Analysis of the Production and Composition of Outer Membrane Vesicles in the Pathogenic Bacterium Neisseria menengitidis for Improved Vaccine Production

Bullen, Anwen Alice (2011). Analysis of the Production and Composition of Outer Membrane Vesicles in the Pathogenic Bacterium Neisseria menengitidis for Improved Vaccine Production. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f1f5

Abstract

Extraction of outer membrane vesicles (OMVs) from Neisseria meningitidis for vaccine production relies exclusively on the use of detergent. Broader protection is induced from naturally formed vesicles but insufficient quantities are generated. Little is known about the mechanisms governing the release, morphology and composition of the OMVs. To identify factors involved in these processes two N. meningitidis proteins, GNA33 and RmpM, were examined. In addition the tol-pal genes from Escherichia coli, a complex which stabilises the outer membrane, were expressed in N. meningitidis. Increased vesicle release could be induced by deletion or mutation of RmpM, the structural protein binding the outer membrane to the peptidoglycan. Regulation of size and shape of vesicles was perturbed by the deletion of the GNA33 protein and by the expression of theTol-Pal proteins. Together these observations indicated a model of vesicle release controlled by selective disconnection of RmpM resulting in separation of the peptidoglycan from the outer membrane. Vesicle size is maintained by the even distribution of proteins that anchored the outer membrane to the peptidoglycan, including GNA33. A strain containing a mutated RmpM protein that lacked only the C-terminal peptidoglycan binding domain demonstrated the greatest increase in vesicle production. In addition, due to the presence of the RmpM N-terminal domain, this mutant maintained interaction with outer membrane proteins and produced vesicles enriched for the immunodominant protein PorA. This combination of PorA enrichment and increased vesicle release makes this strain an excellent candidate for vaccine production.

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