOMVs by mass spectrometry. We confirmed that OMVs produced by this organism contain large quantities of glycosidases and proteases, with most of them being lipoproteins. We found that most of these OMV-enriched lipoproteins are encoded by polysaccharide utilization loci (PULs), such as thesusoperon. We examined the subcellular locations of the components of the Sus system and found a split localization; the alpha-amylase SusG is highly enriched in OMVs, while the oligosaccharide importer SusC remains mostly in the OM. We found that all OMV-enriched lipoproteins possess a lipoprotein export sequence (LES), and we show that this signal mediates translocation of SusG from the periplasmic face of the OM toward the extracellular milieu. Mutations in the LES motif caused defects in surface exposure and recruitment of SusG into OMVs. These experiments link, for the first time, surface exposure to recruitment of proteins into OMVs. We also show that surface-exposed SusG in OMVs is active and rescues the growth of bacterial cells incapable of growing on starch as the only carbon source. Our results support the role of OMVs as “public goods” that can be utilized by other organisms with different metabolic capabilities.IMPORTANCESpecies from theBacteroidesgenus are predominant members of the human gut microbiota. OMVs inBacteroideshave been shown to be important for the homeostasis of complex host-commensal relationships, mainly involving immune tolerance and protection from disease. OMVs carry many enzymatic activities involved in the cleavage of complex polysaccharides and have been proposed as public goods that can provide growth to other bacterial species by release of polysaccharide breakdown products into the gut lumen. This work shows that the presence of a negatively charged rich amino acid motif (LES) is required for efficient packing of the surface-exposed alpha-amylase SusG into OMVs. Our findings strongly suggest that surface exposure is coupled to packing ofBacteroideslipoproteins into OMVs. This is the first step in the generation of tailor-made probiotic interventions that can exploit LES-related sequences to generateBacteroidesstrains displaying proteins of interest in OMVs.

scholarly journals Moraxella catarrhalisOuter Membrane Vesicles Carry β-Lactamase and Promote Survival ofStreptococcus pneumoniaeandHaemophilus influenzaeby Inactivating Amoxicillin

2011 ◽  
Vol 55 (8) ◽  
pp. 3845-3853 ◽  
Author(s):  
Viveka Schaar ◽  
Therése Nordström ◽  
Matthias Mörgelin ◽  
Kristian Riesbeck
Keyword(s):  
Bacterial Species ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Chronic Obstructive ◽  
Respiratory Pathogens ◽  
Upper Respiratory Tract ◽  
Nasopharyngeal Colonization ◽  
Obstructive Pulmonary Disease ◽  
Transmission Electron ◽  
Tract Infections

ABSTRACTMoraxella catarrhalisis a common pathogen found in children with upper respiratory tract infections and in patients with chronic obstructive pulmonary disease during exacerbations. The bacterial species is often isolated together withStreptococcus pneumoniaeandHaemophilus influenzae. Outer membrane vesicles (OMVs) are released byM. catarrhalisand contain phospholipids, adhesins, and immunomodulatory compounds such as lipooligosaccharide. We have recently shown thatM. catarrhalisOMVs exist in patients upon nasopharyngeal colonization. As virtually allM. catarrhalisisolates are β-lactamase positive, the goal of this study was to investigate whetherM. catarrhalisOMVs carry β-lactamase and to analyze if OMV consequently can prevent amoxicillin-induced killing. Recombinant β-lactamase was produced and antibodies were raised in rabbits. Transmission electron microscopy, flow cytometry, and Western blotting verified that OMVs carried β-lactamase. Moreover, enzyme assays revealed thatM. catarrhalisOMVs contained active β-lactamase. OMVs (25 μg/ml) incubated with amoxicillin for 1 h completely hydrolyzed amoxicillin at concentrations up to 2.5 μg/ml. In functional experiments, preincubation of amoxicillin (10× MIC) withM. catarrhalisOMVs fully rescued amoxicillin-susceptibleM. catarrhalis,S. pneumoniae, and type b or nontypeableH. influenzaefrom β-lactam-induced killing. Our results suggest that the presence of amoxicillin-resistantM. catarrhalisoriginating from β-lactamase-containing OMVs may pave the way for respiratory pathogens that by definition are susceptible to β-lactam antibiotics.

scholarly journals New insight into the application of outer membrane vesicles of Gram-negative bacteria

2015 ◽  
Vol 2 (5) ◽  
pp. 93-96 ◽  
Author(s):  
A Fateh ◽  
F Vaziri ◽  
F Rahimi Janani ◽  
S Ahmadi Badi ◽  
M Ghazanfari ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Insight Into

scholarly journals Vesicular delivery of the antifungal antibiotics of Lysobacter enzymogenes C3

2018 ◽  
Author(s):  
Paul R. Meers ◽  
Carol Liu ◽  
Rensa Chen ◽  
William Bartos ◽  
Julianne Davis ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Outer Membrane ◽  
Bacterial Species ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Physical Contact ◽  
Host Cells ◽  
Yeast Cells ◽  
Lysobacter Enzymogenes ◽  
Antifungal Antibiotics

AbstractLysobacter enzymogenes C3 is a predatory strain of gram-negative gliding bacteria that produces antifungal antibiotics by the polyketide synthetic pathway. Outer membrane vesicles (OMV) are formed as a stress response and can deliver virulence factors to host cells. The production of OMV by C3 and their role in antifungal activity are reported here. Vesicles in the range of 130-150 nm in diameter were discovered in the cell-free supernatants of C3 cultures. These OMV contain molecules characteristic of bacterial outer membranes, such as lipopolysaccharide and phospholipids. In addition, they contain chitinase activity and essentially all of the heat stable antifungal activity in cell supernatants. We show here that C3 OMV can directly inhibit growth of the yeast Saccharomyces cerevisiae as well as the filamentous fungus Fusarium subglutinans. The activity is dependent on physical contact between OMV and the cells. Furthermore, fluorescent lipid labeling of C3 OMV demonstrated transfer of the membrane-associated probe to yeast cells, suggesting the existence of a mechanism of delivery for membrane-associated molecules. Mass spectrometric analysis of C3 OMV extracts indicates the presence of molecules with molecular weights identical to some of the previously identified antifungal products of C3. These data together suggest that OMV act as an important remote mobile component of predation by Lysobacter.ImportanceThe data presented here suggest a newly discovered function of outer membrane vesicles (OMV) that are produced from the outer membrane of the bacterial species Lysobacter enzymogenes C3. We show that these OMV can be released from the surface of the cells to deliver antibiotics to target fungal organisms as a mechanism of killing or growth inhibition. Understanding the role of OMV in antibiotic delivery can generally lead to improved strategies for dealing with antibiotic-resistant organisms. These results also add to the evidence that some bacterially produced antibiotics can be discovered and purified using methods designed for isolation of nanoscale vesicles. Information on these systems can lead to better identification of active molecules or design of delivery vehicles for these molecules.

scholarly journals Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies

2018 ◽  
Vol 115 (14) ◽  
pp. E3106-E3115 ◽  
Author(s):  
Taylor C. Stevenson ◽  
Colette Cywes-Bentley ◽  
Tyler D. Moeller ◽  
Kevin B. Weyant ◽  
David Putnam ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protective Immunity ◽  
Membrane Vesicle ◽  
Bacterial Species ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Microbial Pathogens ◽  
Polysaccharide Antigen ◽  
Protein Carriers

Many microbial pathogens produce a β-(1→6)–linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule, including bacterial, fungal, and protozoan cells. Broadly protective immune responses to this single conserved polysaccharide antigen in animals are possible but only when a deacetylated poly-N-acetyl-d-glucosamine (dPNAG; <30% acetate) glycoform is administered as a conjugate to a carrier protein. Unfortunately, conventional methods for natural extraction or chemical synthesis of dPNAG and its subsequent conjugation to protein carriers can be technically demanding and expensive. Here, we describe an alternative strategy for creating broadly protective vaccine candidates that involved coordinating recombinant poly-N-acetyl-d-glucosamine (rPNAG) biosynthesis with outer membrane vesicle (OMV) formation in laboratory strains ofEscherichia coli. The glycosylated outer membrane vesicles (glycOMVs) released by these engineered bacteria were decorated with the PNAG glycopolymer and induced high titers of PNAG-specific IgG antibodies after immunization in mice. When aStaphylococcus aureusenzyme responsible for PNAG deacetylation was additionally expressed in these cells, glycOMVs were generated that elicited antibodies to both highly acetylated PNAG (∼95–100% acetate) and a chemically deacetylated dPNAG derivative (∼15% acetate). These antibodies mediated efficient in vitro killing of two distinct PNAG-positive bacterial species, namelyS. aureusandFrancisella tularensissubsp.holarctica, and mice immunized with PNAG-containing glycOMVs developed protective immunity against these unrelated pathogens. Collectively, our results reveal the potential of glycOMVs for targeting this conserved polysaccharide antigen and engendering protective immunity against the broad range of pathogens that produce surface PNAG.

scholarly journals Gene Transfer Potential of Outer Membrane Vesicles of Acinetobacter baylyi and Effects of Stress on Vesiculation

2014 ◽  
Vol 80 (11) ◽  
pp. 3469-3483 ◽  
Author(s):  
Shweta Fulsundar ◽  
Klaus Harms ◽  
Gøril E. Flaten ◽  
Pål J. Johnsen ◽  
Balu Ananda Chopade ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Zeta Potential ◽  
Outer Membrane ◽  
Bacterial Species ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Hydrodynamic Diameter ◽  
Acinetobacter Baylyi ◽  
Content Type ◽  
Dsdna Antibodies

ABSTRACTOuter membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, including the facilitation of horizontal gene transfer (HGT) processes, have been proposed. In this study, we investigated whether OMVs contribute to the transfer of plasmids between bacterial cells and species using Gram-negativeAcinetobacter baylyias a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations ofEscherichia coliandA. baylyi, including naturally transformation-deficient mutants ofA. baylyi. Anti-double-stranded DNA (anti-dsDNA) antibodies were used to determine the movement of DNA into OMVs. We also determined how stress affected the level of vesiculation and the amount of DNA in vesicles. OMVs were further characterized by measuring particle size distribution (PSD) and zeta potential. Transmission electron microscopy (TEM) and immunogold labeling were performed using anti-fluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-labeled and DNA-containing OMVs. Exposure to OMVs isolated from plasmid-containing donor cells resulted in HGT toA. baylyiandE. coliat transfer frequencies ranging from 10−6to 10−8, with transfer efficiencies of approximately 103and 102per μg of vesicular DNA, respectively. Antibiotic stress was shown to affect the DNA content of OMVs as well as their hydrodynamic diameter and zeta potential. Morphological observations suggest that OMVs fromA. baylyiinteract with recipient cells in different ways, depending on the recipient species. Interestingly, the PSD measurements suggest that distinct size ranges of OMVs are released fromA. baylyi.

scholarly journals FIP200 organizes the autophagy machinery at p62-ubiquitin condensates beyond activation of the ULK1 kinase

2020 ◽  
Author(s):  
Eleonora Turco ◽  
Irmgard Fischer ◽  
Sascha Martens
Keyword(s):  
Kinase Activity ◽  
De Novo ◽  
Super Resolution ◽  
Membrane Vesicles ◽  
Degradation Pathway ◽  
Autophagosome Formation ◽  
Ubiquitinated Proteins ◽  
The Many ◽  
Super Resolution Microscopy ◽  
Insight Into

AbstractMacroautophagy is a conserved degradation pathway, which mediates cellular homeostasis by the delivery of harmful substances into lysosomes. This is achieved by the sequestration of these substances referred to as cargo within double membrane vesicles, the autophagosomes, which form de novo. Among the many cargoes that are targeted by autophagy are condensates containing p62 and ubiquitinated proteins. p62 recruits the FIP200 protein to initiate autophagosome formation at the condensates. How FIP200 in turn organizes the autophagy machinery is unclear. Here we show that FIP200 is dispensable for the recruitment of the upstream autophagy machinery to the condensates, but it is necessary for phosphatidylinositol 3-phosphate formation and WIPI2 recruitment. We further find that FIP200 is required for the activation of the ULK1 kinase. Surprisingly, ULK1 kinase activity is not strictly required for autophagosome formation at p62 condensates. Super-resolution microscopy of p62 condensates revealed that FIP200 surrounds the condensates where it spatially organizes ATG13 and ATG9A for productive autophagosome formation. Our data provide a mechanistic insight into how FIP200 orchestrates autophagosome initiation at the cargo.

scholarly journals Biogenesis of a bacterial metabolosome for propanediol utilization

2021 ◽  
Author(s):  
Mengru Yang ◽  
Nicolas Wenner ◽  
Gregory Dykes ◽  
Yan Li ◽  
Xiaojun Zhu ◽  
...  
Keyword(s):  
De Novo ◽  
Cellular Metabolism ◽  
Structural Analog ◽  
Organelle Biogenesis ◽  
Mechanistic Insight ◽  
Ordered Assembly ◽  
Assembly Pathways ◽  
Enzyme Complexes ◽  
Insight Into

Bacterial metabolosomes are a family of protein organelles in bacteria. Elucidating how thousands of proteins self-assemble to form functional metabolosomes is essential for understanding their significance in cellular metabolism and pathogenesis. Here we investigate the de novo biogenesis of propanediol-utilization (Pdu) metabolosomes and characterize the roles of the key constituents in generation and intracellular positioning of functional metabolosomes. Our results demonstrate that the Pdu metabolosome undertakes both 'Shell first' and 'Cargo first' assembly pathways, unlike the beta-carboxysome structural analog which only involves the 'Cargo first' strategy. Shell and cargo assemblies occur independently at the cell poles. The internal cargo core is formed through the ordered assembly of multiple enzyme complexes, and exhibits liquid-like properties within the metabolosome architecture. Our findings provide mechanistic insight into the molecular principles driving bacterial metabolosome assembly and expand our understanding of liquid-like organelle biogenesis.

scholarly journals A mechanistic overview of ruminal fibre digestion.

2019 ◽  
Author(s):  
Adrian E Naas ◽  
Phillip B Pope
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Rumen Microbiota ◽  
Rumen Microbiome ◽  
Fibre Degradation ◽  
Polysaccharide Utilization Loci ◽  
Insight Into

Ruminants have co-evolved with symbiotic rumen microbiota, which readily convert ingested plant fibres into the nutrients they need to sustain their growth and maintenance. Fibre degradation within the rumen microbiome has been attributed to a limited number of cultivable representatives, which has restricted our ability to understand the different enzymatic machineries that exist. However, via a combination of culturing, meta-omics, bioinformatics, biochemistry and enzymology, we are beginning to expand our insight into the different fibre-digesting strategies that rumen microbiota employ. We discuss findings from studies on well-known Ruminococcus, Fibrobacter and Prevotella isolates, as well as those from poorly understood and as-yet uncultured Bacteroidetes lineages. Collectively, these approaches have revealed new mechanistic information related to the hydrolytic capacity of cellulosomes, free enzymes, outer membrane vesicles, polysaccharide utilization loci and large multi-modular enzymes, which are generating deeper insights into the intricate microbial networks that engage in ruminal fibre digestion.

scholarly journals Protective effects of Helicobacter pylori membrane vesicles against stress and antimicrobial agents

Microbiology ◽  
2020 ◽  
Vol 166 (8) ◽  
pp. 751-758 ◽  
Author(s):  
Benjamin Oliver Murray ◽  
Robin Andrew Dawson ◽  
Lolwah Mohammad Alsharaf ◽  
Jody Anne Winter
Keyword(s):  
Helicobacter Pylori ◽  
Type Species ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Content Type ◽  
Link Type ◽  
H Pylori

Outer-membrane vesicles (OMVs) produced by Helicobacter pylori deliver bacterial components to host cells, provide a mechanism for stabilization of secreted components and may allow the bacteria to exert ‘long-range’ effects in the gastric niche, promoting persistence. In addition to their well-characterized host cell interactions, membrane vesicles improve stress survival in other bacterial species, and are constitutively produced by both pathogenic and non-pathogenic bacteria. We aimed to determine whether OMVs could improve H. pylori survival of a range of stressors. The effects of purified OMVs on the resistance of H. pylori to a range of environmental and antimicrobial stresses were determined using growth curves and survival assays. Addition of purified OMVs to H. pylori cultures provided dose-dependent protection against hydrogen peroxide-mediated killing. Supplementation with OMVs also partially protected H. pylori against the bactericidal effects of the antibiotics clarithromycin and levofloxacin, but not against amoxicillin nor metronidazole. Addition of purified OMVs allowed H. pylori to grow in the presence of inhibitory concentrations of the antimicrobial peptide LL-37. In the presence of 50 µg OMVs ml−1, significantly enhanced H. pylori growth was observed at higher LL-37 concentrations compared with lower LL-37 concentrations, suggesting that OMV–LL-37 interactions might facilitate release of growth-promoting nutrients. Taken together, these data indicate that production of membrane vesicles could help H. pylori to survive exposure to antibiotics and host antimicrobial defences during infection.

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