Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.697752
Title: Structural and functional studies of reduction-modifiable protein from Neisseria meningitidis (RmpM)
Author: Maharjan, Sunil
ISNI:       0000 0004 5993 8482
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2015
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Abstract:
Neisseria meningitidis serogroup B (MenB) is the leading cause of meningococcal disease in the UK. Outer membrane vesicles (OMVs) containing immunogenic outer membrane proteins PorA and PorB are currently the only effective vaccines against MenB. The periplasmic protein RmpM is isolated with OMVs and has been hypothesised to bind peptidoglycan (PGN) through a C-terminal domain (RMc) and the porin (PorA and PorB) complex in the outer membrane via its N-terminal domain (RMn), thus forming a structural link between the outer membrane (OM) and peptidoglycan (PGN). Disruption to this link, by deleting rmpM, increases native OMV formation, and thus a ∆rmpM strain has been considered for use as a vaccine strain for the cost-effective production of native OMVs. However, a lack of RmpM may destabilise PorA/B complexes and affect immunogenicity adversely. The aim of this study was to determine RMn structure, investigate its interaction with PorA and PorB and then gain further insights into the mechanism behind RMc-PGN interaction. Following the expression of recombinant RMn, 1H15N NMR established that this domain is unstructured in an aqueous solution. Size exclusion chromatography and pull-down assays confirmed that RMn is functional and sufficient for binding to PorA and PorB. Semi-native PAGE demonstrated that RMn not only binds native PorA/B, but it also stabilises the porin trimer, although not as efficiently as wild-type RmpM. In contrast, RMn failed to bind recombinant PorA and PorB, possibly due to presence of the N-terminal fusion tag used in expressing the recombinant PorA and PorB. Surface plasmon resonance and pull-down assays established that RmpM, through its C-terminus, exhibits meso-Diaminopimelic acid (DAP)-specific PGN binding. While RmpM exhibited stronger binding affinity to highly cross-linked polymeric PGN, the mutation of conserved residues D120 and R135 in the C-terminal domain reduced affinity. Comparative transcriptome analysis, using Miseq next-generation sequencing, indicated that while the transcription of porA and porB genes in ∆rmpM is significantly down-regulated, their transcription in the ∆C.ter rmpM (expressing only the N-terminal domain of RmpM) mutant did not change significantly. It has been suggested that RmpM may be associated with the integral outer membrane assembly system (the BAM complex) and is therefore involved in the folding and insertion of PorA and PorB into the outer membrane. The lack of RmpM in ∆rmpM may reduce the efficiency of this process, resulting in the accumulation of misfolded PorA/B in the periplasm, while the down-regulation of porA/B genes is probably a feedback mechanism in the ∆rmpM strain, to prevent the accumulation of misfolded PorA/B. A ∆C.ter rmpM strain will retain a hypervesiculating phenotype and, in addition, contain natively assembled PorA/B complexes. This trait combination makes this strain an excellent candidate for native OMV vaccine production.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.697752  DOI: Not available
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