Rakesh Yashroy

Abstract Outer membrane vesicles (OMVs) are 30–200 nm diameter-sized bacterial outer membrane-bounded spherical vesicles containing proteins, virulence signals, DNA fragments, etc., released by all known Gram-negative microorganisms in cultures and natural habitats of soil, water bodies, oceans, animal/plant bodies, etc. The outer membrane of Gram-negative microbes protrudes out in pockets, containing a part of the periplasm, which is then pinched off as OMVs. The membrane enclosing the OMVs contains phospholipids, lipopolysaccharide, and select membrane proteins. Soluble proteins, DNA fragments, virulence determinants, etc. are normally present inside the OMV compartment. OMVs thus traffic a protected repertoire of bacterial molecules into the environment for intraspecies, interspecies, and interkingdom signaling. Transfer of DNA fragments can carry out genetic transformations among microbes. OMVs may signal formation of bacterial biofilms and screen bacteria from phages, antibiotics, antimicrobial peptides, etc. OMVs contain hydrolytic enzymes for breaking down lipids, peptidoglycan, and proteins, thereby enabling bacteria to lyse competing microbes and digest and absorb food reserves available nearby. OMVs are so versatile that bacteria deploy them as combat arsenal for their survival and spread. Isolated OMVs are also being pitted for use as organism-free vaccines. Cite as:YashRoy, R. C. (2017). “Outer membrane vesicles of gram-negative bacteria:
nanoware for combat against microbes and macrobes,” in Nanostructures
for Antimicrobial Therapy, eds A. Ficai and A. H. Grumezescu
(Amsterdam: Elsevier Inc), 341–367. doi: 10.1016/B978-0-323-46152-8.00
015-9

Effect of cell-free extract of Salmonella 3, 10:r organisms on the rabbit ileum was studied 18 hr after treatment by light and electron microscopy. Light microscopic examination revealed severe inflammatory conditions with thickened mucosa, congested blood vessels and focal mononuclear cell infiltration. The mucosal glands showed hyperactivity. The lining epithelium revealed hyperplastic changes and the superficial cell layer appeared denuded at places. Under the transmission electron microscope. the lining epithelial cells at places showed degeneration and accumulation, of membranous bodies. A number of white cells such as macrophages.lymphocytes and neutrophils were also identified as infiltrating the treated ileum. apparently involved in the detoxification process.

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 http://dx.doi.org/10.13070/rs.en.1.1128 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…

ABSTRACT Ultrastructual studies of experimentally infected chicken (n=5) ileum reveal that virulent Salmonella (3,10:r:-) signal their own phagocytosis by tissue macrophages, and are able to dodge host defence mechanisms. The observed fusion of membrane vesicles (MVs) liberated from virulent organisms with closely interacting macrophages is proposed here to constitute a mechanism of type III secretion-like system of gram-negative organisms in general, in order to translocate the necessary biochemical signals into the cytosol of macrophages (eukaryotic host cells). This leads to (1) membrane ruffling culminating in augmented phagocytosis of salmonellae, (2) inhibition of phagosome-lysosome (P-L) fusion for survival and replication of pathogens within the host cytoplasm and (3) initiation of apoptosis of the infected macrophages and ultimate release of replicated salmonellae from the migrating host cells.

Bone is a composite biomaterial, which is formed, when proteins constituting collagen fibers attract calcium, phosphate and hydroxide ions in solution to nucleate atop the fibers. It grows into a hard structure of tiny crystallites of hydroxyapatite, aligned along the long axis of collagen fibers. The present work reports the stimulating effect of static magnetic field on microstructure and mineralization process of bone repair. A unilateral transverse fracture of mid-shaft of metacarpal was surgically created in healthy goats under thiopental sedation and xylocaine analgesia. Two bar magnets (approximately 800 gauss/cm2 field strength) were placed across the fracture line at opposite pole alignment immobilized in Plaster of Paris (POP) splint bandage for static magnetic field stimulation. Radiographs were taken at weekly intervals up to 45 days. Results show that formation of extra-cellular matrix and its microstructure can be influenced by non-invasive physical stimulus (magnetic ...

Spinach chloroplast membranes were studied by natural abundance carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy in their normal state and after heat denaturation of membrane proteins. The membrane proteins were denatured by raising the temperature of the sample to 67 degrees C for 5 minutes [YashRoy, R.C. (1991) J. Biochem. Biophys. Methods 22, 55-59]. Line-broadening of 13C-NMR resonances arising from the 1st (carbonyl), 7th, 9th and 12th carbon atom of fatty-acyl chains with reference to the carbonyl (C-1) group shows increased immobilization of lipid fatty-acyl chains at these locations, obviously caused by changes in interactions between membrane lipids and proteins upon heat denaturation of membrane proteins.

Bone is a composite biomaterial, which is formed, when proteins constituting collagen fibers attract calcium, phosphate and hydroxide ions in solution to nucleate atop the fibers. It grows into a hard structure of tiny crystallites of hydroxyapatite, aligned along the long axis of ...

A new and simple approach for the determination of the temperature of gel-to-liquid crystalline phase transitions (Tc) of biological (chloroplast) membrane lipids from 13C-NMR resonance intensities is proposed. The variation of intensity of a temperature-sensitive NMR resonance is monitored by recording the spectra of the sample at a range of temperatures. From such a series of spectra recorded at different temperatures, a temperature-insensitive resonance is located. Then the ratio of the intensity of the temperature-sensitive to the intensity of the temperature-insensitive resonance is calculated from each spectrum to even out the procedural error, if any. The values of this ratio at different temperatures, when plotted against sample temperature, shows a break at Tc as confirmed by spin label ESR studies.