Study of OMVs reveal abundance of OM proteins (OMPs; OmpA, OmpC, and OmpF), periplasmic proteins (AcrA and alkaline phosphatase) and a series of virulence factors involved in the adhesion and invasion of host tissues. With advancements in the MS-based proteomic profiling technologies, identification of more than 3500 proteins – belonging to diverse functional categories – became possible (Kim et al., 2013). Evidence suggests that protein cellular localization greatly affects its inclusion to OMVs, as observed for H. pylori and Serratia marcescens (Olofsson et al., 2010; McMahon et al., 2012). Periplasmic proteins associated with the inner leaflet of OM exhibit increased incorporation within OMVs, in comparison with the proteins that are tightly bound to inner membrane. Although majority of OMPs from H. pylori load as cargo to OMVs, an anomalous behavior in cargo loading behavior in S. marcescens is also observed (Olofsson et al., 2010; McMahon et al., 2012). In S. marcescens, proteins such as Omps, maltoporin, and TolC, that show abundance on OM, goes undetected in OMVs. Failure in export of MipA in S. marcescens, as a part of OMVs and vice versa, goes unnoticed in the OM (McMahon et al., 2012). Although, actual mechanism for this mysterious behavior remains elusive, the decision of secretion or cellular retention is believed to occur via a series of protein and lipid recruitment factors. Contrastingly, shiga toxin of S. dysenteriae promotes its secretion by enhancing OMV production (Yokoyama et al., 2000). Upholding strain-specific characteristics, preferential packaging of proteins to OMVs is influenced by protein content of OMVs. As packaging process depends on the concentration ratio of OMV with its cellular contents, enrichment of proteins to OMVs occurs when protein contents of OMVs, that are normalized to OM, are significantly higher with respect to their cellular concentration (Bonnington and Kuehn, 2014).

Lipids represent important structural components of Gram-negative OMVs. Sharing similarity with OM (Chowdhury and Jagannadham, 2013; Kulkarni and Jagannadham, 2014), there are reports of some lipids being present in OMVs but not as part of OM (Kato et al., 2002). Horstman and Kuehn (2000) reported that enterotoxigenic E. coli OMVs have glycerophospholipids, phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin as its major lipids, which are associated with the curvature of OMVs (Horstman and Kuehn, 2000). Chowdhury and Jagannadham, (2013) revealed fine structural characterization of OMVs while performing mass spectrometric (MS) studies on P. syringae (Chowdhury and Jagannadham, 2013). They found, phosphatidylglycerol and phosphatidylethanolamine are the major lipid components in the OMVs. Similarly, other studies also reveal that phosphatidylglycerols are major constituents in OMVs, however, it was also seen that phosphatidylethanolamines compose the major portion of OM (Tashiro et al., 2011). Moreover, higher proportion of saturated fatty acids in OMVs attributes them with a rigid structure.

Performing functions of adhesion in biofilms, LPS (characteristic of Gram negative OM) are also present as component to OMVs. Similar to proteins, only a fractional amount of parent LPS are present in the OMVs (Kulkarni and Jagannadham, 2014). Expressing two types of O-antigen side chains, P. aeruginosa show enrichment of negatively charged B-band forms of LPS in OMVs, compared to neutral A-band (Kadurugamuwa and Beveridge, 1995). Enrichment of OMVs with B-band LPS is believed to be the consequence of the charge repulsive forces between adjacent B-band molecules in OM. Contrastingly, enrichment with A-band LPS of OMVs has been observed in the dental pathogen, Porphyromonas gingivalis (Haurat et al., 2011).

Outer Membrane Vesicles carry both luminal and surface associated DNA. A clear distinction between them arises on DNase treatment; luminal DNA being resistant persists even after the treatment process (Renelli et al., 2004). With this, several different forms of luminal DNA have been reported from E. coli, N. gonorrhoeae, P. aeruginosa, and H. influenza (Dorward and Garon, 1989; Kolling and Matthews, 1999; Mashburn-Warren and Whiteley, 2006; Lee et al., 2008). In addition to DNA, RNA, plasmid and DNA of phage and chromosomal DNA, has also been reported in OMVs. Although actual mechanisms of nucleic acid incorporation remain unclear, but it is believed that nucleic acid incorporation into the interior of OMVs happens from the lysed remains of cells that are present in the milieu, during the process of biogenesis (Yaron et al., 2000; Renelli et al., 2004).

Sorting of individual cellular components and their loading as cargo, controls composition and distribution of OMVs, and as such their specificity in counteracting the immune defense system of host. Acting as a signature to bacterial fitness, characteristic enrichment of proteins, lipids and nucleic acids to lumen or localization to OMV membrane, sheds light on the physiological functioning of OMVs and their association in increasing the bacterial survivability in their niche.

Differences in peptidoglycan composition makes bacteria prone to death by OMVs. The killing effect is prominent for bacterial types that show similarity in peptidoglycan composition, as that of OMV donors (Kulkarni and Jagannadham, 2014). Neutralization of bacteria is compromised due to the presence of similar degradative enzymes in OMVs and bacteria, making bacteria less susceptible to degradation. Moreover, fusion of OMVs with a non-self-strain increases their susceptibility toward degradative enzyme machinery (Beveridge, 1999). The enzyme cargo of OMVs administers bacteria with the capability of making distinction between self and non-self populations, enabling target specific killing of non-self-bacteria (Vasilyeva et al., 2008). An excellent example of this system is operational in the Lysobacter sp. – that secretes endopeptidase L5 – which is capable of degrading competing Gram-negative bacteria (Vasilyeva et al., 2008). A similar mechanism also operates for PG hydrolases. OMVs that possess PG hydrolases produce extermination effects after making a clear distinction for non-self-bacteria (Kadurugamuwa and Beveridge, 1996; MacDonald and Kuehn, 2012).

Of the versatile roles, packaging of enzymes such as proteases and glycosidases as cargoes to OMVs, plays a prominent role in the acquisition of nutrients for bacterial communities. OMV associated DNA and proteins function as a source of carbon and nitrogen during the bacterial growth. OMVs of Myxococcus xanthus carry alkaline phosphatase, which upon act on competitive bacteria cause release of phosphate that promotes development of multicellular community (Evans et al., 2012; Berleman et al., 2014). Phosphoenolpyruvate, a catalytic product of OMVs carrying enolase, converts plasminogen to plasmin. Phosphoenolpyruvate also aids in the bacterial colonization of the host, following the degradation of matrix proteins (Toledo et al., 2012). The scarcity of metal ions in the bacterial habitats leads to inter- and intra-species competition (Kulp and Kuehn, 2010). In addition to serving as an arsenal in interspecies competition, enrichment of OMVs with rare metal ions makes them available for easy disposal to microbial use.

Mutation in the stress responsive genes increases production of OMVs in the bacteria. Exposure of cells to environmental contaminants (antibiotics) has potentially evolved bacterial OMVs, either with multidrug efflux pumps capabilities or with ability to catalyze degradation by sequestering antibiotics from the extracellular milieu (Ciofu et al., 2000; Manning and Kuehn, 2011). Acting as nutrient sensors and transporters of essential molecules, enhanced expression of surface receptors and ABC transporter in OMVs increases bacterial survival. The release of unfolded and misfolded proteins to OMVs gives an impression of its development, by sensing a stress responsive state (Baumgarten et al., 2012).

Biofilms are surface adhering structures produced in response to stress by bacteria (Kulp and Kuehn, 2010; Manning and Kuehn, 2011). Matrix assisted biofilms contain polysaccharides, lipids, nucleic acids and protein entities like flagella, pili and OMVs. OMVs mediate delivery of growth factors and components of the extracellular matrix (Schooling and Beveridge, 2006; Klimentova and Stulik, 2015). Release of exopolysaccharides through OMVs increases co-aggregation of cells in the biofilms. Shift from free living to the sedentary state of the bacterial population in biofilms protects cells from desiccation, starvation and adverse effects of antimicrobial agents (Klimentova and Stulik, 2015). Production of OMV helps bacteria to overcome the effects of antimicrobial peptides. Acting as carriage of resistant determinants like β-lactams, and enzymes such as protease, endopeptidases, etc., OMVs give survival advantage to bacteria due to antibiotic resistance traits via biofilms, thereby protects bacteria from antibiotic carnage (Beveridge et al., 1997). Represented as decoy entities, vesicle associated multidrug efflux pumps contribute in transient survival of susceptible bacteria in their surroundings. Association of OMVs with the biofilms of P. aeruginosa has been already reported (Beveridge et al. (1997), subsequent studies suggest intimidate relation between stress and the production of OMV on observing increase in OMV production during stress conditions (Beveridge et al., 1997; Baumgarten et al., 2012; Fulsundar et al., 2014).

Interaction of bacteria with host triggers release of OMVs, carrying proteins (OspA and OspB in B. burgdorferi) and other adhesion molecules (SabA, BabA, and VacA in H. Pylori, UspA1 in M. catarrhalis and aminopeptidase in P. aeruginosa) on their surface (MacDonald and Kuehn, 2012). OMVs acting as a bridge to increase adhesion of bacteria with the host tissues. They are also known to increase adherence of bacteria with epithelial linings of gut or the respiratory tract which helps bacteria resist their physical elimination. Association of OMVs with pathogen associated molecular patterns (PAMPs) such as LPS and porins, mediates strong immune response in endothelial cells and induces expression of pattern recognizing receptors on macrophages (Ellis and Kuehn, 2010; Kim et al., 2013). OMVs associated toxins such as leukotoxin, LPS, and ClyA are more potent than their soluble forms (Wai et al., 2003; Kuehn and Kesty, 2005). Secretion of shiga toxin from enterohemorrhagic E. coli O157:H7 and stx1 and stx2 of S. dysenteriae as cargo to OMVs efficiently inhibits eukaryotic protein synthesis (Yokoyama et al., 2000; Dutta et al., 2004). OMVs harboring multiple virulence factors such as Cif, PlcH, LPS, and alkaline phosphatase produce pronounced effect on host cells (Bomberger et al., 2009; Ellis and Kuehn, 2010). Secretion of toxins and virulence factors help bacteria to invade host cells, hijack host machinery for nutrient acquisition and also evade host defense system by modulating host immune response, which is pivotal for their fecundity and survival in the host.

It is well established that OMVs have diverse biological functions i-e. from short range intercellular communications, to their long distance mode of action. They also perform immunomodulatory function by regulating delivery of different constituents to recipient cells (Corrado et al., 2013). Internalization of OMVs is achieved by four different pathways i-e. Macropinocytosis, Clathrin, Caveolin, and Lipid raft mediated endocytosis. Macropinocytosis is an actin driven process, which allows internalization of OMVs after sampling of extracellular medium at the cell surface. Macropinocytosis is reported from S. flexneri, where it helps to establish infection on host tissues (O’Donoghue and Krachler, 2016; Weiner et al., 2016). Clathrin mediated endocytosis via, clathrin coated pits occurs following ligand binding to cell surface receptor, triggering internalization of OVMs (Rewatkar et al., 2015). Unlike macropinocytosis that allows internalization of vesicles of 1 μm diameter, clathrin dependent endocytosis allows internalization of cargo, with a maximum of 120nm diameter. Clathrin mediated endocytosis is adapted for internalization of shiga toxin, CT, and cytotoxic virulence factor, VacA of H. pylori (Boisvert and Duncan, 2008; Parker et al., 2010). Caveolin mediated endocytosis occurs as cave shaped invagination (80nm) of membrane rich in cholesterol, sphingolipids, and caveolin. Despite sluggish internalization speed (five times less than clathrin mediated), it leads in the efficient delivery of cargo to the cytosol (Ritter et al., 1995; Mulcahy et al., 2014; Rewatkar et al., 2015). This type of transfer is utilized by H. influenza. Lipid raft (plasma membrane domains enriched in cholesterol and sphingolipids) mediated endocytosis is known for its involvement in the OMVs entry to host cells (Furuta et al., 2009; Jin et al., 2011; Sharpe et al., 2011; Mulcahy et al., 2014; Mondal et al., 2016). It is hypothesized that clustering of cholesterol rich regions causes curvature in membrane that drives movement of molecules as invagination (90nm) to host cell.