Histone modulation by nuclear localized meningococcal autotransporters

Dutta, Shoma (2020) Histone modulation by nuclear localized meningococcal autotransporters. PhD thesis, University of Nottingham.

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Abstract

The meningococcal autotransporters Adhesion and penetration protein (App) and Meningococcal serine protease A (MspA) are secreted S6-peptidase family autotransporters which have previously been demonstrated to have various roles in meningococcal virulence including functioning as adhesins. One study from our group showed previously that FITC-labelled recombinant App or MspA passenger domain could be taken up by dendritic cells (DCs) and localised to the nucleus; App and MspA can also bind to, and cleave, recombinant histone H3. It was hypothesized the nuclear localisation of App and MspA occurred via nuclear-localisation sequences (NLS) present within them. This study aimed to shed more light on the nuclear localization of these autotransporters, and to identify the NLS of App and MspA.

In this study, nuclear localization was studied by expressing fluorescently labelled App and MspA fusion proteins using the expression plasmid pDsRed. Before identifying the NLS sequence of App and MspA, the nuclear localisation of DsRedApp and DsRedMspA were determined by confocal laser scanning microscopy. To achieve this, plasmids encoding DsRed-tagged proteins were transfected into Hep-2 cells (human epithelial carcinoma cell line) and HEK293T cells (human embryonic kidney cells with SV40 T-antigen) by transient transfection. The data confirmed that App and MspA were localized to the nucleus when expressed in either cell type. Proteolytic activity was not required for their nuclear localisation, as mutant proteins DsRedAppS267A and DsRedMspAS241A were also translocated.

In order to identify the motifs required to direct App and MspA to the nuclear compartment, various deletions were introduced into DsRedApp and DsRedMspA, and the impact on nuclear localisation assessed. Three different mutants of App and two different mutants of MspA were expressed and their nuclear localisation properties assessed. Differences in the nuclear localisation patterns were observed between the wild-type and mutant App fusion proteins, with the mutant DsRedApp molecules lacking the putative NLS not showing nuclear localisation but remaining in the cytosol. Subsequent site-directed mutagenesis of serine residues within the deleted region also resulted in disruption of nuclear trafficking, confirming the localisation of a potential NLS within App between residues 933R and 940R.

In contrast, no classical NLS was predicted in MspA and a number of deletion mutants behaved like the wild type protein with respect to their nuclear localisation patterns. However, deletion of 933D to 1073A of MspA resulted in a different pattern of localisation (punctuate cytoplasmic localisation) which was not observed with the other MspA mutants. The reason for this difference was unclear.

An additional aim of this study was to further characterise the proteolytic activity of App and MspA against histones, in terms of specificity and the requirement for histone post-translational modifications (PTMs), as it is known that histone clipping can influence chromatin structure and function. In histone clipping assays, a wide range of recombinant histones and epithelial cell-derived histones were used as cleavage substrates and the clipping products visualised by immunoblot analysis. Prior to these experiments, recombinant App and MspA were shown to have proteolytic activity against recombinant H1, H2B and H3 and against Hep-2 cell-derived H1, H2B and H3 (which possess post-translational modifications not found on the recombinant versions), demonstrating that histone PTMs do not effect App- and MspA-mediated cleavage. Furthermore, by adding recombinant App or MspA to host cells in vitro and detecting histone cleavage products, we show that histone clipping occurs in intact cells following uptake and nuclear localization of the meningococcal autotransporters. Moreover, histone clipping was also observed in histones derived from sub-cellular chromatin preparations, indicating that the App and MspA-mediated proteolytic processing of histones occurs within intact chromatin.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Wooldridge, Karl
Oldfield, Neil
Keywords: Histone modulation, Histones, Meningococcal autotransporters
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 61429
Depositing User: Dutta, Shoma
Date Deposited: 31 Dec 2020 04:40
Last Modified: 31 Dec 2020 04:40
URI: http://eprints.nottingham.ac.uk/id/eprint/61429

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