* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Biogenesis and multifaceted roles of outer membrane
Survey
Document related concepts
Theories of general anaesthetic action wikipedia , lookup
Magnesium transporter wikipedia , lookup
Lipid bilayer wikipedia , lookup
Cell nucleus wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Chemical synapse wikipedia , lookup
Membrane potential wikipedia , lookup
Cell encapsulation wikipedia , lookup
Model lipid bilayer wikipedia , lookup
Cytokinesis wikipedia , lookup
Type three secretion system wikipedia , lookup
Signal transduction wikipedia , lookup
SNARE (protein) wikipedia , lookup
Lipopolysaccharide wikipedia , lookup
List of types of proteins wikipedia , lookup
Cell membrane wikipedia , lookup
Transcript
Microbiology (2014), 160, 2109–2121 Review DOI 10.1099/mic.0.079400-0 Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria Heramb M. Kulkarni and Medicharla V. Jagannadham Correspondence CSIR – Centre for Cellular and Molecular Biology, Tarnaka, Hyderabad-500007, India Medicharla V. Jagannadham [email protected] Received 24 March 2014 Accepted 25 July 2014 Outer membrane vesicles (OMVs) released from Gram-negative bacteria consist of lipids, proteins, lipopolysaccharides and other molecules. OMVs are associated with several biological functions such as horizontal gene transfer, intracellular and intercellular communication, transfer of contents to host cells, and eliciting an immune response in host cells. Although hypotheses have been made concerning the mechanism of biogenesis of these vesicles, research on OMV formation is far from complete. The roles of outer membrane components, bacterial quorum sensing molecules and some specific proteins in OMV biogenesis have been studied. This review discusses the different models that have been proposed for OMV biogenesis, along with details of the biological functions of OMVs and the likely scope of future research. Introduction The perception that prokaryotic organisms exhibit a simple structure as compared with eukaryotes prevailed for a long time. The prokaryotes, including bacteria, were thought to be a collection of independently surviving autonomous cells with few interactions, and almost no compartmentalization (Manning & Kuehn, 2013). Various new findings have shifted the paradigm, and prokaryotes are now known to possess a very ordered structure and organization, finely tuned physiology, and numerous social interactions which give rise to collective solutions for their survival (Raymond & Bonsall, 2013; Spitzer & Poolman, 2013). Different classes of prokaryotes have evolved different organelles and strategies, of which many are analogous to those in eukaryotic cells. The Gram-negative bacteria are known to release extracellular membrane-bound vesicles, which have been compared with the exosomes produced by unicellular and multicellular eukaryotes. They are named outer membrane vesicles (OMVs) and are the subject of this review. OMVs The reasons for the term OMV are mainly historical. It was first seen that a cell-free filtrate of Vibrio cholerae was able to elicit an immune response in rabbits (De, 1959). As the outer membrane (OM) components of Gram-negative bacteria were known to be immunogenic, it was suspected that the components of the OM were somehow leaching into the medium. Later it was shown that Escherichia coli cultures grown on lysine-limiting medium showed secretion of LPS in the form of spherical ‘bags’ (Bishop & Work, 1965; Work et al., 1966). Soon after that, V. cholerae was Abbreviations: OM, outer membrane; OMV, outer membrane vesicle; PQS, 2-heptyl-3-hydroxy-4-quinolone. 079400 G 2014 The Authors found to release spherical membrane-bound vesicles which were produced by pinching off of the OM (Chatterjee & Das, 1967). It was also observed that the density of some granular material was higher near the site where the ‘pinching off’ event was taking place on the OM, and some granules were entering into the OMVs, suggesting possible sorting of cytosolic contents to these vesicles. Later, many different Gram-negative bacteria were shown to release similar vesicles, ranging in size from 20 to 250 nm (Kulp & Kuehn, 2010). Studies of the chemical composition of the OMVs showed the presence of components from the OM as well as other cellular compartments. Therefore, these structures were called ‘membrane vesicles’, ‘extracellular vesicles’, ‘outer membrane fragments’ or ‘blebs’ (Mayrand & Grenier, 1989), although the historical term ‘outer membrane vesicles’ is now generally accepted for Gramnegative bacteria and is used throughout this review. Gram-positive bacteria and Archaea are also reported to exhibit the presence of membrane vesicles produced from cell surfaces (Manning & Kuehn, 2013), but this review mainly focuses on OMVs produced by Gram-negative bacteria. Functional studies have shown that OMVs have a multifaceted role in the physiology of bacteria. OMVs were found to be released in all growth phases of the bacterial culture (Manning & Kuehn, 2011) although their amount and composition may be dependent upon the growth conditions. Attempts to isolate a non-vesiculated mutant have been unsuccessful so far, although many genes have been identified whose alteration changes the number of OMVs released (McBroom et al., 2006). As the OMVs are known to provoke the immune system, they have been recognized as promising agents to be used as vaccines. Many efforts in this direction are being carried out (Aoki et al., 2007), one successful example being a vaccine for Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 Printed in Great Britain 2109 H. M. Kulkarni and M. V. Jagannadham meningitidis caused by Neisseria meningitidis (Findlow et al., 2006; Boutriau et al., 2007; Williams et al., 2007). Efforts to prepare a vaccine for other infectious diseases continue (Keenan et al., 2003; Zhu et al., 2005; Schild et al., 2009; Nieves et al., 2014), and OMVs hold great promise to combat these diseases. Besides direct medical applications, the study of OMVs is also shedding new light on the physiology of Gram-negative bacteria. increases their efficacy. Furthermore, if multiple enzymes are needed for a distant activity, for example degradation of complex molecules from a solid surface, then OMVs can co-transport proteins so that they reach the remote site simultaneously. Thus, the energy expenditure of the cell in making OMVs is justified by their action on remote targets with high efficiency. The Gram-negative bacteria possess sophisticated machineries for secretion of various biologically important molecules. Secretion systems designated type I to type VI have been identified and studied in Gram-negative bacteria. OMVs are proposed to be a different secretion system, ubiquitously present in all Gram-negative bacteria, which are independent of the conventional systems (Kadurugamuwa & Beveridge, 1995; Kuehn & Kesty, 2005). For example, the cytotoxic protein cytolysin A (ClyA) is secreted via OMVs in E. coli (Bendtsen et al., 2005). Secretion via OMVs has many important advantages over the other secretion systems. Structural components of OMVs Exploration of the chemical components of OMVs showed that they contain phospholipids, lipopolysaccharides, proteins and in rare cases nucleic acids. They are also known to transport metabolites, quorum sensing signals and other small molecules. The structural studies of OMVs invariably showed that they were spherical and enclosed with a membrane bilayer, and had a strain-specific characteristic size distribution. Proteins Characteristics of OMV-mediated secretion OMVs facilitate the secretion of insoluble or hydrophobic materials such as lipids, membrane proteins and signalling molecules. The success of pathogenic bacteria lies in the successful colonization of host tissue. Integral membrane proteins called adhesins mediate co-aggregation of bacterial cells and attachment to host surfaces. In Porphyromonas gingivalis, OMVs contain multivalent complexes of adhesins which cause cellular aggregation and the formation of dental plaque biofilms (Grenier & Mayrand, 1987; Inagaki et al., 2006). The role of OMVs in the formation of biofilms will be discussed in greater detail later. OMVs were also found to transport the hydrophobic quorum sensing molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) from Pseudomonas aeruginosa, and it has been shown that PQS causes the biogenesis of OMVs (MashburnWarren et al., 2008). In other secretion systems, soluble proteins are secreted into the medium and so are exposed to the potentially harsh conditions of the extracellular environment. OMVs may be a means by which soluble proteins and nucleic acids can be encapsulated in a bilayer membrane and released in a protective structure. Soluble proteins are transported in the OMV lumen (Lee et al., 2007). Proteins inside the OMV lumen, as well as soluble proteins externally associated with the surface of OMVs, are highly resistant to external proteases (Kesty & Kuehn, 2004). Nucleic acids encapsulated into OMVs are similarly protected from external nucleases. So, OMV-mediated transport can allow less stable soluble molecules, such as protease-susceptible toxins, and nucleic acids, to reach their destinations undamaged. OMVs may even behave like time-release capsules, which provide beneficial activity a period of time after they were released. OMVs allow proteins to be delivered at high local concentrations and in proximity of the target site, which 2110 The protein content of OMVs has been studied thoroughly by many groups using different bacterial strains. The presence of virulence factors was shown in the OMVs produced by different pathogenic bacteria (Kadurugamuwa & Beveridge, 1997; Chatterjee & Chaudhury, 2011). Wensink & Witholt (1981) showed that the protein profile of OMVs of E. coli is different from that of other subcellular fractions, which was later confirmed in other organisms. Advances in the MS-based proteomics have given momentum to the high-throughput profiling of OMV proteins (Lee et al., 2007). Many protein constituents have also been identified by classical Western blotting and functional experiments. The proteins found in OMVs belong to the OM and periplasm. Some researchers have used the presence of outermembrane proteins (e.g. OmpW) as markers of the OMVs (McBroom & Kuehn, 2007; Manabe et al., 2013). Many cytoplasmic and inner-membrane proteins were also found in OMVs, and these were thought to be contaminants from lysed cells in the cultures (Berlanda Scorza et al., 2008; Kulp & Kuehn, 2010). To resolve this issue, a density-gradient centrifugation step was added to the protocol for preparation of OMVs, to separate OMVs from proteins which were free or loosely associated with OMVs. Even after this stringent preparation, the OMVs from multiple bacterial strains showed the presence of cytosolic and inner membrane proteins (Lee et al., 2007). So it appears that there must be a machinery that sorts cytosolic proteins into the OMVs. Several functional studies also supported this view. In some strains, DNA fragments were found in OMVs, as we discuss in a subsequent section. The presence of DNA in OMVs proved that cytosolic contents can, in principle, be sorted into OMVs. The proteins from these OMVs belonged to diverse functional categories, including OM structural proteins, porins, ion channels, transporters for different molecules, periplasmic and cytoplasmic enzymes, and proteins related to stress responses (Lee et al., 2007, 2008). Not all outer membrane proteins from a given strain were Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 Microbiology 160 Studies on outer membrane vesicles Table 1. Proteomics studies on OMVs of different Gram-negative bacteria Study no. Source for OMVs Sample description 1 Neisseria meningitidis serogroup B Wild-type 2 Neisseria meningitidis MC58 Dgna33 3 Neisseria meningitidis NZ98/254 Dgna33 4 5 6 7 8 9 10 11 12 Wild-type Wild-type Wild-type Wild-type Wild-type Vegetative cell Developmental cell Wild-type Wild-type 13 14 15 16 17 18 19 20 21 Escherichia coli DH5a Legionella pneumophila Acinetobacter baumannii DU202 Helicobacter pylori J99 Helicobacter pylori NCTC 11637 Myxococcus xanthus DK1622 Myxococcus xanthus DK1622 Francisella philomiragia ATCC 25015 Francisella tularensis subsp. novicida ATCC 15482 Edwardsiella tarda ED45 Pseudomonas aeruginosa PAO1 Staphylococcus aureus 06ST1048* Acinetobacter baumannii ATCC 19606T Klebsiella pneumoniae ATCC 13883 Pseudomonas aeruginosa PAO1 Pseudomonas aeruginosa PAO1 Pseudomonas aeruginosa PAO1 Pseudomonas aeruginosa PAO1 22 Acinetobacter baumannii AbH12O-A2 Wild-type Wild-type Wild-type Wild-type Wild-type Wild-type DlexAN Wild-type biofilm Wild-type planktonic culture Wild-type 23 Pseudomonas syringae pv. tomato T1 Wild-type 24 Francisella novicida ATCC 15482 25 Francisella novicida ATCC 15482 26 Neisseria meningitidis H44/76 (serogroup B) Neisseria gonorrhoeae strains FA1090, F62, MS11, 1291 Wild-type exponential phase Wild-type early stationary phase Wild-type 27 28 29 30 31 32 33 34 Escherichia coli Nissle 1917 (EcN) Porphyromonas gingivalis Campylobacter jejuni 11168 Bacteroides fragilis Bacteroides thetaiotaomicron Pseudomonas syringae Lz4W Vibrio cholerae Wild-type Wild-type Wild-type Wild-type Wild-type Wild-type Wild-type Wild-type sorted to OMVs (Lee et al., 2008), although membrane proteins make up a significant portion of the OMV proteome. Table 1 summarizes proteomic analyses of OMVs and the number of proteins that were identified in each case. Enzymes present in OMVs include proteases, peptidases, nucleases and b-lactamases (Lee et al., 2007; Schaar et al., http://mic.sgmjournals.org Proteomics strategy No. of identified proteins Reference (1) 1DE-MALDI-MS/MS, (2) 1DE-LC-ESI-MS/MS (1) 1DE-MALDI-MS/MS, (2) 2DE-MALDI-MS/MS (1) 1DE-MALDI-MS/MS, (2) 2DE-MALDI-MS/MS LC-ESI-MS/MS 2DE-MALDI-MS/MS 1DE-LC-ESI-MS/MS 1DE-LC-ESI-MS/MS 1DE-LC-ESI-MS/MS 1DE-LC-MALDI-MS/MS 1DE-LC-MALDI-MS/MS 1DE-LC-ESI-MS/MS 1DE-LC-ESI-MS/MS 50 Post et al. (2005) 63 Ferrari et al. (2006) 63 Ferrari et al. (2006) 132 181 131 86 61 429 218 238 415 Lee et al. (2007) Galka et al. (2008) Kwon et al. (2009) Mullaney et al. (2009) Mullaney et al. (2009) Kahnt et al. (2010) Kahnt et al. (2010) Pierson et al. (2011) Pierson et al. (2011) 1DE-LC-ESI-MS/MS LC-ESI-MS/MS 1DE-LC-ESI-MS/MS 1DE-LC-ESI-MS/MS 1DE-LC-ESI-MS/MS 1DE-LC-ESI-MS/MS 1DE-LC-ESI-MS/MS 1DE-LC-ESI-MS/MS 1DE-LC-ESI-MS/MS 72 314 143 113 159 105 71 76 193 Park et al. (2011) Choi et al. (2011) Gurung et al. (2011) Jin et al. (2011) Lee et al. (2012) Maredia et al. (2012) Maredia et al. (2012) Toyofuku et al. (2012) Toyofuku et al. (2012) (1) 1DE-LC-MALDI-MS/MS, (2) LC-MALDI-MS/MS 1DE-LC-ESI-MS/MS 175 Mendez et al. (2012) 139 LC-ESI-MS/MS 99 Chowdhury & Jagannadham (2012) McCaig et al. (2013) LC-ESI-MS/MS 286 McCaig et al. (2013) LC-ESI-MS/MS 140 LC-MALDI-MS/MS, quantitative study by isobaric tagging (iTRAQ) 1DE-LC-ESI-MS/MS 1DE-LC-ESI-MS/MS LC-ESI-MS/MS 1DE-Q-TOF 1DE-Q-TOF 1DE-LC-ESI-MS/MS In-solution digestion-LCESI-MS/MS 308 van de Waterbeemd et al. (2013) Zielke et al. (2014) 192 151 134 115 58 429 90 Aguilera et al. (2014) Veith et al. (2014) Jang et al. (2014) Elhenawy et al. (2014) Elhenawy et al. (2014) Kulkarni et al. (2014) Altindis et al. (2014) 2011, 2013, 2014). OMVs may also contain toxins and other virulence factors (Kulp & Kuehn, 2010). The makeup of the OMV proteome may help to shed light on the physiological functions served by OMVs. The exact process by which proteins are sorted to OMVs is not yet known. Protein profiles of OMVs harvested at different growth phases or Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 2111 H. M. Kulkarni and M. V. Jagannadham after imposition of different stresses were found to be different (Berlanda Scorza et al., 2012). OMVs have been used successfully as delivery vehicles for recombinant proteins (Chen et al., 2010). Phospholipids Similar to the OM, the outer leaflet of OMVs is predominantly composed of LPSs in addition to lipids. In an early study, the OMVs of E. coli were found to have a similar phospholipid profile to the OM (Hoekstra et al., 1976). Various studies have subsequently been carried out to compare the phospholipids from the OM and OMVs, mostly using classic techniques such as TLC (Horstman & Kuehn, 2000; Kato et al., 2002; Nevot et al., 2006). Apparently, OM phospholipids are present in the membranes of OMVs (Tashiro et al., 2011; Chowdhury & Jagannadham, 2013; Kulkarni et al., 2014), and there is evidence that OMVs contain some lipids that are not detected in the OM (Kato et al., 2002). There are some reports of the fine structural characterization of the phospholipids present in OMVs using MS (Chowdhury & Jagannadham, 2013; Kulkarni et al., 2014), and fatty acid analysis by GC-MS (Tashiro et al., 2011; Fulsundar et al., 2014). However, a quantitative lipidomic analysis of the different kinds of phospholipids present has not yet been done using modern high-throughput MS-based techniques. The curvature of the OMV membrane is far higher than that of the parent cell and therefore it is likely to be characterized by a different composition of various phospholipids. The phospholipid composition of the OM and OMVs from Pseudomonas aeruginosa was found to be different. In the OM, phosphatidylethanolamine was the abundant species while in OMVs it was phosphatidylglycerol (Tashiro et al., 2011). Furthermore, the relative amount of saturated fatty acyl chains in OMVs was higher as compared with the OM (Tashiro et al., 2011) making the OMVs more rigid than the OM. Tashiro et al. (2012) later proposed that the release of OMVs leaves the bacterial OM more fluid, and cells may shed OMVs to maintain the fluidity optimum for the prevailing growth conditions. LPSs LPSs found in the OM are a characteristic feature of the Gram-negative cell envelope. They are also known to have endotoxin and pyrogenic activity in mammalian systems (Gu & Tsai, 1991) and they are important adhesins for biofilm formation. LPS molecules were also found in OMVs (Bishop & Work, 1965; Work et al., 1966). As for proteins, not all LPSs of the parent cell appear in OMVs; rather only a fraction of the LPS complement seems to be sorted into OMVs (Li et al., 1996; Chowdhury & Jagannadham 2013). OMVs from Pseudomonas aeruginosa are mainly composed of the negatively charged B-band of LPS instead of the more neutral A-band (Kadurugamuwa & Beveridge, 1995). LPS may be an important player in the biogenesis of OMVs (Tashiro 2112 et al., 2012), and this point is further clarified below. MS studies on the sorting of the Lipid A component of LPS from the OM to OMVs have been carried out by Feldman’s group. They showed that in the case of the dental pathogen Porphyromonas gingivalis, deacetylated Lipid A accumulated in OMVs (Haurat et al., 2011) but in the related species Bacteroides fragilis the Lipid A profiles of the OM and OMVs were not different (Elhenawy et al., 2014). Nucleic acids The presence of plasmid DNA was shown for the first time in the OMVs of Haemophilus parainfluenzae (Kahn et al., 1982). Later, other OMVs were found to contain RNA and DNA (Dorward et al., 1989; Mayrand & Grenier, 1989); they belonged to the genera Escherichia, Pseudomonas, Haemophilus, Neisseria and others. DNA fragments from lysed cells could potentially bind to the positively charged surface of OMVs. Treatment of OMVs with DNase proved that some DNA fragments were trapped in the lumen of the OMVs (Renelli et al., 2004). A detailed study of the presence of nuclease-resistant DNA in membrane vesicles and OMVs from Gram-positive and Gram-negative bacteria, respectively, was carried out by Dorward & Garon (1990). They observed that DNA in the form of circular plasmids, linear plasmids and chromosomal fragments is likely to be present in Gram-negative OMVs but not in Gram-positive membrane vesicles. Very little is known regarding the sorting of DNA to the OMVs. It has been shown that OMVs can pick up free DNA fragments present in their environment, and that DNA can enter through the periplasm during their biogenesis (Renelli et al., 2004). The nucleic acids from an infecting bacteriophage might be thrown out from the cell by means of OMVs (Loeb & Kilner, 1978). The DNA fragments found in the OMVs were of plasmid, phage or chromosomal origins (Yaron et al., 2000). They can be linear or circular. The OMVs of Neisseria gonorrhoeae were found to contain RNA in addition to DNA (Dorward et al., 1989). DNA was not found in the OMVs of many other strains (Zhou et al., 1998). Three different strains of Porphyromonas gingivalis were found to produce OMVs that did not contain any DNA (Zhou et al., 1998). Although DNA was present in some OMVs, not all of them could transform other cells (Yaron et al., 2000; Renelli et al., 2004). Other molecules OMVs also transport a variety of ions, quorum sensing signals and metabolites (Schertzer & Whiteley, 2012; Biller et al., 2014). The appearance of these molecules in OMVs has not been studied extensively to date. There is speculation that many other types of bioactive molecules will continue to be found in OMVs. There have been some reports describing the presence of cell wall components such as peptidoglycan and muramic acid in OMVs (Kadurugamuwa & Beveridge, 1995), but this aspect has not since been revisited. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 Microbiology 160 Studies on outer membrane vesicles Biogenesis of OMVs The formation of OMVs was initially thought to be a physical process associated with the routine wear and tear of the OM of the cell (Haurat et al., 2011). Although this idea is still held by some, recent discoveries show that there is likely to be an elaborate mechanism behind the biogenesis of OMVs. Also, the formation of OMVs is an active resource-depleting process, which suggests that OMVs are not products of simple material shearing of the membrane. Although there is no direct evidence of an energy requirement for OMV biogenesis, there is likely to be energy expenditure required for the sorting of biomolecules to OMVs (Kulp & Kuehn, 2010). The exact pathway of OMV biogenesis is not currently known. However, many mechanisms have been proposed, and it may be possible that all of them work together for the formation of OMVs. OMVs as a product of cell wall turnover The earliest attempts to decipher the mechanism of biogenesis of OMVs were those of Burdett & Murray (1974) and Hoekstra et al. (1976). It was noted that OMVs are shed from the OM, despite the presence of covalent linkages between the peptidoglycan layer and OM. Later, OMVs of E. coli were found to contain a relatively low amount of lipoproteins, as compared with the OM (Wensink & Witholt 1981). It was further proposed that OMVs form due to the faster growth of the OM compared with the cell wall and associated lipoproteins. Zhou et al. (1998) studied the biogenesis of OMVs from Porphyromonas gingivalis and proposed a model that suggested that OMV production was due to turnover of the cell wall. This model was further revisited by showing that autolysin mutants of Porphyromonas gingivalis produced fewer OMVs than wild-type strains (Hayashi et al., 2002). During cell wall synthesis, peptidoglycan and muramic acid exert a turgor pressure onto the OM. The blebs thus produced ultimately pinch off from the membrane and relieve the cell of the pressure caused by excess wall materials. Biochemical methods showed that the OMVs contain muramic acid, but not nucleic acids or cytoplasmic proteins. The OM protein profile was comparable with that of OMVs (Zhou et al., 1998). This model does not explain how cytosolic molecules may be packaged into OMVs. In later experiments with other organisms, nucleic acids were found in OMVs, and the presence and possibly selective sorting of proteins from other compartments of the cell were demonstrated (Haurat et al., 2011). OMV production as a stress response It is expected that any external physical and chemical stress would be felt on the OM of the Gram-negative cell. It has been found in a number of studies that the presence of stress increases the production of OMVs (Kulp & Kuehn, 2010). Careful experiments using lysed cells as a control showed that the observed increase in OMV production during stress occurred without any increase in cell lysis (MacDonald & Kuehn, 2013; Schwechheimer et al., 2013). http://mic.sgmjournals.org Therefore, it has been thought that the biogenesis of OMVs from bacteria is primarily a stress response. It was seen in the case of OMVs of Pseudomonas putida DOT-T1E that chemical stresses such as toxic concentrations of long chain alcohols and EDTA (which chelates ions needed for the normal growth of bacterial cells), as well as physical stresses such as osmotic pressure and heat shock, caused the cells to release OMVs (Baumgarten et al., 2012). Antibiotic stress has also been studied in this context (Maredia et al., 2012). The antibiotic ciprofloxacin disrupts DNA replication and leads to the SOS response. The SOS response was found to be involved in increasing OMV production. It was also observed that the shedding of OMVs makes the cells more prone to form biofilms, possibly as they increase the hydrophobic nature of the bacterial cell surface (Tashiro et al., 2012). However, the fact that OMVs are released even when cells grow without external stress makes it difficult to accept that OMV biogenesis is exclusively a stress response. McBroom et al. (2006) showed that membrane instability cannot be correlated with OMV biogenesis. In their study, many of the genes responsible for the overproduction of OMVs were found to be related to peptidoglycan synthesis, OM proteins and the sigma E stress response pathway. However, instability of the OM was probably not the cause of the production of OMVs (McBroom et al., 2006). McBroom & Kuehn (2007) found that temperature stress in E. coli leads to accumulation of misfolded proteins that ultimately are thrown out by being packaged in OMVs. Although they did not discuss it, the increased fluidity of the OM at higher temperatures may also have partially contributed to their observations. They recognize OMV production as a novel stress response, but also assert that it is a vital physiological process found in all Gram-negative bacteria. The most interesting observation, however, is that mutants producing higher amounts of OMVs could better resist temperature stress. Considering all of these observations, we conclude that OMV biogenesis occurs and is presumably important in non-stressed cells, but is also a component of multiple stress responses. A bilayer-couple model for biogenesis The process of making of OMVs, which leaves the integrity and functions of the bacterial OM intact, hints that there must be some well-defined process with many steps. The membrane curvature of the OMVs is far higher than that of the OM. The cell wall determines the size and shape of bacteria. As OMVs are devoid of cell wall components or any similar structure, the curvature required for their structure calls for a different explanation. Mashburn & Whiteley (2005) found that, in Pseudomonas aeruginosa, PQS is needed for OMV production. They further showed that PQS induces membrane curvature by introducing itself into the membrane. The extent of curvature was found to be in proportion to the local concentration of PQS. They proposed a ‘bilayer-couple model’, in which the secreted PQS becomes inserted into the OM of the bacterial cell, inducing curvature that ultimately leads to Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 2113 H. M. Kulkarni and M. V. Jagannadham the formation of OMVs (Mashburn-Warren et al., 2009; Schertzer & Whiteley, 2012). This model has not yet been extrapolated to other organisms. However, it accepts the production of OMVs to be a fundamental physical process, and identifies a direct cause which marks the start of OMV biogenesis. PQS may also be involved in the destabilization of Mg2+ and Ca2+ salt bridges in the OM, which leads to the negative charge repulsion between LPS molecules. As quinolones are known to sequester positive charges (Marshall & Piddock, 1994), and chelating agents such as EDTA are known to increase OMV production (Eagon & Carson, 1965), a specific role for PQS becomes possible. The observation that external supplementation of Mg2+ ions in the growth medium suppressed the formation of OMVs in Pseudomonas aeruginosa (MashburnWarren & Whiteley, 2006) also supports this hypothesis. In further research, Tashiro et al. (2010) showed that the production of PQS itself is further controlled by bicyclic compounds such as 1-naphthol, 2-naphthol, 2, 3-dihydroxynaphthalene, 1-aminonaphthalene and 8-quinolinol. These compounds inhibited the production of PQS, which further repressed the production of OMVs. This effect is significant as it shows that certain chemicals can directly affect OMV production and therefore the pathogenicity of bacteria, which may be applicable in medicine. Proteins playing a role in OMV biogenesis The OMVs of Salmonella enterica contain the proteins PagC and OmpX, whose overexpression is known to accelerate the production of OMVs (Kitagawa et al., 2010). Similarly, the E. coli inner membrane protein NlpA was found to regulate the biogenesis of OMVs (Bodero et al., 2007). The presence of curvature-inducing proteins has been found to be important in vesicle formation in many eukaryotic cells (Graham & Kozlov, 2010), although similar observations have not yet been made for prokaryotes. Previously, it was shown that lipoproteins are specifically depleted in OMV membranes (Hoekstra et al., 1976). Subsequently, Wensink & Witholt (1981) also showed a decreased amount of lipoproteins in the OMVs of E. coli. Since then, disruptions in several other tethering proteins (e.g. Braun’s lipoprotein, OmpA and the Tol–PAL complex) have also been shown to be associated with increased OMV production. Note that Pseudomonas aeruginosa possesses fewer lipoproteins than E. coli (Martin et al., 1972), which may provide an explanation for the observation that P. aeruginosa usually produces significantly higher levels of OMVs than E. coli (MashburnWarren & Whiteley, 2006). Some OM proteins such as Omps, proteins related to Tol–Pal systems and YbgF are suspected to have a role in the bulging of the bacterial OM which marks the onset of OMV production (Bernadac et al., 1998). Very recently it has been shown that in V. cholerae the DegP protein influences OMV contents, and the abundance of several other functionally significant proteins (Altindis et al., 2014). 2114 OMV production through an interplay of peptidoglycan, LPS and the OM Some early studies performed with Salmonella and Pseudomonas aeruginosa showed that strains containing truncated LPS exhibited enhanced membrane blebbing (Smit et al., 1975; Meadow et al., 1978). A model for Pseudomonas aeruginosa OMV formation has been proposed in which the electronegative charge of the B-band LPS causes charge-to-charge repulsion and membrane instability, which results in outward membrane blebbing and a random trapping of periplasmic components within OMVs (Kadurugamuwa & Beveridge, 1995). This model is supported by the observation that growth under conditions that enrich the B-band of LPS enhances OMV formation by Pseudomonas aeruginosa (Sabra et al., 2003). Deatherage et al. (2009) showed that some of the connections between the peptidoglycan layer and the OM (OM-PG) and those which connect OM–peptidoglycan–inner membrane (OM-PG-IM) are associated with the release of OMVs from Salmonella. They also suggested that, during various physiological processes, whenever the density of these connections reaches some threshold, OMVs pinch out as a result. Based on these results, Kuehn & Kesty (2005) have proposed a model of OMV biogenesis. Nanopods for OMV biogenesis A strikingly different phenomenon was seen to work for OMV production and delivery in a soil bacterium, Delftia sp. Cs1-4. Cells of this strain were found to form long tubes projecting from the cell surface and OMVs were siphoned out of these tubes (Shetty et al., 2011). The tubes have been termed ‘nanopods’ by the authors. The tube-like structures are also found associated with the OMVs of Francisella novicida (McCaig et al., 2013), but their connection with the biogenesis of OMVs has not been studied. A mechanism of this kind is suspected to be operative only in strains living in habitats such as soil where the availability of water is scarce. In other habitats, secreted OMVs quickly spread over long distances through water. So, the long-distance transfer of OMVs requires physical transfer to the site of delivery (Sanchez, 2011). Functions of OMVs Many different functions have been demonstrated for OMVs, making them an important player in the physiology of Gram-negative bacteria. The prevalent idea is that OMVs are indispensable for the survival of these bacteria. Over many decades, different roles played by the OMVs were found; some of the prominent functions are discussed below. Assistance in biofilm formation Biofilms are surface-adhering structures made from a matrix consisting of exopolysaccharides, DNA, proteins and many other molecules, in which bacterial cells remain embedded. In general, bacteria form biofilms as a response to stress. OMVs were found to be associated with the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 Microbiology 160 Studies on outer membrane vesicles biofilms of Pseudomonas aeruginosa (Beveridge et al., 1997). The number of OMVs released from bacteria is found to increase during stress, which suggests a relationship between OMVs and biofilm formation (Baumgarten et al., 2012; Fulsundar et al., 2014). OMVs and the proteins they contain have been shown to play an important role in the biofilm formation of Helicobacter pylori (Yonezawa et al., 2009, 2011). Biofilm formation is characterized by the expression of genes responsible for exopolysaccharide production and co-aggregation of cells. OMVs were found to be involved in co-aggregation of cells (Grenier & Mayrand, 1987; Whitchurch et al., 2002; Kamaguchi et al., 2003; Inagaki et al., 2006; Ellis et al., 2010). Also, it has been suggested that OMVs provide a platform for the interactions of exopolysaccharides, DNA, proteins and the attachment surface, along with the bacterial cells (Schooling & Beveridge, 2006; Schooling et al., 2009). Although OMVs showed a role in biofilm formation, the detailed mechanism of their mode of action remains to be investigated. Delivery of biomolecules between cells OMVs, as described earlier in this review, encapsulate a variety of biomolecules. Horizontal transfer of genes among bacterial species has been attributed to OMVs (Yaron et al., 2000; Biller et al., 2014). The OMVs from Acinetobacter baylyi were found to transfer small DNA fragments to E. coli (Fulsundar et al., 2014). Transfer of autolysins to competing bacterial species (Li et al., 1998), and virulence factors or cytotoxins to host eukaryotic cells (Furuta et al., 2009a, b; Alaniz et al., 2007) have also been described. Haurat et al. (2011) showed that virulence factors are specifically sorted into OMVs. The first described function of OMVs is to provoke the mammalian immune system, as they deliver the antigenic OM components (De, 1959; Kondo et al., 1993), and LPS, which is pyrogenic, along with other endotoxins. Cell-free preparations of OMVs create infection-like symptoms in the host (Bauman & Kuehn, 2006; Ellis et al., 2010). Thus, OMVs seem to spearhead the infection process. The soluble luminal part of the OMV cargo is delivered to target cells by two mechanisms. First, the OMVs burst open or lyse in the vicinity of the target cells, shedding their contents at very high local concentrations. Second, the OMVs bind to the surface of target cells and deliver the content via proximal lysis, phagocytic internalization or membrane fusion (Kadurugamuwa & Beveridge, 1996). OMVs are known to be stable (Kadurugamuwa & Beveridge, 1999; Post et al., 2005), and there is almost no evidence for spontaneous lysis. Renelli et al. (2004) proposed that OMVs can open transiently, internalize DNA fragments from the medium and reseal. Although this does not give direct evidence for the spontaneous lysis theory of cargo transport, it shows that OMVs can open and reseal without external stimulus. Lysis of OMVs near the target site triggered by external stimuli has been demonstrated (Li et al., 1998). OMVs were found to lyse near Gram-positive cells, supposedly triggered by interaction of the OMVs with http://mic.sgmjournals.org the positively charged peptidoglycan, releasing the autolysins that kill these cells. In the same study, the OMVs were also found to kill Gramnegative cells, where they were found to transfer their contents to the cells by membrane fusion, highlighting the second proposed mechanism of transport. OMVs were also shown to transfer their contents to eukaryotic cells via membrane fusion (Kadurugamuwa & Beveridge, 1999). The particular fusion of these OMVs suggests a possible self–non-self distinguishing mechanism at work. OMVs are also known to take advantage of a natural process of endocytosis to deliver cargo to eukaryotic cells (Furuta et al., 2009a, b; Bomberger et al., 2009). This pathway does not depend upon the fusion of heterotypic membranes of OMVs and eukaryotic cells; rather, the OMVs enter into the target cells as a whole entity. In immune cells, the delivery of bacterial antigens to the antigen-presenting cells resulted in a display of a cohort of bacterial epitopes to the host immune system (Alaniz et al., 2007). The fusion of the OMVs with the eukaryotic cell membranes has also been shown by Bomberger et al. (2009), who used microscopic studies using fluorescently labelled OMVs. OMVs from enterotoxicogenic E. coli are known to elicit enterotoxic and proinflammatory responses (Kesty & Kuehn, 2004). The OMVs are known to transport cell surface attachment protein and haemolysin, which greatly increases the efficiency of haemolysis (Aldick et al., 2009). Killing of competing microbial cells OMVs produced from one bacterium can kill other competing microbes in the vicinity. The OMVs of various Gram-negative strains were found to kill many Grampositive and Gram-negative bacteria (Li et al., 1998). Their study investigated the effectiveness of OMVs in killing target bacteria with differing peptidoglycan chemotypes. Killing was most effective when the target possessed peptidoglycan that was similar to the OMV donor. The self–non-self recognition of cells by the OMVs may even be simpler. If the peptidoglycan hydrolases present in the OMVs are the same as those of the target strain then they cannot cleave the peptidoglycan layer. However, if they fuse with cells of a non-self strain, then they degrade the cell wall, killing the target cell (Beveridge, 1999). In a more recent study, Vasilyeva et al. (2008) found that Lysobacter sp. XL1 secreted bacteriolytic enzymes in OMVs. This activity of OMVs shows that they might be capable of distinguishing between self and non-self cells in a mixed community. The idea of using OMVs as a new antimicrobial agent has been explored. However, the process does not seem to be commercially feasible. It was found that OMVs can package antibiotics and deliver them at a very high concentration at the target (Kadurugamuwa & Beveridge, 1995). This phenomenon can be exploited to kill intracellular pathogens that are not reached by the antibiotics in external body fluids (Kadurugamuwa et al., 1998). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 2115 H. M. Kulkarni and M. V. Jagannadham Response to physical and chemical stress As discussed previously, bacteria tend to produce more OMVs as a response to stress (McBroom et al., 2006). It was also shown that hyper-vesiculating mutants were better able to adapt to stress conditions (Kulp & Kuehn, 2010; Manning & Kuehn, 2011). During temperature stress, OMVs were found to remove the misfolded proteins (Baumgarten et al., 2012). In cells exposed to antibiotics, OMVs were found to sequester (Manning & Kuehn, 2011) or to degrade (Ciofu et al., 2000) the antibiotics. When stress is induced by antibodies or bacteriophages, OMVs can act as decoy targets protecting cells by titration of the antibodies or phages (Manning & Kuehn, 2011). The production of OMVs is also found to increase with nutrient stress, which shows that OMVs have a role in bacterial nutrition (further explained in the next subsection). OMV production increased when the genes for the envelope stress response were mutated, showing that OMVs can compensate for the decreased stress handling by other pathways (McBroom et al., 2006). Nutrition of bacterial cells In natural bacterial environments, metal ions are typically scarce, leading to interspecies and intraspecies competition for them. OMVs are found to be enriched with these ions, and therefore are thought to collect and concentrate the rare ions for the consumption of bacterial cells (Kulp & Kuehn, 2010). Proteomics studies on different OMVs have shown the presence of different metal ion binding proteins, and the machinery for ATP synthesis (Lee et al., 2007). Enzymes found in OMVs may be involved in degrading complex biomolecules in the medium to make nutrients available. Biller et al. (2014) showed that, in marine bacteria, OMVs may be a significant player in the carbon flux between several species of bacteria and cyanobacteria. Thus, OMVs perform a role in intraspecies nutrient transfer. Defence and resistance A role for OMVs in the defence and resistance of bacteria is a recently discovered phenomenon. The production of OMVs requires resources, and the expenditure of biomolecules by bacteria would be justified if OMVs play a role in stress resistance. OMVs are used to remove misfolded proteins and toxins (McBroom & Kuehn, 2007). External stress factors initially impact the cell envelope. OMVs are able to remove agents that potentially harm the cell surface. For example, OMV production increases the survival of bacterial cells treated with lytic bacteriophages (Manning & Kuehn, 2011). It has been shown that, during phage adhesion, vesiculation increases and OMVs act as decoy targets for the phages (Loeb & Kilner, 1978; Manning & Kuehn, 2011). It was also shown recently that OMVs can alleviate the stress caused by membrane-targeted peptide antibiotics, such as polymyxin B, by acting as decoy targets, and transporting these molecules away from the cell (Manning & Kuehn, 2011). The absorption by OMVs of 2116 antibiotics and other molecules such as complement has also been shown (Tan et al., 2007). Inactivation of antibiotics by OMVs has been studied by Ciofu et al. (2000). The presence of various proteases, peptidases and enzymes such as b-lactamases has also been widely demonstrated in OMVs (Lee et al., 2007; Schaar et al., 2011, 2013). The OMVs produced by one bacterial species were found to protect other bacterial species from the antibiotic stress, emphasizing the ecological role of OMVs in microbial niches (Mashburn & Whiteley, 2005; Schaar et al., 2014; our unpublished data, see Fig. 1). Manning & Kuehn (2011) called OMVs the ‘innate defence’ of bacterial cells. The offensive and defensive roles of these ‘innate defence’ players have been described by MacDonald & Kuehn (2012). Conclusion and future directions OMVs seem to be fundamental for the survival of Gramnegative bacteria. Understanding the biogenesis of OMVs will allow us to engineer them for potential applications. To understand the genetic basis and the exact pathway of biogenesis of OMVs, a high-throughput approach should be applied. As described above, many genes are known to play an important role in increasing or decreasing the yield of OMVs. In a high-throughput approach, all the viable single gene mutants of a bacterial species should be examined for the production and yield of OMVs. Gramnegative bacteria may have 4000–6000 genes, and the biggest hurdle is the development of techniques that will enable the researcher to prepare and quantify OMVs from a large number of bacterial cultures. With the availability of the technologies required for investigation of genes one by one in a planned fashion in a short time, global study may be possible. The information obtained from such experiments will hopefully help to elucidate the genetic basis of OMV biogenesis. The organization and the quantity of OMVs are suspected to be responsive to the growth conditions, stress factors and growth phases of bacterial cultures. How some proteins are selectively sorted into OMVs is currently unknown. Quantitative proteomics studies of OMVs prepared from cultures grown under different conditions or of different mutants have not been undertaken. Information from such experiments would help to show how dynamic the contents of OMVs are, and, more importantly, what are the common minimum components of OMVs. Comparative proteomics of OMVs from mutants may provide hints about the genes and pathways that affect sorting of certain classes of proteins to OMVs. This would help in preparing engineered OMVs. Quantitative lipidomics is still an emerging field; it may prove useful in the study of OMVs as a complement to proteomic studies. The curvatures of the surface of OMVs, the properties of the OMV membrane and the factors which determine the size of OMVs have been studied only sporadically. The function of OMVs in consortia of different bacterial species is an emerging area of investigation. In a natural Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 Microbiology 160 Studies on outer membrane vesicles Outer leaflet (lipopolysaccharides) Inner leaflet (glycerophospholipids) OMVs interacting with: Membrane active peptides (a) (b) Membrane active peptide β -Lactam antibiotics β -Lactam antibiotic molecules (c) Bacteriophages β -Lactamasedegrading antibiotic molecules Bacteriophage Protease/ peptidase Nuclease Outer membrane porin Fig. 1. Schematic depiction of the role of OMVs as an innate defence of bacterial cells against antibacterials and other toxins. The mode of action of OMVs is depicted. (a) The interaction of membrane active peptides with OMVs. The OMVs can sequester or degrade membrane active peptides. (b) The degradation of the b-lactam antibiotics by b-lactamase in the lumen of OMVs. (c) The fate of bacteriophages, as they are either titrated away from bacteria or possibly inactivated by OMVs. niche, numerous bacterial species generally coexist and individuals are probably exposed to a pool of OMVs from a variety of other bacterial cells. OMVs have been described to kill competing microbes, as well as to protect members of the producer species. These contradictory roles of OMVs imply their multifaceted influence in bacterial ecology. Knowledge of the ecological role of OMVs may help the design of probiotic drugs to combat various bacterial pathogens. The preparation of vaccines using OMVs has been well studied. High-yield OMVs with consistent composition is a key factor in developing OMV-based vaccines. Some bacterial genera, such as Pseudomonas, are known to naturally produce higher yields of OMVs than others. Expressing antigenic proteins of interest in a species with higher yields of OMVs may be advantageous, provided the antigen is sorted into the OMVs. Engineered OMVs containing human proteins that act as sites for virus attachment can be used as decoy targets for human viral diseases. The viral particles may inject their genetic material into these decoy targets, which would degrade it by using bacterial nucleases in their lumen. There is a resurgence of classical bacterial diseases with a steep decline in the efficacy of antibiotics. New bacterial and viral diseases are emerging, for which rapid methods for vaccine development would be important. The application of OMVs holds some promise in this context. The availability of high-throughput ‘omics (genomics, proteomics, lipidomics) technologies, automation in microbiological http://mic.sgmjournals.org techniques and support from bioinformatics makes the exploration of this promise more practical. Apart from potential applications, the study of OMVs will also improve our overall understanding of bacterial physiology. Acknowledgements We thank DST, New Delhi, for the financial support to carry out the research work. H. M. K is a recipient of a CSIR senior research fellowship. References Aguilera, L., Toloza, L., Giménez, R., Odena, A., Oliveira, E., Aguilar, J., Badia, J. & Baldomà, L. (2014). Proteomic analysis of outer membrane vesicles from the probiotic strain Escherichia coli Nissle 1917. Proteomics 14, 222–229. Alaniz, R. C., Deatherage, B. L., Lara, J. C. & Cookson, B. T. (2007). Membrane vesicles are immunogenic facsimiles of Salmonella typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo. J Immunol 179, 7692–7701. Aldick, T., Bielaszewska, M., Uhlin, B. E., Humpf, H. U., Wai, S. N. & Karch, H. (2009). Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin. Mol Microbiol 71, 1496–1508. Altindis, E., Fu, Y. & Mekalanos, J. J. (2014). Proteomic analysis of Vibrio cholerae outer membrane vesicles. Proc Natl Acad Sci U S A 111, E1548–E1556. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 2117 H. M. Kulkarni and M. V. Jagannadham Aoki, M., Kondo, M., Nakatsuka, Y., Kawai, K. & Oshima, S. (2007). Stationary phase culture supernatant containing membrane vesicles induced immunity to rainbow trout Oncorhynchus mykiss fry syndrome. Vaccine 25, 561–569. Bauman, S. J. & Kuehn, M. J. (2006). Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response. Microbes Infect 8, 2400–2408. Baumgarten, T., Sperling, S., Seifert, J., von Bergen, M., Steiniger, F., Wick, L. Y. & Heipieper, H. J. (2012). Membrane vesicle formation as a multiple-stress response mechanism enhances Pseudomonas putida DOT-T1E cell surface hydrophobicity and biofilm formation. Appl Environ Microbiol 78, 6217–6224. Bendtsen, J. D., Kiemer, L., Fausbøll, A. & Brunak, S. (2005). Non- classical protein secretion in bacteria. BMC Microbiol 5, 58. Berlanda Scorza, F., Doro, F., Rodrı́guez-Ortega, M. J., Stella, M., Liberatori, S., Taddei, A. R., Serino, L., Gomes Moriel, D., Nesta, B. & other authors (2008). Proteomics characterization of outer mem- brane vesicles from the extraintestinal pathogenic Escherichia coli DtolR IHE3034 mutant. Mol Cell Proteomics 7, 473–485. engineered outer membrane vesicle vaccines. Proc Natl Acad Sci U S A 107, 3099–3104. Choi, D. S., Kim, D. K., Choi, S. J., Lee, J., Choi, J. P., Rho, S., Park, S. H., Kim, Y. K., Hwang, D. & Gho, Y. S. (2011). Proteomic analysis of outer membrane vesicles derived from Pseudomonas aeruginosa. Proteomics 11, 3424–3429. Chowdhury, C. & Jagannadham, M. V. (2013). Virulence factors are released in association with outer membrane vesicles of Pseudomonas syringae pv. tomato T1 during normal growth. Biochim Biophys Acta 1834, 231–239. Ciofu, O., Beveridge, T. J., Kadurugamuwa, J., Walther-Rasmussen, J. & Høiby, N. (2000). Chromosomal b-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa. J Antimicrob Chemother 45, 9–13. De, S. N. (1959). Enterotoxicity of bacteria-free culture-filtrate of Vibrio cholerae. Nature 183, 1533–1534. Deatherage, B. L., Lara, J. C., Bergsbaken, T., Rassoulian Barrett, S. L., Lara, S. & Cookson, B. T. (2009). Biogenesis of bacterial membrane vesicles. Mol Microbiol 72, 1395–1407. Berlanda Scorza, F., Colucci, A. M., Maggiore, L., Sanzone, S., Rossi, O., Ferlenghi, I., Pesce, I., Caboni, M., Norais, N. & other authors (2012). High yield production process for Shigella outer membrane membrane derived vesicles of Gram-negative but not Gram-positive bacteria. Appl Environ Microbiol 56, 1960–1962. particles. PLoS ONE 7, e35616. Dorward, D. W., Garon, C. F. & Judd, R. C. (1989). Export and Bernadac, A., Gavioli, M., Lazzaroni, J. C., Raina, S. & Lloubès, R. (1998). Escherichia coli tol-pal mutants form outer membrane vesicles. intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae. J Bacteriol 171, 2499–2505. J Bacteriol 180, 4872–4878. Eagon, R. G. & Carson, K. J. (1965). Lysis of cell walls and intact cells Beveridge, T. J. (1999). Structures of gram-negative cell walls and their derived membrane vesicles. J Bacteriol 181, 4725–4733. of Pseudomonas aeruginosa by ethylenediamine tetraacetic acid and by lysozyme. Can J Microbiol 11, 193–201. Beveridge, T. J., Makin, S. A., Kadurugamuwa, J. L. & Li, Z. (1997). Elhenawy, W., Debelyy, M. O. & Feldman, M. F. (2014). Preferential Interactions between biofilms and the environment. FEMS Microbiol Rev 20, 291–303. packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles. MBio 5, e00909–e00914. Biller, S. J., Schubotz, F., Roggensack, S. E., Thompson, A. W., Summons, R. E. & Chisholm, S. W. (2014). Bacterial vesicles in Ellis, T. N., Leiman, S. A. & Kuehn, M. J. (2010). Naturally produced marine ecosystems. Science 343, 183–186. Bishop, D. G. & Work, E. (1965). An extracellular glycolipid produced by Escherichia coli grown under lysine-limiting conditions. Biochem J 96, 567–576. Bodero, M. D., Pilonieta, M. C. & Munson, G. P. (2007). Repression of the inner membrane lipoprotein NlpA by Rns in enterotoxigenic Escherichia coli. J Bacteriol 189, 1627–1632. Bomberger, J. M., MacEachran, D. P., Coutermarsh, B. A., Ye, S., O’Toole, G. A. & Stanton, B. A. (2009). Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles. PLoS Pathog 5, e1000382. Boutriau, D., Poolman, J., Borrow, R., Findlow, J., Domingo, J. D., Puig-Barbera, J., Baldó, J. M., Planelles, V., Jubert, A. & other authors (2007). Immunogenicity and safety of three doses of a bivalent (B : 4:p1.19,15 and B : 4:p1.7-2,4) meningococcal outer membrane vesicle vaccine in healthy adolescents. Clin Vaccine Immunol 14, 65–73. Burdett, I. D. & Murray, R. G. (1974). Electron microscope study of septum formation in Escherichia coli strains B and B-r during synchronous growth. J Bacteriol 119, 1039–1056. Chatterjee, D. & Chaudhuri, K. (2011). Association of cholera toxin with Vibrio cholerae outer membrane vesicles which are internalized by human intestinal epithelial cells. FEBS Lett 585, 1357–1362. Chatterjee, S. N. & Das, J. (1967). Electron microscopic observations on the excretion of cell-wall material by Vibrio cholerae. J Gen Microbiol 49, 1–11. Chen, D. J., Osterrieder, N., Metzger, S. M., Buckles, E., Doody, A. M., DeLisa, M. P. & Putnam, D. (2010). Delivery of foreign antigens by 2118 Dorward, D. W. & Garon, C. F. (1990). DNA is packaged within outer membrane vesicles from Pseudomonas aeruginosa elicit a potent innate immune response via combined sensing of both lipopolysaccharide and protein components. Infect Immun 78, 3822–3831. Ferrari, G., Garaguso, I., Adu-Bobie, J., Doro, F., Taddei, A. R., Biolchi, A., Brunelli, B., Giuliani, M. M., Pizza, M. & other authors (2006). Outer membrane vesicles from group B Neisseria meningitidis Dgna33 mutant: proteomic and immunological comparison with detergent-derived outer membrane vesicles. Proteomics 6, 1856–1866. Findlow, J., Taylor, S., Aase, A., Horton, R., Heyderman, R., Southern, J., Andrews, N., Barchha, R., Harrison, E. & other authors (2006). Comparison and correlation of Neisseria meningitidis serogroup B immunologic assay results and human antibody responses following three doses of the Norwegian meningococcal outer membrane vesicle vaccine MenBvac. Infect Immun 74, 4557–4565. Fulsundar, S., Harms, K., Flaten, G. E., Johnsen, P. J., Chopade, B. A. & Nielsen, K. M. (2014). Gene transfer potential of outer membrane vesicles of Acinetobacter baylyi and effects of stress on vesiculation. Appl Environ Microbiol 80, 3469–3483. Furuta, N., Tsuda, K., Omori, H., Yoshimori, T., Yoshimura, F. & Amano, A. (2009a). Porphyromonas gingivalis outer membrane vesicles enter human epithelial cells via an endocytic pathway and are sorted to lysosomal compartments. Infect Immun 77, 4187–4196. Furuta, N., Takeuchi, H. & Amano, A. (2009b). Entry of Porphy- romonas gingivalis outer membrane vesicles into epithelial cells causes cellular functional impairment. Infect Immun 77, 4761–4770. Galka, F., Wai, S. N., Kusch, H., Engelmann, S., Hecker, M., Schmeck, B., Hippenstiel, S., Uhlin, B. E. & Steinert, M. (2008). Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles. Infect Immun 76, 1825–1836. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 Microbiology 160 Studies on outer membrane vesicles Graham, T. R. & Kozlov, M. M. (2010). Interplay of proteins and lipids in generating membrane curvature. Curr Opin Cell Biol 22, 430–436. Grenier, D. & Mayrand, D. (1987). Functional characterization of extracellular vesicles produced by Bacteroides gingivalis. Infect Immun 55, 111–117. Gu, X. X. & Tsai, C. M. (1991). Purification of rough-type lipopolysaccharides of Neisseria meningitidis from cells and outer membrane vesicles in spent media. Anal Biochem 196, 311–318. development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles. J Proteome Res 9, 5197–5208. Kamaguchi, A., Ohyama, T., Sakai, E., Nakamura, R., Watanabe, T., Baba, H. & Nakayama, K. (2003). Adhesins encoded by the gingipain genes of Porphyromonas gingivalis are responsible for co-aggregation with Prevotella intermedia. Microbiology 149, 1257–1264. Kato, S., Kowashi, Y. & Demuth, D. R. (2002). Outer membrane-like Gurung, M., Moon, D. C., Choi, C. W., Lee, J. H., Bae, Y. C., Kim, J., Lee, Y. C., Seol, S. Y., Cho, D. T. & other authors (2011). vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin. Microb Pathog 32, 1–13. Staphylococcus aureus produces membrane-derived vesicles that induce host cell death. PLoS ONE 6, e27958. Keenan, J. I., Rijpkema, S. G., Durrani, Z. & Roake, J. A. (2003). Haurat, M. F., Aduse-Opoku, J., Rangarajan, M., Dorobantu, L., Gray, M. R., Curtis, M. A. & Feldman, M. F. (2011). Selective sorting of cargo proteins into bacterial membrane vesicles. J Biol Chem 286, 1269–1276. Hayashi, J., Hamada, N. & Kuramitsu, H. K. (2002). The autolysin of Porphyromonas gingivalis is involved in outer membrane vesicle release. FEMS Microbiol Lett 216, 217–222. Hoekstra, D., van der Laan, J. W., de Leij, L. & Witholt, B. (1976). Differences in immunogenicity and protection in mice and guinea pigs following intranasal immunization with Helicobacter pylori outer membrane antigens. FEMS Immunol Med Microbiol 36, 199–205. Kesty, N. C. & Kuehn, M. J. (2004). Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles. J Biol Chem 279, 2069–2076. Kitagawa, R., Takaya, A., Ohya, M., Mizunoe, Y., Takade, A., Yoshida, S., Isogai, E. & Yamamoto, T. (2010). Biogenesis of Salmonella Release of outer membrane fragments from normally growing Escherichia coli. Biochim Biophys Acta 455, 889–899. enterica serovar typhimurium membrane vesicles provoked by induction of PagC. J Bacteriol 192, 5645–5656. Horstman, A. L. & Kuehn, M. J. (2000). Enterotoxigenic Escherichia Kondo, K., Takade, A. & Amako, K. (1993). Release of the outer coli secretes active heat-labile enterotoxin via outer membrane vesicles. J Biol Chem 275, 12489–12496. membrane vesicles from Vibrio cholerae and Vibrio parahaemolyticus. Microbiol Immunol 37, 149–152. Inagaki, S., Onishi, S., Kuramitsu, H. K. & Sharma, A. (2006). Kuehn, M. J. & Kesty, N. C. (2005). Bacterial outer membrane vesicles Porphyromonas gingivalis vesicles enhance attachment, and the leucine-rich repeat BspA protein is required for invasion of epithelial cells by ‘‘Tannerella forsythia’’. Infect Immun 74, 5023–5028. Kulkarni, H. M., Swamy, ChV. & Jagannadham, M. V. (2014). Jang, K. S., Sweredoski, M. J., Graham, R. L., Hess, S. & Clemons, W. M., Jr (2014). Comprehensive proteomic profiling of outer membrane vesicles from Campylobacter jejuni. J Proteomics 98, 90–98. Jin, J. S., Kwon, S. O., Moon, D. C., Gurung, M., Lee, J. H., Kim, S. I. & Lee, J. C. (2011). Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles. PLoS ONE 6, e17027. Kadurugamuwa, J. L. & Beveridge, T. J. (1995). Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion. J Bacteriol 177, 3998–4008. Kadurugamuwa, J. L. & Beveridge, T. J. (1996). Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics. J Bacteriol 178, 2767–2774. Kadurugamuwa, J. L. & Beveridge, T. J. (1997). Natural release of virulence factors in membrane vesicles by Pseudomonas aeruginosa and the effect of aminoglycoside antibiotics on their release. J Antimicrob Chemother 40, 615–621. Kadurugamuwa, J. L. & Beveridge, T. J. (1999). Membrane vesicles derived from Pseudomonas aeruginosa and Shigella flexneri can be integrated into the surfaces of other gram-negative bacteria. Microbiology 145, 2051–2060. and the host–pathogen interaction. Genes Dev 19, 2645–2655. Molecular characterization and functional analysis of outer membrane vesicles from the Antarctic bacterium Pseudomonas syringae suggest a possible response to environmental conditions. J Proteome Res 13, 1345–1358. Kulp, A. & Kuehn, M. J. (2010). Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol 64, 163–184. Kwon, S. O., Gho, Y. S., Lee, J. C. & Kim, S. I. (2009). Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate. FEMS Microbiol Lett 297, 150–156. Lee, E. Y., Bang, J. Y., Park, G. W., Choi, D. S., Kang, J. S., Kim, H. J., Park, K. S., Lee, J. O., Kim, Y. K. & other authors (2007). Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli. Proteomics 7, 3143–3153. Lee, E. Y., Choi, D. S., Kim, K. P. & Gho, Y. S. (2008). Proteomics in gram-negative bacterial outer membrane vesicles. Mass Spectrom Rev 27, 535–555. Lee, J. C., Lee, E. J., Lee, J. H., Jun, S. H., Choi, C. W., Kim, S. I., Kang, S. S. & Hyun, S. (2012). Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response. FEMS Microbiol Lett 331, 17–24. Li, Z., Clarke, A. J. & Beveridge, T. J. (1996). A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division and secretion in surface membrane vesicles. J Bacteriol 178, 2479–2488. Kadurugamuwa, J. L., Mayer, A., Messner, P., Sára, M., Sleytr, U. B. & Beveridge, T. J. (1998). S-layered Aneurinibacillus and Bacillus Li, Z., Clarke, A. J. & Beveridge, T. J. (1998). Gram-negative bacteria spp. are susceptible to the lytic action of Pseudomonas aeruginosa membrane vesicles. J Bacteriol 180, 2306–2311. produce membrane vesicles which are capable of killing other bacteria. J Bacteriol 180, 5478–5483. Kahn, M. E., Maul, G. & Goodgal, S. H. (1982). Possible mechanism Loeb, M. R. & Kilner, J. (1978). Release of a special fraction of the outer membrane from both growing and phage T4-infected Escherichia coli B. Biochim Biophys Acta 514, 117–127. for donor DNA binding and transport in Haemophilus. Proc Natl Acad Sci U S A 79, 6370–6374. Kahnt, J., Aguiluz, K., Koch, J., Treuner-Lange, A., Konovalova, A., Huntley, S., Hoppert, M., Søgaard-Andersen, L. & Hedderich, R. (2010). Profiling the outer membrane proteome during growth and http://mic.sgmjournals.org MacDonald, I. A. & Kuehn, M. J. (2012). Offense and defense: microbial membrane vesicles play both ways. Res Microbiol 163, 607– 618. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 2119 H. M. Kulkarni and M. V. Jagannadham MacDonald, I. A. & Kuehn, M. J. (2013). Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa. J Bacteriol 195, 2971–2981. Manabe, T., Kato, M., Ueno, T. & Kawasaki, K. (2013). Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110. Biochem Biophys Res Commun 441, 151– 156. Manning, A. J. & Kuehn, M. J. (2011). Contribution of bacterial outer membrane vesicles to innate bacterial defense. BMC Microbiol 11, 258. Manning, A. J. & Kuehn, M. J. (2013). Functional advantages conferred by extracellular prokaryotic membrane vesicles. J Mol Microbiol Biotechnol 23, 131–141. Maredia, R., Devineni, N., Lentz, P., Dallo, S. F., Yu, J., Guentzel, N., Chambers, J., Arulanandam, B., Haskins, W. E. & Weitao, T. (2012). Vesiculation from Pseudomonas aeruginosa under SOS. Burkholderia pseudomallei outer membrane vesicle vaccine provides protection against lethal sepsis. Clin Vaccine Immunol 21, 747– 754. Park, S. B., Jang, H. B., Nho, S. W., Cha, I. S., Hikima, J., Ohtani, M., Aoki, T. & Jung, T. S. (2011). Outer membrane vesicles as a candidate vaccine against edwardsiellosis. PLoS ONE 6, e17629. Pierson, T., Matrakas, D., Taylor, Y. U., Manyam, G., Morozov, V. N., Zhou, W. & van Hoek, M. L. (2011). Proteomic characterization and functional analysis of outer membrane vesicles of Francisella novicida suggests possible role in virulence and use as a vaccine. J Proteome Res 10, 954–967. Post, D. M., Zhang, D., Eastvold, J. S., Teghanemt, A., Gibson, B. W. & Weiss, J. P. (2005). Biochemical and functional characterization of membrane blebs purified from Neisseria meningitidis serogroup B. J Biol Chem 280, 38383–38394. ScientificWorldJournal 2012, 402919. Raymond, B. & Bonsall, M. B. (2013). Cooperation and the Marshall, A. J. & Piddock, L. J. (1994). Interaction of divalent cations, quinolones and bacteria. J Antimicrob Chemother 34, 465–483. evolutionary ecology of bacterial virulence: the Bacillus cereus group as a novel study system. Bioessays 35, 706–716. Martin, H. H., Heilmann, H. D. & Preusser, H. J. (1972). State of the Renelli, M., Matias, V., Lo, R. Y. & Beveridge, T. J. (2004). DNA- rigid-layer in celll walls of some gram-negative bacteria. Arch Mikrobiol 83, 332–346. containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential. Microbiology 150, 2161–2169. Mashburn, L. M. & Whiteley, M. (2005). Membrane vesicles traffic Sabra, W., Lünsdorf, H. & Zeng, A. P. (2003). Alterations in the signals and facilitate group activities in a prokaryote. Nature 437, 422–425. Mashburn-Warren, L. M. & Whiteley, M. (2006). Special delivery: vesicle trafficking in prokaryotes. Mol Microbiol 61, 839–846. Mashburn-Warren, L., Howe, J., Garidel, P., Richter, W., Steiniger, F., Roessle, M., Brandenburg, K. & Whiteley, M. (2008). Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation. Mol Microbiol 69, 491–502. Mashburn-Warren, L., Howe, J., Brandenburg, K. & Whiteley, M. (2009). Structural requirements of the Pseudomonas quinolone signal for membrane vesicle stimulation. J Bacteriol 191, 3411–3414. Mayrand, D. & Grenier, D. (1989). Biological activities of outer membrane vesicles. Can J Microbiol 35, 607–613. McBroom, A. J. & Kuehn, M. J. (2007). Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. Mol Microbiol 63, 545–558. McBroom, A. J., Johnson, A. P., Vemulapalli, S. & Kuehn, M. J. (2006). Outer membrane vesicle production by Escherichia coli is independent of membrane instability. J Bacteriol 188, 5385–5392. McCaig, W. D., Koller, A. & Thanassi, D. G. (2013). Production of outer membrane vesicles and outer membrane tubes by Francisella novicida. J Bacteriol 195, 1120–1132. Meadow, P. M., Wells, P. L., Salkinoja-Salonen, M. & Nurmiaho, E. L. (1978). The effect of lipopolysaccharide composition on the ultrastructure of Pseudomonas aeruginosa. J Gen Microbiol 105, 23–28. Mendez, J. A., Soares, N. C., Mateos, J., Gayoso, C., Rumbo, C., Aranda, J., Tomas, M. & Bou, G. (2012). Extracellular proteome of a formation of lipopolysaccharide and membrane vesicles on the surface of Pseudomonas aeruginosa PAO1 under oxygen stress conditions. Microbiology 149, 2789–2795. Sanchez, C. (2011). Cellular microbiology: bacterial pea shooters. Nat Rev Microbiol 9, 562. Schaar, V., Nordström, T., Mörgelin, M. & Riesbeck, K. (2011). Moraxella catarrhalis outer membrane vesicles carry b-lactamase and promote survival of Streptococcus pneumoniae and Haemophilus influenzae by inactivating amoxicillin. Antimicrob Agents Chemother 55, 3845–3853. Schaar, V., Paulsson, M., Mörgelin, M. & Riesbeck, K. (2013). Outer membrane vesicles shield Moraxella catarrhalis b-lactamase from neutralization by serum IgG. J Antimicrob Chemother 68, 593–600. Schaar, V., Uddbäck, I., Nordström, T. & Riesbeck, K. (2014). Group A streptococci are protected from amoxicillin-mediated killing by vesicles containing b-lactamase derived from Haemophilus influenzae. J Antimicrob Chemother 69, 117–120. Schertzer, J. W. & Whiteley, M. (2012). A bilayer-couple model of bacterial outer membrane vesicle biogenesis. MBio 3, e00297-11. Schild, S., Nelson, E. J., Bishop, A. L. & Camilli, A. (2009). Characterization of Vibrio cholerae outer membrane vesicles as a candidate vaccine for cholera. Infect Immun 77, 472–484. Schooling, S. R. & Beveridge, T. J. (2006). Membrane vesicles: an overlooked component of the matrices of biofilms. J Bacteriol 188, 5945–5957. Schooling, S. R., Hubley, A. & Beveridge, T. J. (2009). Interactions of DNA with biofilm-derived membrane vesicles. J Bacteriol 191, 4097– 4102. highly invasive multidrug-resistant clinical strain of Acinetobacter baumannii. J Proteome Res 11, 5678–5694. Schwechheimer, C., Sullivan, C. J. & Kuehn, M. J. (2013). Envelope Mullaney, E., Brown, P. A., Smith, S. M., Botting, C. H., Yamaoka, Y. Y., Terres, A. M., Kelleher, D. P. & Windle, H. J. (2009). Proteomic and control of outer membrane vesicle production in Gram-negative bacteria. Biochemistry 52, 3031–3040. functional characterization of the outer membrane vesicles from the gastric pathogen Helicobacter pylori. Proteomics Clin Appl 3, 785–796. Shetty, A., Chen, S., Tocheva, E. I., Jensen, G. J. & Hickey, W. J. (2011). Nanopods: a new bacterial structure and mechanism for Nevot, M., Deroncelé, V., Messner, P., Guinea, J. & Mercadé, E. (2006). Characterization of outer membrane vesicles released by the deployment of outer membrane vesicles. PLoS ONE 6, e20725. Smit, J., Kamio, Y. & Nikaido, H. (1975). Outer membrane of psychrotolerant bacterium Pseudoalteromonas antarctica NF3. Environ Microbiol 8, 1523–1533. Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J Bacteriol 124, 942–958. Nieves, W., Petersen, H., Judy, B. M., Blumentritt, C. A., RussellLodrigue, K., Roy, C. J., Torres, A. G. & Morici, L. A. (2014). A Spitzer, J. & Poolman, B. (2013). How crowded is the prokaryotic 2120 cytoplasm? FEBS Lett 587, 2094–2098. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 Microbiology 160 Studies on outer membrane vesicles Tan, T. T., Morgelin, M., Forsgren, A. & Riesbeck, K. (2007). Haemophilus influenzae survival during complement-mediated attacks is promoted by Moraxella catarrhalis outer membrane vesicles. J Infect Dis 195, 1661–1670. Tashiro, Y., Toyofuku, M., Nakajima-Kambe, T., Uchiyama, H. & Nomura, N. (2010). Bicyclic compounds repress membrane vesicle production and Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa. FEMS Microbiol Lett 304, 123–130. Tashiro, Y., Inagaki, A., Shimizu, M., Ichikawa, S., Takaya, N., Nakajima-Kambe, T., Uchiyama, H. & Nomura, N. (2011). Characterization of phospholipids in membrane vesicles derived from Pseudomonas aeruginosa. Biosci Biotechnol Biochem 75, 605–607. Tashiro, Y., Uchiyama, H. & Nomura, N. (2012). Multifunctional membrane vesicles in Pseudomonas aeruginosa. Environ Microbiol 14, 1349–1362. Toyofuku, M., Roschitzki, B., Riedel, K. & Eberl, L. (2012). Identification of proteins associated with the Pseudomonas aeruginosa biofilm extracellular matrix. J Proteome Res 11, 4906–4915. van de Waterbeemd, B., Mommen, G. P., Pennings, J. L., Eppink, M. H., Wijffels, R. H., van der Pol, L. A. & de Jong, A. P. (2013). Quantitative proteomics reveals distinct differences in the protein content of outer membrane vesicle vaccines. J Proteome Res 12, 1898–1908. Vasilyeva, N. V., Tsfasman, I. M., Suzina, N. E., Stepnaya, O. A. & Kulaev, I. S. (2008). Secretion of bacteriolytic endopeptidase L5 of Lysobacter sp. XL1 into the medium by means of outer membrane vesicles. FEBS J 275, 3827–3835. Veith, P. D., Chen, Y. Y., Gorasia, D. G., Chen, D., Glew, M. D., O’Brien-Simpson, N. M., Cecil, J. D., Holden, J. A. & Reynolds, E. C. (2014). Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors. J Proteome Res 13, 2420–2432. Wensink, J. & Witholt, B. (1981). Outer-membrane vesicles released by normally growing Escherichia coli contain very little lipoprotein. Eur J Biochem 116, 331–335. Whitchurch, C. B., Erova, T. E., Emery, J. A., Sargent, J. L., Harris, J. M., Semmler, A. B., Young, M. D., Mattick, J. S. & Wozniak, D. J. http://mic.sgmjournals.org (2002). Phosphorylation of the Pseudomonas aeruginosa response regulator AlgR is essential for type IV fimbria-mediated twitching motility. J Bacteriol 184, 4544–4554. Williams, J. N., Skipp, P. J., Humphries, H. E., Christodoulides, M., O’Connor, C. D. & Heckels, J. E. (2007). Proteomic analysis of outer membranes and vesicles from wild-type serogroup B Neisseria meningitidis and a lipopolysaccharide-deficient mutant. Infect Immun 75, 1364–1372. Work, E., Knox, K. W. & Vesk, M. (1966). The chemistry and electron microscopy of an extracellular lipopolysaccharide from Escherichia coli. Ann N Y Acad Sci 133, 438–449. Yaron, S., Kolling, G. L., Simon, L. & Matthews, K. R. (2000). Vesicle- mediated transfer of virulence genes from Escherichia coli O157 : H7 to other enteric bacteria. Appl Environ Microbiol 66, 4414–4420. Yonezawa, H., Osaki, T., Kurata, S., Fukuda, M., Kawakami, H., Ochiai, K., Hanawa, T. & Kamiya, S. (2009). Outer membrane vesicles of Helicobacter pylori TK1402 are involved in biofilm formation. BMC Microbiol 9, 197. Yonezawa, H., Osaki, T., Woo, T., Kurata, S., Zaman, C., Hojo, F., Hanawa, T., Kato, S. & Kamiya, S. (2011). Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori. Anaerobe 17, 388–390. Zhou, L., Srisatjaluk, R., Justus, D. E. & Doyle, R. J. (1998). On the origin of membrane vesicles in gram-negative bacteria. FEMS Microbiol Lett 163, 223–228. Zhu, W., Thomas, C. E., Chen, C. J., Van Dam, C. N., Johnston, R. E., Davis, N. L. & Sparling, P. F. (2005). Comparison of immune responses to gonococcal PorB delivered as outer membrane vesicles, recombinant protein, or Venezuelan equine encephalitis virus replicon particles. Infect Immun 73, 7558–7568. Zielke, R. A., Wierzbicki, I. H., Weber, J. V., Gafken, P. R. & Sikora, A. E. (2014). Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets. Mol Cell Proteomics 13, 1299–1317. Edited by: S. Spiro Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 09:31:15 2121