Investigation of the role of the non-integrin laminin receptor in the pathogenesis of bacterial meningitis

Qarani, Sozan (2016) Investigation of the role of the non-integrin laminin receptor in the pathogenesis of bacterial meningitis. PhD thesis, University of Nottingham.

[img] PDF (Thesis - as examined) - Repository staff only - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
Download (5MB)

Abstract

The human non-integrin laminin receptor (LamR) is a multifunctional protein which is localised to a number of sub-cellular locations. LamR is a component of the ribosome and has a number of intracellular functions; it also acts as an extracellular receptor for laminin, growth factors, pathogenic microorganisms, prion proteins, toxins and the anticarcinogen epigallocatechin gallate (ECGC). Although LamR is present in most cellular compartments, its overexpression in many types of cancer cells suggests a vital role for LamR in tumor-cell migration and invasion. There are two isoforms of laminin receptor: the monomeric 37-kDa laminin receptor precursor (37LRP) and the mature 67-kDa laminin receptor (67LR). Although the precise molecular nature of 67LR is unclear, accumulating evidence strongly suggest that 37LRP can undergo homo- and/or hetero-dimerization with Galectin-3 (Gal-3) to form mature 67LR. A recent study demonstrated that both homo- and heterodimer LamR forms are present on the cell surface, where they form distinct populations.

Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) are major causes of bacterial meningitis. The contribution of LamR in traversal of the blood brain barrier (BBB) by neurotropic viruses is well established and interaction with LamR was recently found to be critical for initiation of bacterial contact with the blood brain barrier (BBB). These bacteria bind LamR via the surface protein adhesins: meningococcal PilQ and PorA; pneumococcal CbpA; and H. influenzae OmpP2.

Further investigations showed that the fourth and second extracellular loops of meningococcal PorA and OmpP2 of H. influenzae, respectively, are responsible for LamR binding.

The work presented here consists of two complementary projects in which a number of approaches were taken to characterise the ligand-binding domains of LamR. The first project aimed to identify sites on LamR that are critical for binding the ligands of bacterial meningeal pathogens. The second project aimed to identify residues that contribute to the stability of LamR homodimers and the heterodimer with Gal-3.

Several mutations were introduced into full-length human LamR, either by deletion mutations within the C-terminal domain (CTD) of LamR using inverse polymerase chain reaction (IPCR), or by single-amino acid substitution in the N-terminal domain (NTD) of LamR using site-directed mutagenesis. Protocols for large-scale expression of full-length and truncated LamR proteins in human cells were developed, as well as non-denaturing purification protocols.

We hypothesised that bacteria-binding domains could be located on both the NTD and CTD of LamR. In vivo examination using ELISA assays, in which the interaction of LamR and whole bacteria or purified recombinant PorA or PilQ were tested identified several novel sites on LamR that contributed significantly to binding of the bacterial ligands. These sites include amino acids 206-229 and 263-282, located within the CTD, and Tyrosine 156 in the NTD, each of which contributed to the binding of meningococcal PorA, and more specifically it’s fourth extracellular loop. Furthermore, three sites on LamR comprising amino acids 206-229 and 263-282 within the CTD and Tyrosine 139 in the NTD were shown to contribute to binding pneumococcal CbpA, OmpP2 and Loop two of OmpP2 of H. influenzae. These results indicate that the three neuroinvasive bacteria share the same binding sites on LamR.

Bimolecular fluorescence complementation (BifC) coupled with flow cytometry and confocal microscopy revealed the vital contribution of amino acid residues Arginine 155, Tyrosine 156 and Lysine 166 (all within the NTD of LamR) for the homodimerization and heterodimerization of LamR with Gal-3. The dimerization of two meningococcal host receptors, LamR and Gal-3, may help to extend spectrum of their bacterial adhesins, which may act cooperatively or synergistically at different stages of infection. Information about the residues in LamR that contribute to the stabilization of LamR dimers has implications for the role of LamR dimers in the pathogenesis of bacterial meningitis. Identification of bacteria-binding domains on LamR is of particular interest for development of vaccines or therapeutic interventions that provide protection against the three major meningitis-causing bacteria.

The aim of the current work was first to localise the domains of LamR responsible for binding with PorA and PilQ of N. meningitidis; CbpA and OmpP2 of S. pneumonia, and OmpP2 of H. influenzae. Also, previous studies have shown conspicuous homodimerisation of 37LRP and heterodimerisation with Gal-3. Our second aim was to identify of amino acid residues involved in 37LRP self-association and heterodimer formation with Gal-3.

In current work, several regions of LamR were hypothesised to constitute the binding site for the bacterial ligands; these predictions were based on previous studies on LamR binding domains and the crystal structure of laminin receptor. Also, to investigate both homo and heterodimerisation of LamR, the involvement of several putative amino acid residues within 37LRP in LamR dimerisation was was hypothesised.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Wooldridge, K.G.
Oldfield, N.
Keywords: Bacterial meningitis, Human non-integrin laminin receptor, LamR, Homodimers
Subjects: R Medicine > RC Internal medicine
W Medicine and related subjects (NLM Classification) > WC Communicable diseases
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 31739
Depositing User: Qarani, Sozan
Date Deposited: 27 Sep 2018 09:26
Last Modified: 08 Feb 2019 10:17
URI: http://eprints.nottingham.ac.uk/id/eprint/31739

Actions (Archive Staff Only)

Edit View Edit View