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Dufailu, Osman Adamu (2017) Role of meningococcal Neisserial autotransporter lipoprotein (NalP) in host pathogenesis. PhD thesis, University of Nottingham.

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Abstract

Neisseria meningitidis (Nm) can cause life-threatening bacterial meningitis and septicaemia. The high mortality of meningitis is associated with meningococcal invasion of the central nervous system (CNS) by breaching of the blood-brain barrier (BBB). Nm elaborates several cell-surface and secreted virulence factors that promote host invasion and colonisation. One important class are the autotransporter proteins. Adhesion and penetration protein (App), meningococcal serine protease A (MspA), Immunoglobulin A1 protease (IgA1p) and Neisserial autotransporter lipoprotein (NalP) are serine protease autotransporters of Nm; all have previously been demonstrated to have roles in meningococcal virulence.

For App and MspA, uptake by host cells, nuclear localisation, histone clipping and induction of apoptosis have been described in our laboratory. However, a time course for the efficient uptake and nuclear localisation of these proteins into human brain microvascular endothelial cells (HBMECs) and details regarding the mechanism for histone clipping remained unknown. To address these points, both App and MspA and proteolytically-inactive mutant derivatives were expressed using the pColdTF vector system and purified under native conditions. Using confocal scanning microscopy optimal uptake and nuclear localisation of recombinant fusion proteins containing the functional passenger domains of both App and MspA in HBMECs was shown to occur 8 h-post exposure. Furthermore, the requirement for the active site serine residue in both autotransporters for H3.1 clipping was demonstrated, and human coagulation factor V (FV) was shown to be an additional substrate for both proteins.

NalP is a cell-surface maturation protease which processes App, MspA and other meningococcal surface proteins such as IgA1P, LbpB and NhbA, and thus modulates the cell surface and secretome of the organism. Previous studies aimed at functional characterisation of NalP have typically relied on the phenotypic comparison of wild-type and nalP-mutant derivatives. Here an active recombinant NalP was expressed and purified, and used to investigate the interaction of NalP with host cells in order to more comprehensively elucidate the role of NalP during meningococcal interaction with the host. A recombinant NalP (rNalP) passenger domain was purified under non-denaturing conditions using immobilized nickel chromatography. Although rNalP had apparent molecular weight 8-10 kDa less than that of NalP secreted by wild-type meningococci, it was functional as determined by its ability to process human complement 3 (C3). rNalP was shown to cleave human coagulation factor V (FV), a proteolytic event which is likely to contribute to bacterial pathogenesis.

Binding and uptake of rNalP into human cells was demonstrated by flow cytometry and confocal microscopy. Interestingly, rNalP was differentially localised to different cellular compartments in different cell types. Treatment of HBMECs with rNalP resulted in increased levels of IL-6 and IL-8, and decreased levels of TNF-α in culture supernatants. rNalP was shown to clip histone 2B but not other histone proteins. Using a re-tagging approach a number of rNalP-interacting host proteins were identified. These included proteins of the candidate membrane, cytoplasm, cytoskeleton endoplasmic reticulum, mitochondrion, and nucleus. Some of these proteins are likely to be involved in trafficking of NalP and the cellular response to this protein.

Overall, the findings of this study expand our knowledge on the contribution of three autotransporters to meningococci pathogenesis and provide a platform to further explore the host response to NalP uptake and the epigenetic changes associated with autotransporter host interactions, which may guide the development of future therapeutic interventions.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Wooldridge, K.G. Oldfield, N.
Keywords:	Neisseria meningitidis, Autotransporters, Neisserial autotransporter lipoprotein Host-pathogen interaction, Complement 3, Coagulation factor V, Histones and cytokines, Receptor, Retagging
Subjects:	Q Science > QR Microbiology QS-QZ Preclinical sciences (NLM Classification) > QW Microbiology. Immunology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	41127
Depositing User:	Dufailu, Osman
Date Deposited:	17 Jul 2017 04:40
Last Modified:	07 Feb 2019 18:03
URI:	https://eprints.nottingham.ac.uk/id/eprint/41127

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