Gram Negative Bacteria Produce Membrane Vesicles Which Are Capable of Killing Other Bacteria

GRAM NEGATIVE BACTERIA PRODUCE MEMBRANE VESICLES WHICH ARE CAPABLE OF KILLING OTHER BACTERIA

Many gram negative bacteria produce external membrane vesicle (MV). During their formation, MV's entraps several periplasmic components such as alkaline phosphatise, phospholipase c, proelastase, protease, and peptidoglycan hydrolase. Several of these components are virulence factors including the lipopolysacharide contained in the MV membrane itself. The MVs may be important during the preliminary phase of infection, as they concentrate such factor and transport them to host tissue. This cell wall degrading enzyme (peptidoglycan hydrolase) is used to lyse surrounding disparate bacteria in the donor bacterium's environment. Thus, releasing organic compounds for growth of the MV. An earlier study showed that MV's from P.aeruginosa PAO1 were capable of lysing Staphylococcus aureus, Escherichia coli, and another pseudomonas strains.

The MV's would affix to the cell wall surface where they break open the membrane cell. In turn, the MV lyses gram negative bacteria by a subtly different mechanism. MV membrane fuses into the outer membrane of the host bacterium and introduces the MV's luminal contents, which include peptidoglycan hydrolase into the host's periplasm. In the periplasm, hydrolase will diffuse around the murrain sacculus and absorb it at a number of different sites, causing multiple-site lysis. MV's are able to adhere to s-layered gram positive bacteria and peptidogykan hydrolase migrates through the s-layer and attacks the underlying wall. It is fully composed of protein or glycoprotein that arranged in oblique, square, or hexagonal lattice formats. Thus, rendering it unable to inhibit MV mediated lysis. The evidence is gram negative bacteria will produce MV's and that MV's are able to lyse other bacteria.

This group o-g gram negative and positive bacterium was chosen for two prime reasons to be descriptive of target cell peptidoglycan chemotypes in order to determine a bacteriolysis range and a broad spectrum of gram negative. Occurrence of MV's, all of the gram negative bacteria produced MVs when grown in broth medium that used in laboratory for non-fastidious cultures such as Trypticase soy broth is used for the growth of E.coli, P.vulgaris, P.aeruginosa, and S.marcescens and it will be isolated by combination of differential centrifugation and filtration by using the ultracentrifugation to concentrate the MVs.

MV formation could be monitored by electron microscopy and was similar to that previously seen with P.aeruginosa. By using a negative stain, could be seen that the outer membrane blebbed outwards. The isolated MVs from the various strain revealed pliable spherically shaped vesicles about 50 to 250 nm in diameter which were easily malformed by surface tension during drying in the negative stain. MVs from each stain possessed a relative constant diameter. Freeze substitution will confirm that the MVs were bi-layered structures and that during their formation, periplasm was entrapped. MVs produced by P.aeruginosa established that it existence and MV formation in all strains used in this study was consistent.

Killing was measured by using an agar plate assay in which suspensions of the various host bacteria were mixed with molten agar and host bacteria were not actively growing and dividing. No nutrients were added to the agar but only 20mm sodium phosphate buffered saline, ph 7.0 was used. Under this condition at 37oC, bacteriolysis in control plate was not seen after overnight incubation. Electron microscopy of bacteria in these zone of lysis supported the view that the bacteria which were subjected to the MV's had been killed. Gram stain bacteria will performed on gram positive bacteria in these zones and it become gram negative.

Electron microscopy was used in an effort to determine how MVs killed the bacteria. For gram positive, cell wall immediately underlying and attached MV was digested and these single hit sites lysed the cell and similar to that detected by Kadurugamuwa. For gram negative, MVs attached to and fused into the outer membrane, liberating their luminal contents into the periplasm of the targeted cells. Multiple sites on the murrain sacculus were digested and multisite lysis.

MVs that used in present study did not lyse bacteria possessing peptidoglycan chemotypes removed from the A1 chemotype of the donor strains. A1ƴ was far the most easily attacked. MV encapsulates peptidoglycan hydrolase be components of the donor bacterium's autolysin system. Autolysins are used during peptidoglycan metabolism as the parent cell grows and divides. MVs attach to foreign cell and liberate their luminal constituent to a substrate, the substrate must be recognizable to the released enzyme and A1 peptidoglycan will be the closest fit. A2, A3, A4, and B1 chemotypes dissimilar peptidoglycans may not be recognize, there would be no cell wall digestion and no lysis.

Some lysis was seen with A1α and A2α strains. The chemotypes A1 in A1α an L-lysine replaces meso-diaminopimelic acid at position 3 of the peptide stem and is directly linked to the terminal D-alanine at position 4 on the adjacent peptide stem.

The detection hydrolysis of peptidoglycan by MV peptidoglycan hydrolase could be done by using an SDS-page Zymogram System. Polyacrylamide gel electrophoresis zymogram system is used to detect and separate peptidoglycan hydrolase. Gels that were used in zymogram system contain isolated peptidoglycan sacculli. Hydrolase will separate into distinct bands in the gel by electrophoresis and it re-natured and allowed to digest incorporated sacculli and gels are then stained to emphasize regions of clearing due to peptidoglycans hydrolysis. MVs from each of the donor bacteria were run in gels containing A1 peptidoglycan sacculi from P.aeruginosa; one or more zymogram bands were seen. Production of MVs by gram negative is commonly seeing MVs blebbing from bacteria. Usually MVs contain hydrolytic enzymes and that these include peptidoglycan hydrolase. MVs bleb from the cell surface, they entrap periplasmic constituents within their lumen. MVs also are structures with gram negative bacteria use to partition and concentrate periplasmic components so they can be sent to perform particular functions. Bacteria are usually secreting a number of soluble extracellular enzymes and diffusion must quickly dilute enzyme concentrations as they move from the cell. MVs are concentrated and remain so until they reach a particulate substrate. MVs systems have evolved over time to increase delivery efficiency. These peptidoglycan hydrolyses appear to be normal components donor cell's autolysin system.

REFERENCES

1. Bernadsky, G., T. J. Beveridge, and