Identification of Polymorphic and Conserved Genes - Report Example

Introduction Vesicles can form naturally during the process of secretion, uptake and transport of materials within the cytoplasm. On the other hand artificially formed vesicles are called liposomes. The membrane enclosing the vesicle resembles that of the plasma membrane. Furthermore they can fuse with plasma membrane to release their contents outside of the cell. Vesicles can also fuse with other organelles within the cell. Vesicles are small membranous compartments travelling between cellular compartments. They serve to transport soluble molecules dissolved in the aqueous interior of the vesicle membrane and molecules forming part of the vesicle membrane itself such as lipids, channels or receptors. Once vesicles are formed in the source chamber and are loaded with those molecules which have to be transported they are released into the cytosol and directed toward the target organelle, which recognize and to finally merge. The transported molecules then form part of the organelle and will be responsible for its function. However, other molecules are only passing through this compartment and will be repackaged into other vesicles to go to another cellular compartment. It’s what happens to some proteins that travel from the endoplasmic reticulum; pass through the Golgi apparatus, where they are packaged into other compartments (MICKLOS & FREYER, 2002). What are MV Membrane vesicle release is a conserved feature of pathogenic microbes and contributes to the adaptive capabilities of the microbial cell. Support for this has been observed in salmonella Typhimurium which is a gram negative bacterium that release membrane vesicles during its intracellular growth in the epithelial cells and in macrophages. Helicobacter Pylori has also been observed to release virulence factors in the form of membrane vesicles upon its interaction with the gastric tissue of the human stomach. The biochemical and functional properties of pathogen-derived vesicles reveal their potential to critically impact disease. In almost every case, virulence factors of Gram negative pathogens are secreted products enhancing the survival of the bacteria and/or damage the host. What does MV CONTAIN? The formation of a vesicle to a source compartment is complex. Numerous molecules involved: defining the site of vesicle formation, which selected molecules have to be transported, involved in formation and cleavage, virulence factors for bacterial survival. Bacterial surface proteins are integral to the function and structure of its cell and play a role in membrane vesicle release, as in gram negative bacteria they release surface-derived membrane vesicles. Secretion of virulence factors of Gram-negative pathogens is complicated by the fact that the bacteria envelope consists of two lipid bilayers, the inner and outer membrane, and the periplasm in between. Why do bacteria make MV Gram negative bacteria naturally secrete outer membrane (OM) vesicles and once a portion of the outer-membrane along with periplasmic content is selectively “blebbed” off, the vesicle is formed. Production of membrane vesicles by bacteria is needed to perform a number of diverse roles such as to directly transport virulence factors to its target host cells, facilitate in the modification of microbial surface to avoid immune detection, interact with tissues or bloodstream of the host organism. A key conserved feature of membrane vesicle production for bacterial cells is to communicate with one another as well as with the host. This intermicrobial communication can either stimulate negative or positive interactions and is significant in the management of pathogenic mechanisms and demolition of rival species. For example, a quorum sensing signalling molecules pqs (an intracellular communication between bacteria) is release by pseudomonas aeuroingosa via MV’s. What is known about HP MV – contain – effects on cell The micro-aerophillic, gram-negative bacterium; helicobacter pylori is a gastric pathogen that resides in the human stomach and colonises within the gastric mucosa. If left untreated infection can persist for the lifetime of the host and induce chronic gastric inflammation, leading to a range of diseases (NOBLE, 1993: 100). It has been hypothesised that molecules secreted by the vesicles of helicobacter pylori is what help it to continually persist and it has been proposed that strain-specific genetic diversity is involved in the bacteria’s capability to cause different diseases. Many molecules have been identified in the membrane vesicles released by helicobacter pylori. Similarly to most other gram negative bacteria the OMV’s released by the bacterium contain lipopolysaccharides (LPS) and outer membrane proteins (OMP). Vacuolatingcytotoxin(VacA), cytotoxin-associated protein (CagA) and neutrophil-activating protein (NapA) are all known virulence factors present in membrane vesicles of helicobacter pylori, suggesting the membrane vesicles play an important role in delivering toxins to gastric epithelium (MEANS, 1995: 41). Many molecules have been identified in membrane vesicles released by helicobacter pylori. Similarly to most other gram negative bacteria the OMV’s released by the bacterium contain Lipopolysaccharides (LPS), peptidoglycan (PG) and outer membrane proteins (OMP’s). Outer membrane proteins of helicobacter pylori include; blood group antigen binding adhesion (BaBa), sialic acid-binding adhesion (SaBa), and adherence associated lipoprotein A (AlpA) and Oipa. These adhesions are what enable binding of the helicobacter pylori MV to human gastric epithelium. LPS is present in all gram negative bacteria and provides stability to structures. The O-chain of LPS can vary in length amongst different strains of helicobacter pylori on the outer membrane of vesicles, of which Lewis Blood Group (LBG) antigens are expressed. The composition of the vesicles indicates an essential role of membrane vesicles in pathogenesis as they serve to transfer and deliver toxins, the lewis antigens serve as decoys to avoid immune detection and all functions aid in the virulence and bacterial persistence of helicobacter pylori. It is known that helicobacter pylori affect the gastric epithelial cells, to stimulate a production of interleukin-8 (IL-8) and the toxic effects of VacA upon epithelial cells. In the epithelial cell lines in vitro the toxins induce apoptosis and vascuolation. Why helpful to know how bacteria make MV? Process of biogenesis? Genes/proteins involved? The process of membrane vesicle biogenesis is of great interest and continual research is being done into revealing similar and pathogen-specific roles of membrane vesicles in bacterial virulence of different bacterium. Research will provide knowledge into the pathogenicity process and thus provide a gap to develop treatment such as discovering new antibiotic targets in order to prevent the effect of vesicles and block vesiculation (PERGOLIZZI, 1982: 10). Outer membrane vesicles produced by Gram-negative bacteria contain active proteins and also perform diverse biological processes. OMVs allow enzymes to reach far-targets in a concentrated, targeted and protected form. They also play roles in bacterial survival. Produced as a bacterial stress response, they assist in nutrient acquisition, biofilm development together with biogenesis (AMERICAN SOCIETY FOR MICROBIOLOGY., 1976: 3318). What genes known involved in MV biogenesis of other gram negative bacteria? Research has been conducted in many other gram negative bacteria such as, Escherichia coli, Neisseria meningitides, Pseudomonas aeruginosa, salmonella enterca and the gram positive bacteria; staphylococcus epidermidis in the midst to identify genes involved in there membrane vesicle biogenesis (JARRELL, 2009: 185). As part of this research project, genes identified to play a role in membrane vesicle biogenesis of the above named bacterium will be tested to see whether the same genes are also present in helicobacter pylori MV biogenesis (ZANNONI, 2004: 71). The genes will be checked for homologues and identified whether they are polymorphic or conserved. In the context of treatment it is hopeful for the genes to be conserved as they are easy to target (TALBOT, 2004: 17). Methods Natural membrane vesicles(n-MVs) produced by Pseudomonas aeruginosa PAO1 and PAO1 laden plasma pAK1900(p-MVs),after purification were analysed for the content of DNA. Further, to ensure that no contamination from parent MV-producing cells; MVs were consequently isolated.7.80ng DNA (20 microg MV protein) (-1) were thereafter revealed to be present in p-Mvs after Fluorometry analysis. PCR examination by means of precise primers for pAK1900 series and a chromosomal aim, oprL, pointed towards barely plasmid the DNA to be enclosed inside the lumen of p-MVs after exogenous DNA was assimilated by DNase. Previous research revealed that MVs were capable of fusion with the outer membrane (OM) of PAo1 and Escherichia coli DH5 alpha. As a matter of fact, p-MVs deliver plasmid into the periplasm by by-passing the plasma membrane (PM) to effectively transform. Importantly, contrary to speculationsp-Mvs do not increase transformation efficiency. In fact, beneath a variety of conditions-MVs were found not to transform PAO1 nor did they transform DH5.To distinguish p-MVs and to make certain that membrane-encapsulated pAK1900 was not copied as of a small segment of lysed cells inside the culture and bound by PM as an alternative of Om, which characteristically shapes n-MVs, the corporeal and ultra structural dissimilarity sandwiched between n- and p-MVs were unwavering. Cryo-transmission electron microscopy (cryo-TEM)exposed that n-MVs and p-MVs personally look like isolated OM. floating compactness capacity by means of isopycnic sucrose inclined on secluded PM,OM, n- and p-MVs established that inaccessible OM and n-MVs both fractioned into two bands [rho=1.240 and 1.260 g ml (-1)].p-Mvs also fashioned two groups but at two dissimilar compactness (rho=1.250 and 1.265 g ml(-1)) which may be credited to the turnout of DNA.SDS-PAGE demonstrated that p-MVs crazed most leading OM proteins and also contained 43.70 nmol 3-deoxy-d-manno-octulosonic acid(KDO) (mg protein) (-1) as an LPS indicator. The quantity of NADH oxidase action, a PM enzyme, in the p-MVs was scarcely obvious. Bibliography MICKLOS, D. A., & FREYER, G. A. (2002). DNA science: a first course. New York, NY, Cold Spring Harbor Laboratory Press. DEL GIUDICE, G., & RAPPUOLI, R. (2003). Helicobacter pylori: epidemiology, virulence, immune responses, and vaccine approaches : special issue. OFEK, I., & DOYLE, R. J. (1994). Bacterial adhesion to cells and tissues. London, Chapman and Hall. JARRELL, K. F. (2009). Pili and flagella: current research and future trends. Norfolk, UK, Caister Academic Press.http://public.eblib.com/EBLPublic/PublicView.do?ptiID=238429. ZANNONI, D. (2004). Respiration in archaea and bacteria. Dordrecht, Kluwer Academic. PERGOLIZZI, R. G. (1982). Genetic engineering/biotechnology sourcebook. Washington, D.C., McGraw-Hill. AMERICAN SOCIETY FOR MICROBIOLOGY. (1991). Abstracts of the ... General Meeting of the American Society for Microbiology. Washington, DC, American Society for Microbiology. AMERICAN SOCIETY FOR MICROBIOLOGY. (1976). Applied and environmental microbiology. [Washington], American Society for Microbiology. NOBLE, D. (1993). Ionic channels and effect of taurine on the heart. Boston u.a, Kluwer Academic Publ. MEANS, A. R. (1995). Calcium regulation of cellular function. New York, Raven Press. http://search.ebscohost.com/login.aspx?direct=true&scope=site&db=nlebk&db=nlabk&A N=210056. Read More