Alsuwat, Meshari
(2022)
Investigating the interaction between Neisseria meningitidis and human FGFR1.
PhD thesis, University of Nottingham.
Abstract
Neisseria meningitidis is one of the most common bacterial causes of meningitis. This organism colonises the nasopharyngeal epithelium of the upper respiratory tract, where it is considered to be a commensal species. It can subsequently traverse the nasopharyngeal epithelium and enter the bloodstream, which can lead to disease including sepsis and meningitis. To cause meningitis, N. meningitidis must further cross the blood–brain barrier (BBB) to reach the meninges. This requires it to first bind to endothelial cells in a process that is incompletely understood at present. However, previous reports have shown that epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) might be involved.
Earlier research in our group has established that N. meningitidis interacts directly and specifically with the IIIc splice variant of FGFR1, a new cellular receptor for pathogenic meningococci. That study employed a PilQ-deficient meningococcal strain (ΔpilQ) with mutation of a key component of type IV pili (TFP), thereby revealing that these bacterial structures are important for FGFR1-mediated adhesion of N. meningitidis to host endothelial cells. However, other adhesins have been implicated in invasion of the host, and multiple virulence factors are evidently involved in the invasion of host tissues that occurs in meningococcal disease. Thus, as yet unidentified adhesins might play an important role in the pathogenesis of meningococcal disease through their interaction with host cell receptors. This study aimed to identify the specific bacterial ligands responsible for FGFR1-dependent attachment of N. meningitidis to human brain microvascular endothelial cells (HBMECs).
Discovery of several potentially important novel meningococcal ligands was enabled by searching for and comparing homologues in the closely-related commensal species N. lactamica. After analysing an initial set of 41 candidate meningococcal ligands, 18 proteins were identified without close homologues in N. lactamica, and this set was filtered down to a final set of four priority candidates as the focus of further study (MafA, Opc5, FadL and NMB0506). Knockout strains of N. meningitidis were produced for all four target genes. Interaction of the wild type and mutant meningococcal strains with HBMECs was compared in cellular adhesion and invasion assays. Of the genes targeted as potential FGFR1 ligands, only fadL showed a significant contribution towards attachment of N. meningitidis MC58 to the host cells, whereas all four genes of interest were found to play a significant role in meningococcal invasion of HBMECs.
A plasmid for production of recombinant Fc-FGFR1 IIIc was validated (pEF-Bos-ssFc-extFGFR1IIIc-ires-TPZ) and used to transfect HEK293T cells using the GeneJuice reagent. This permitted successful expression and purification of Fc-FGFR1 IIIc, along with a control (Fc-stop; i.e., Fc-tag only) as verified by SDS–PAGE and immunoblotting, and allowed for further investigation of the FGFR1-binding properties of the proposed bacterial ligands by whole-cell ELISA. Significantly lower binding to Fc-FGFR1 IIIc was seen for N. lactamica and MC58ΔpilQ/ΔporA mutant, compared to the wild type MC58 strain. However, no significant difference was evident for interaction of the mutants lacking mafA, fadL, opc5 or NMB0506, relative to wild type.
For a more direct assessment of the interactions involved, the proteins encoded by the targeted bacterial genes were overexpressed in E. coli in order to evaluate their specific interactions with FGFR1 IIIc. Due to time constraints, expression and purification were only attempted for three of the original four proteins of interest (MafA, Opc5 and FadL). Whereas purified recombinant MafA was obtained successfully, purification of Opc5 and FadL under native conditions was not achieved since these proteins were not readily expressed in soluble form and were instead found in inclusion bodies. Nonetheless, far-western blotting with solubilised cell lysates from overexpression cultures showed no evidence of an interaction between either Opc5 or FadL and Fc-FGFR1 IIIc. However, an interaction was observed between MafA and the latter. This result was confirmed with a protein–protein interaction ELISA experiment conducted using purified recombinant proteins. Significant binding was observed between Fc FGFR1 IIIc and MafA relative to control (1% BSA/PBS + MafA).
In summary, this study has demonstrated a novel role for FadL in attachment of N. meningitidis to human brain endothelial cells and established contributions of MafA, Opc5, FadL and NMB0506 to cellular invasion. This work has also provided evidence supporting a role for MafA as a meningococcal FGFR1 ligand. In the future, it is hoped that building on these findings will result in more effective therapeutic approaches against the disease.
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