Mechanism of infection of a human isolate Salmonella (3,10:r:-) in chicken ileum: ultrastructural study.

Biophysics & Electron Microscopy Section, Indian Veterinary Research Institute, Izatnagar India.
The Indian Journal of Medical Research (Impact Factor: 1.66). 01/2008; 126(6):558-66.
Source: PubMed

ABSTRACT Originally isolated from severe human food-poisoning cases, Salmonella (3,10:r:-), a monophasic variety of otherwise diphasic serotypes such as S. weltevreden and S. simi, causes serious infections in man, animals and poultry. Mechanism of infection of this versatile and deadly organism is important to understand for its control. The objective of this study was to enhance our understanding of infection of Salmonella (3,10:r:-) in vivo at cellular level.
Aliquots of 10(9) cfu of Salmonella (3,10:r:-) organisms were injected intra-ileally in 24 h pre-fasted 3 month old broiler chickens by standard ligated ileal loop method. After 18 h, the fluid accumulated in the ileum was drained and small tissue pieces were fixed in 2.5 per cent buffered (pH 7) glutaraldehyde and subsequently in 1 per cent aqueous osmium tetraoxide. Ultra-thin sections of araldite-embedded tissue pieces were examined under transmission electron microscope operated at 100 KV after staining with uranyl acetate and lead citrate.
Over 70 per cent of salmonellae interacting within 300 nm with ileal epithelial cells developed numerous surface blebs of periplasmic extensions designated "periplasmic organelles" (POs). Large sized POs were apparently pinched off as outer membrane vesicles (OMVs), 50-90 nm in diameter. Type III secretion needle complex-like "rivet complexes" (RCs) were viewed to rivet the bacterial outer and inner membranes together, allowing only pockets of periplasm to expand/inflate in order to liberate OMVs. Many OMVs were found visibly docked on the plasma membrane of host epithelial cells. The invading organisms appeared to leave the epithelial cells so as to find entry into the lymphatic vessels, where, they again appeared to be closely interacting with ileal macrophages, by forming numerous POs and concomitantly liberating OMVs. Inside the cytoplasm of macrophages, numerous tight phagosomes were seen, each containing two organisms. The final stage appeared to contain replicated salmonellae, four in each loose phagosome and, at the same time, macrophages also showed signs of apoptotic disintegration, culminating in the release of replicated salmonellae.
Outer membrane vesicles released from a fiercely virulent human isolate, Salmonella 3,10:r:- pathogens have been implicated in translocating biochemical signals from the host-interactive organisms to the eukaryotic cells at both stages of invasion leading to epithelial cell and macrophage infection in vivo, in the chicken ileal model. A comprehensive cellular mechanism at ultrastructural level is outlined for typhoid-like Salmonella infections caused by this humans-infecting organism.


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    ABSTRACT: Research journal logo Membrane vesicle trafficking in prokaryotes: molecular biomechanics of biogenesis of outer membrane vesicles of gram negative (Salmonella 3,10:r:-) microbes in chicken ileal invasion model in vivo Rakesh C YashRoy (rakeshyashroy at gmail dot com) Formerly of Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India; Executive 9-D, Suncity Vistar, Bareilly, Uttar Pradesh 243001, India DOI Date 2014-10-29 Cite as Research 2014;1:1128 License CC-BY Abstract Introduction & Methods: Human isolate, Salmonella 3,10:r:- (a monophasic variety of the otherwise diphasic serotypes such as Salmonella weltevreden and Salmonella simi), known for causing serious food poisoning infections in man, animals and birds was introduced into chicken ileum by standard ligated ileal loop methodology, injecting an aliquot containing 109 cfu, in 24-hr prefasted 3-month old broiler chickens. Eighteen hours later, exudate/fluid that accumulated in the experimental and control loops was drained and ileal tissue pieces suitable fixed. Thereafter, ultrathin sections were examined by transmission electron microscopy to study ultrastructure of biogenesis of bacterial outer membrane vesicles at host-pathogen interface in vivo. Results: Treated ileal sections revealed altered surface ultra-structure of several interactive salmonellae, marked by the presence of numerous pockets or extensions of bacterial periplasm, which also appeared to be liberate 80-90 nm diameter bacterial outer membrane-bound spherical vesicles, in proximity of specialized protruding needle structures (about 100 nm in length) embedded in bacterial cell and outer membranes. A molecular biomechanical model for biogenesis of outer membrane vesicles (OMVs) in Salmonella organisms, which may also apply to other gram-negative bacteria, is proposed. This model involves a specialized role assigned to supramolecular protein rivet complexes (RCs), suggestively akin to type III secretion nano-machines. RCs are implicated in riveting together, the bacterial inner and outer membranes, thereby, mediating a ‘bubble-off’ process from pockets of extended bacterial periplasm as OMVs in analogy to soap bubble release with a ‘bubble tube’. At Stage I, heavy secretion of bacterial proteins via well-known general secretory pathway (accompanied by secretion of other virulence and allied factors), fills pockets of bacterial periplasm making them protrude out as periplasmic organelles (POs). This is aided by rivet complexes involved in situ riveting of bacterial cell and outer membranes together at the base of POs. Stage II: High concentration of solutes inside POs causes osmotic inflow of water to bring into action a turgor pressure resulting in gradual inflation of POs. Expansion of POs generates a stretch force along the extending bacterial outer membrane at their base, implicated in ‘pulling’ RCs together, as if ‘directing’ laterally diffusing RCs to mutually align into a bubble tube-like assembly. Stage III: Slight tilting of slender extending needle portions of RCs is allowed by existing smaller diameter of outer hollow rings vis a vis larger inner hollow rings of RCs, coming in physical contact by lateral diffusion. This narrows the ‘orifice’ at the level of tips of the optimally-extended slender needles, constituting the bubble tube assembly so as to pinch off an OMV from the inflated PO. Discussion & Conclusion: The observed process of trafficking of membrane vesicles from gram-negative micro-organisms (Salmonella) to animal host cells is being viewed as a novel cell-cell signaling process, for translocation of bacterial bio-chemicals to animal host or target cells in vivo. A molecular biomechanical model is presented here for OMV biogenesis and discussed in light of other models for this process available in current scientific literature. Introduction Gram negative bacterial outer membrane vesicles (OMVs) have been known to exist in bacterial cultures, for about forty-seven years [1]. However, their biological role remained unclear until they were implicated in translocating bacterial secretions to animal host cells in vivo [2]. Now, practically all gram negative organisms are known to secrete OMVs, and these have been assigned varied physiological functions, including virulence and immuno-modulatory roles during host-pathogen interactions [3]. In addition, OMVs have been implicated in intra- and inter-species bacterial signaling for symbiotic or competitive relationships. OMVs of coccobacillus, P. gingivalis and putative ‘outer sheath vesicles’ of helical-shaped spirochete T. denticola have been implicated in inter-species signaling for a metabolic symbiotic relationship between these two organisms, leading to enhanced synergistic virulence and co-infection into subgingival plaque formation in chronic periodontitis infections [4]. E. coli OMVs, whether from avirulent or pathogenic strains, are found to be potentially genotoxic, causing double-stranded DNA breaks producing multinucleate cells when internalized by Caco-2 cells [5]. From intracellular compartments within infected host cultured epithelial cells, Salmonella typhimurium releases lipopolysaccharide (LPS), which accumulates in host cell vesicles, suggestive of existence of a signaling mechanism from pathogen to host cells, before salmonellae start to proliferate [6]. Also, OMVs shed by intracellular Legionella pneumophila organisms from within the macrophage phagosomes, intercalate with phagosomal membrane to not only inhibit phagosome-lysosome fusion, but also promote remodeling of its surface to render it conducive for intracellular replication of the pathogen [7]. Biogenesis of OMVs of gram negative micro-organisms has also generated a keen interest of researchers. A N. meningitidis-infected septic-shock patient’s plasma also contained multiple bacterial outer membrane protrusions, suggesting involvement of such blebs in disease production in humans [8]. Similar structures were seen on surface of virulent Salmonella (3,10:r:-) organisms in experimental infection of chicken ileum and these bacterial outer membrane-bound protrusions were suggested to contain bacterial toxins [9]. Further electron microscopic studies suggested that these blebs/protrusions were precursors of 50-90 nm diameter spherical balls, now called bacterial outer membrane vesicles (OMVs). Further, these OMVs were also seen as apposed in a close ‘fusion-like’ membrane-to-membrane contact with chicken ileal epithelial cell microvilli, in vivo [2]. These surface protrusions/blebs were thus considered to be ‘extended storage-spaces’ and designated as ‘periplasmic organelles’ (POs). A structural model for these organelles, as well as for biogenesis of OMVs was also thus proposed, and likened to type III secretory system of gram-negative bacteria [10]. An earlier model for biogenesis of OMVs implicated few outer membrane lipoprotein (Lpp) linkages to underlying layers of bacterial cell wall but, however, without any electron microscopic evidence [11]. Wensink and Wikot [12] proposed that outer membrane growth exceeding that of peptidoglycan layer, causes OMV biogenesis. Complete loss of interaction between outer membrane protein A (OmpA) and peptidoglycan (PG) layer, enhances release of OMVs without loss of membrane integrity. In Salmonella, modulation of envelope proteins through gene regulation, was proposed to result in different ratios of interconnected proteins such as OmpA and Lpp, which can work as a tunable (with environmental pH, ionic strength, temperature, etc) system to stimulate OMV biogenesis [13]. Another study proposes a ‘bilayer-couple model’, in which, a bacterial signaling molecule, PQS (3-hydroxy-4-quinolone) stimulates OMV biogenesis through direct interaction with the outer leaflet of bacterial outer membrane. In this, low rate of inter-leaflet flip-flop forces PQS to accumulate in and expand the outer leaflet, relative to the inner leaflet, thereby, inducing a membrane curvature [14]. Biogenesis and multifaceted roles of OMVs from Gram-negative microbes has been recently reviewed [15]. The current report presents a comprehensive biomechanical model for biogenesis of gram negative bacterial outer membrane vesicles with special reference to Salmonella pathogens at the animal host-pathogen interface, invoking additional processes of membrane dynamics, lateral diffusion, turgor pressure, etc. Material & Methods The microbial pathogen, Salmonella 3,10:r:- (a monophasic variety of otherwise diphasic serotypes such as Salmonella weltvreden and Salmonella simi), originally isolated from human food poisoning cases, was obtained from National Salmonella Centre at the Indian Veterinary Research Institute, Bareilly, UP, India. This particular strain has been typed distinctly from Salmonella weltvreden [16] [17] and is maintained at this Centre. Cultures of the Salmonella 3,10:r:- organisms, originally isolated from cases of human food poisoning [3] with 109 cfu were injected into chicken ileum in 24 h pre-fasted 3 month old five broiler chickens (obtained from Central Avian Research Institute, Bareilly, UP, India and specifically maintained under suitable laboratory conditions), using standard ligated ileal loop methodology [18] [19]. Briefly, the procedures used employ thorough washing-out of the ileum of its contents (including naturally inhabiting organisms). In this procedure, the experimental (injected with the dose of organisms) and control ileal loops (injected with sterile medium without organisms) were located in the same animal(s) so as to allow for excellent control versus experimental sampling. A fluid was found to be exsorbed only in the experimental loops after 18 h of injection, and their contents were tested for presence of injected organism to make sure that reaction was indeed caused by Salmonella (3,10:r:-) pathogens. The fluid accumulated in the ileal loops was drained and ileal tissue pieces (size approximately 1-2 mm3) of the experimental and control loops were fixed in 2.5 per cent glutaraldehyde in phosphate buffer (pH 7) at 5 deg C for 6 h, and subsequently post-fixed in aqueous 1 per cent osmium tetroxide for 6 h at room temperature following standard methodology [20] [21]. The fixed tissue blocks were embeded to make in arlaldite blocks and ultra-thin sections (approximately 500 Ao in thickness) were cut using glass knife with an ultramicrotome (LKB Ultrotome III, Sweden). Ultra-thin araldite-embedded sections obtained on 3 mm diameter copper grids, were stained with uranyl acetate and lead citrate stains for contrast and examined under JEOL JEM 1200EX electron microscope (Japan) working in transmission mode operated at 100 kilovolts. [enlarge] Membrane vesicle trafficking in prokaryotes: molecular biomechanics of biogenesis of outer membrane vesicles of gram negative (Salmonella 3,10:r:-) microbes in chicken ileal invasion model in vivo figure 1 Figure 1. OMVs released from a cross-sectionally cut organism (Sal.) revealing a pair of ‘needle-like’ structures, proposed to rivet bacterial inner and outer membranes together and designated as rivet complexes. Two more of such rivet complexes are envisioned to be present at this location in the opposite plane (not seen), so as to constitute a functional ‘bubble tube’-like orientation to ‘pinch off’(see Figures 2 and 3) an outer membrane vesicle (OMV) from a protruded pocket of periplasm, designated periplasmic organelle (PO). Globular particles (suggestively, proteins) are discernibly present in OMV lumen, as well as in OMV membrane envelope. Scale bar, 200 nm. Results Figure 1 reveals in vivo needle-like structures called rivet complexes seen ‘anchored’ in the cell wall of the OMV-secreting Salmonella 3,10:r:- organisms, as revealed by transmission electron microscopy, 18-hr after experimental infection of chicken ileum in ligated ileal loop model. About 100 nm in length, each of these complexes appear to be embedded in bacterial outer and inner membranes, and thus they are thought to rivet the bacterial inner and outer membranes together, as if, to allow only pockets of periplasm to blow or bleb out as several periplasmic organelles (POs). Though, these needle complexes appear to be hollow, yet not wide-enough to allow globular proteins to pass through. However, granular/globular structures are seen both inside the OMVs, as well as in OMV-membrane. [enlarge] Membrane vesicle trafficking in prokaryotes: molecular biomechanics of biogenesis of outer membrane vesicles of gram negative (Salmonella 3,10:r:-) microbes in chicken ileal invasion model in vivo figure 2 Figure 2. Proposed molecular bio-mechanical model for release of outer membrane vesicles (OMVs) by Gram-negative micro-organisms. STAGE-I involves, the role of bacterial general secretory pathway in secretion of proteins across cell membrane into periplasmic space. Inflating periplasm thus blows out in pockets/blebs due to scattered presence of rivet complexes, riveting together bacterial inner and outer membranes. Lateral diffusion of riveting multi-protein complexes is envisaged to be directed inwards due to an expected biophysical stretch-force arising from elongation of bacterial outer membrane vis a vis stable peptidoglycan layer and cytoplasmic membrane. STAGE-II: Mounting turgor pressure due to water inflow caused by increasing solute concentration of secretory materials accumulating in expanding periplasmic pockets/periplasmic organelles ‘guides’ lateral diffusion of rivet complexes so as to align them parallel in analogy of a ‘bubble tube’, alongwith concomitant lengthening of the externally extendable needles of the rivet complexes (also, see ultra-structure in figure 1). Inner leaflets of tightly interdigitating outer membrane is expected to undergo lipid mixing in the needle zone of closely aligned rivet complexes, leading to sealing of vesicle being liberated. STAGE-III: The outer membrane vesicle (OMV) may be ‘pinched off’ due to forced narrowing of an orifice created at the level of tips of the ‘bubble tube’ assembly resulting from differential lateral diffusion of rivet complexes in bacterial inner and outer membranes, as further explained in Figure 3. Biomechanical model Figures 2 & 3 presents a biomechanical model for biogenesis of outer membrane vesicles of Gram negative micro-organisms based on ultra-structural information gathered from Salmonella-animal host interactions in vivo, as described above. However, current information from scientific literature on the ultra-structure of type III secretion system (T3SS) of protein secretion by gram negative microbes is being used for this model (references in Discussion). Close similarity of the structure of ‘needle-like’ rivet complexes of Salmonella 3,10:r:- organisms, shown in electron micrograph (Figure 1) and T3SS needle complexes seen in literature suggests that they may be very similar or even the same structures. The molecular biomechanical model (a) conceives a motional freedom allowing lateral diffusion of rivet complexes, in spite of their being embedded in both the cell membrane and the outer membrane of the gram negative microbes. Further, this lateral diffusion may not be totally random, in that it can be somewhat directed due to a presumed stretch force acting on stretched portions of bacterial outer membrane covering the fast inflating POs. (b) This model presumes that a system of general secretory pathway, which is present in the bacterial cell membrane, secretes proteins, which are synthesized in the bacterial cytoplasm, into the periplasm of gram negative microbes. (c) This model conceives existence of a turgor pressure being exerted on portions of membrane surrounding periplasmic blebs, effecting water-inflow, to osmotically compensate for increasing solute concentration in POs due to active secretion of secretory products across the bacterial cell membrane into periplasm. (d) This model also foresees the likelihood of stimulated synthesis of bacterial secretory products with the role of internal or external inducers like change in temperature, pH, ionic composition, host’s own or environmental signals. [enlarge] Membrane vesicle trafficking in prokaryotes: molecular biomechanics of biogenesis of outer membrane vesicles of gram negative (Salmonella 3,10:r:-) microbes in chicken ileal invasion model in vivo figure 3 Figure 3. Molecular biomechanics of Gram negative bacterial outer membrane vesicle pinch off (diagrammatic view). Rivet Complexes (RCs) of the gram negative bacteria are proposed to be equivalent to multi-protein needle complexes or syringes of Type 3 secretory system (T3SS) of these microbes for protein secretion targeted to be delivered directly into the cytosol of host/target cell. They consist of a four-ring hollow connected by a middle cylindrical base: the upper small pair of rings is embedded in outer membrane and the large lower pair of rings is embedded in bacterial cell membrane. These pairs of rings are thus proposed to rivet the bacterial outer and inner membranes together, linked by middle hollow cylindrical base and terminating in an extendable slender needle. Due to inwardly directed lateral diffusion (explained in Figure 2), four larger rings are proposed to impede further lateral movement due to their coming in physical contact with one another in the bacterial cell membrane, whereas, the upper smaller rings allow further inward diffusional movement of RCs within the bacterial outer membrane. The four tips of the extendable needles thus tilt inwards to cut the fused outer membrane so as to pinch off an outer membrane vesicle, at the level of a narrowing orifice created by assembly of needle tips. Figure 2 shows a proposed flow diagram of steps (stages I to III) of molecular biomechanics of OMV biogenesis. STAGE-I implicates the role of bacterial general secretory pathway (GSP) in secretion of proteins across the bacterial cell membrane into periplasmic space. Inflating periplasm thus ‘bloats out’ blebs of POs in pockets studded by random presence of supra-molecular RCs, riveting together bacterial inner and outer membranes. Lateral diffusion of riveting multi-protein complexes in the two membrane is proposedly directed inwards due to an expected biophysical stretch-force arising from elongation-stretch of bacterial outer membrane vis a vis stable peptidoglycan layer and cytoplasmic membrane. STAGE-II: Building turgor pressure due to water inflow caused by increased solute concentration of secretory materials accumulating in expanding periplasmic pockets/POs, sort of ‘guides’ lateral diffusion of RCs so as to align them parallel in analogy of a ‘bubble tube’, with concomitant lengthening of the externally extendable needles of the rivet complexes ( ultra-structure in Figure 1). Inner leaflets of tightly interdigitating outer membrane is expected to result in membrane lipid mixing in the needle zone of closely aligned rivet complexes, thereby sealing the vesicle being liberated. STAGE-III: Outer membrane vesicle (OMV) may be ‘pinched off’ due to inward tilting of extended slender needles and forced narrowing of the orifice at the tip of the ‘bubble tube’ due to differential lateral diffusion of rivet complexes in bacterial inner and outer membranes, as explained in Figures 2 and 3. Figure 3 explains the proposed detailed biomechanics of how gram negative bacterial OMVs can be released or pinched off the secretion-active organisms. Each rivet complex, when fully formed is presumed to be composed of four ring hollows connected by a single cylindrical base, on the lines of T3SS needle complexes, already known for gram negative organisms, in general. The pair of hollow rings embedded in bacterial outer membrane is smaller in diameter than the pair of hollow rings embedded in bacterial cell membrane. Expansion of pocket-like periplasmic organelles (PO), is thought to stretch the influenced portion of the bacterial outer membrane and this ‘stretch-force’ is presumed to draw the rivet complexes at the base of POs closer (from stage I to stage II in figure 2), as allowed by well-established phenomenon of lateral diffusion of proteins in fluid mosaic model of membranes. While the extendable needle elongates with a single constituent protein ‘threading out’ of the central hollow conduit in the RCs to an optimal length, the multi-protein complexes (RCs) move by lateral diffusion, as if directed by a force arising from stretching of outer membrane, driven by turgor pressure exerted on the inflating periplasmic organelles (study changes between stage I and II, figure 2). Roughly, four RCs may cooperate in impeding such a stretch force-directed lateral diffusional movement any further, with their coming in direct physical contact at the level of the approaching larger hollow rings embedded within the bacterial cell membrane. However, upper smaller hollow rings embedded in bacterial outer membrane may continue their further movement thus closing up to narrow the orifice at the level of external tips the four ‘tilting’ slender needles of the ‘bubble tube’, so as to pinch off the PO as an OMV (stage III, Figure 2; ultrastructure in Figure 1), as detailed in Figure 3. Discussion Gram negative micro-organisms differ from their gram-positive counterparts in their cell surface presentation to outside. Gram-negative microbes contain an extra compartment known as periplasm, which is bounded by an additional membrane called outer membrane. Periplasm, thus provides an extra space for storage of bacterial secretions, outside the cell membrane and its adjoining peptidoglycan layer of gram negative organisms. Since year 1967, gram negative microbes in cultures, have been known to release nano-scale spherical vesicles bounded by bacterial outer membrane [1]. Current literature uses the term, bacterial outer membrane vesicles, for these structures, which are found to be released by all known gram negative micro-organisms [15]. Although, these bacterial OMVs have been implicated in inter-bacterial, inter-species and even inter-kingdom cell-cell signaling, this paper discusses about their biogenesis inside animal host during infection in vivo. OMVs are liberated by secretion-active Salmonella 3,10:r:- organisms during invasion of chicken ileum [2] [39]. Figure 1 shows the presence of two ‘needle-like’ structures, about 100 nm in length, located close to a section micro-location where OMVs appear to be liberated from Salmonella pathogens, at the host-pathogen interface. As these OMVs are also seen docked on the host ileal epithelial cell microvilli , on the other side, the OMVs were thus implicated in the translocation of bacterial toxins into eukaryotic host cells in vivo [2]. The observation of translocation of gram negative bacterial secretory products as OMVs to another target/host cell marked a novel ‘discovery’ of vesicular exocytosis in prokaryotes [22] as it implied a paradigm shift of membrane vesicle trafficking in prokaryotes [2], which was traditionally regarded as a prerogative of eukaryotic cells [23]. Figure 1 shows ultra-structure of the above-mentioned needle structures revealing a central hollow space through the length of these needles. These structures were named as rivet complexes (RCs), as they are considered to rivet bacterial outer and inner membranes together [10]. The ‘channel’ constituted by the central hollow of these RCs is thought to allow thread-like passage of single molecules of un-conformed protein(s), synthesized in the bacterial cytosol. Such a ‘needle-threading’ passage of the protein molecules is opined to increase length of the extendable slender-needle component of rivet complex. This proposal has an oblique reference to the ‘threading the needle’ concept developed for assembly of type III secretion system (T3SS) of gram negative microbes [24]. This model supports the earlier presumption [10] that the rivet complex structures seen in Figure 1 are same as gram negative bacterial T3SS multi-protein ‘syringe-needle injectisomes’ [25] [26] [27], however, with a different modus operandi, in the present paper. Figure 2 proposes a comprehensive molecular model for biogenesis of gram negative bacterial outer membrane vesicles with the background of structure of T3SS [25] [26] [27] and that of fluid-dynamic model of biological membranes [28] as well as, ultra-structure of cell wall of gram negative microbes [29]. This model is developed further from our earlier proposal [10] with the information that T3SS of Salmonella typhimurium does not require contact with eukaryotic host [30]. It presumes existence of a general secretory pathway (GSP) system in the cell membrane of the gram negative microbes, by which selected bacterial proteins may be transported across the bacterial cell membrane for creation of and storage into cell surface appendages/POs or for release into extracellular medium [31]. Synthesis and secretion of bacterial proteins may be stimulated by some environmental signals, which may also determine the type and amounts of these secretory proteins. It was proposed that anti-microbial peptides (CAMPs) of host could stimulate synthesis of virulence proteins and allied factors in the host-interactive gram negative microbes [22], and later experimental evidence was found for such a process in Salmonella typhimurium as CAMPs, indeed were recognized by bacterial pathogens for colonizing animal tissues [32]. It is also known that Phop/PhoQ regulators of Salmonella typhimurium sense host environments to promote re-modeling of bacterial envelope by including enzymes that modify lipopolysaccharide (LPS). Modified LPS has increased resistance to CAMPS due to altered host recognition of LPS; this promotes bacterial survival [33]. Virulence proteins and allied virulence factors are proposed to accumulate in bacterial outer membrane bound blebs or pockets, named as periplasmic organelles (POs). POs are proposed to inflate under the influence of a turgor pressure-sustained water-inflow, necessary to counter osmotic-imbalance created due to increasing solute concentrations in POs with heavy secretion (Figure 3, stage I). Outer membrane proteins (OMPs) and some specialized proteins (e. g., membrane fusion proteins) may get incorporated in the LPS-rich outer membrane, during the time its ‘bloating out’ as blebs or POs. Such specialized fusion proteins present in OMV membrane have been implicated in effecting a focal fusion of OMVs with cell membrane of host/target cells e.g. at cholesterol-rich rafts leading to endocytosis or direct injection-like translocation of OMV contents subsequent to docking of OMVs on host/target cells with the help of chelating Ca++/Mg++ ions [10]. Existence of such a process has some experimental evidence in that H. pylori vacuolating toxin (Vac A) was indeed localized by immunochemistry in the periplasm, bacterial outer membrane, bacterial surface blebs (POs), as well as in OMVs. Also, OMV-enclosed Vac A was internalized by MKN28 cells, and that too, was detectable in gastric mucosa of H. pylori-infected humans [34]. Earlier, OMVs were found in exponentially growing cultures of Vibrio cholera and Vibrio parahaemolyticus; they were bounded by bacterial outer membrane and filled with electron dense mass [35]. Universality of OMVs is quite appealing in that, virulence factors were found to be released in association with such vesicles from Pseudomonas syringae pv. Tomato T1, during normal growth [36]. OMVs of Bacteroides gingivalis contain multivalent adhesions which cause aggregation of cells to form biofilms resulting in formation of dental plaques [37]. OMVs were also shown to transport PQS, the hydrophobic quorum sensing molecules from P. aeruginosa and that, PQS also causes biogenesis of OMVs [38]. Figure 2 (stage II) proposes that at least 3 or 4 rivet complexes (RCs) align together as close as possible to form a bubble tube like assembly as shown in this diagram, as per interpretation of electron microscopic structure seen in Figure 1. Here, the extendable needle portions of the RCs are optimally elongated to compress the apposed two inner leaflets of the stretched and extended outer membrane of the optimally inflated PO. It is believed that multi-protein supra-molecular rivet complexes are functionally the same as T3SS needle complexes and their assembly and extension has been studied to great details already [24] [25] [26] [27]. However, an additional role to this complex has been assigned and that is to rivet bacterial outer and inner membranes together at scattered places so that controllable periplasmic extensions occur as blebs or POs, rather than the whole periplasm is ‘blown off’ as one balloon [10]. It is also believed that both outer and inner membranes of gram negative organisms, allow lateral diffusion of these RCs as per basic tenets of fluid mosaic model of cell membranes [28]. Additionally, limitations on rate of fresh synthesis of LPS and other outer membrane components of rapidly extending POs, is likely to stretch the outer membrane to a reasonable extent in order to quickly inflate POs and secrete bacterial bio-chemicals as OMVs. Such demand and supply constraints are proposed to produce a directed stretch force on the bacterial outer membrane at the base of inflating POs. Such a stretch force is visualized to direct lateral diffusion of RCs at the base around the growing centre of a PO towards this centre. Such a stretch force is viewed to favor collection of three or more of RCs into an assembly analogous to a soap bubble tube (Figure 3, stage II). Theoretically, at least 3 RCs are required to constitute a bubble tube to ‘blow off’ a small OMV. Likewise 4 RCs are likely to align to secrete medium sized OMVs. Large and larger known OMVs are likely to be secreted via four or more RCs aligned together as a tube. It is known that OMVs are secreted in widely varied sizes of 20-250 nm [15]. Figure 2 (stage III) visualizes how an optimally inflated bleb/PO may be facilitated to be pinched off with the ‘cutting edge’ made of tips of the optimally extended slender needles of the rivet complexes/RCs, aligned in the tubular assembly. This biomechanical model for OMV secretion, uses the basic knowledge of structure of T3SS machinery available from scientific literature, in that the diameter of hollow pair of rings embedded in the bacterial outer membrane is much smaller than the other hollow pair of rings embedded in the bacterial inner membrane [24] [25] [26] [27]. The new concept of directed lateral diffusion of RCs, as discussed in stages I and II of Figure 2, is visualized to culminate when the hollow pairs of rings in bacterial inner membrane stop their further travel upon touching one another in the mutually aligned group of four RCs (Figure 3). However, continued stretch-directed lateral movement of yet mutually un-touching smaller hollow pairs of rings in the bacterial outer membrane shall tilt the optimally extended slender needles to help ‘pinch off’ and OMV at the narrowing orifice created by their ‘closing’ tips (Figure 2, stage III; Figure 3)). Electron microscope data (Figure 1) strongly suggests such a tilt of needles, in micro-location(s) closely above the real OMVs released from the organism(s). Figure 3 shows a diagrammatic model of final pinch off stage, as per discussion above. A multi-probe analysis at the level of single molecule dynamics to prove this model is apparently not currently available in any known single laboratory. However, this model shall stimulate focused experimental work, as OMVs are turning into a hot topic of current research, and bacterial OMVs represent a paradigm shift in biology, highlighting membrane vesicle trafficking and vesicular exocytosis in prokaryotes [22]. Their significance in host-pathogen interactions, disease production [39] [3], inter-bacterial inter-relationships [4], quorum sensing, bacterial biofilm formation [15], etc., has immense value in dealing with cell-cell communications in the whole living world. Declarations Electron microscope facilities of Regional Sophisticated Instruments Centre, Chandigarh, India are acknowledged. Expertise and cooperation of National Salmonella Centre, and guidance and help of Professor of Bacteriology (retired) B R Gupta, Indian Veterinary Research Institute are duly acknowledged. This study does not violate ethics of animal care and upkeep for use in research work. This research-work, including the proposed model was presented and discussed with eminent international biophysicists at the National Symposium on Biophysics (Biophysics in Medicine and Biology) as an invited lecture IL-11 and abstract published in its proceedings as: “Biomechanics of outer membrane vesicle-exocytosis from Gram negative organisms” – R C YashRoy, ; including other references of the author cited in the present research article can be accessed free from author’s page at ResearchGate, References Chatterjee S, Das J. 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    10/2014; Research(October 2014 issue):1.1128. DOI:10.13070/rs.en.1.1128
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