Cellular response to PorA-Loop4-derived peptides of Neisseria meningitidis

Neisseria meningitidis (the meningococcus) is a major meningitis-causing bacterium and is known for its ability to breach the blood-brain barrier (BBB). Meningococci bind to the Laminin receptor (LAMR) on the surface of the vascular endothelium, which is part of the BBB. In a previous study, the meningococcal surface proteins PorA and PilQ were identified as the bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). PorA preferentially targets the 37 kDa laminin receptor precursor (37LRP) rather than the 67 kDa laminin receptor form (67LR) on the cell surface. It was confirmed that the PorA:37LRP interaction is mediated by PorA-Loop4 as deletion of this loop abrogates recruitment of 37LRP under meningococcal colonies. Using a cyclised peptide corresponding to PorA-Loop4 from N. meningitidis MC58, the PorA-Loop4:37LRP interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1-phase cell cycle arrest. The interaction of PorA-Loop4:37LRP was cytostatic, not cytotoxic. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells at S-phase and a corresponding increase in the G1 population. Based on these results, it is hypothesised that PI3K/Akt pathway was affected by the interaction resulting in G1 arrest. The current study aimed to investigate the perturbations of key proteins’ expression and activation involved in the PI3K/Akt pathway (including Akt, GSK-3β, Cyclin D1, and CDK4) after the treatment of PorA-Loop4 peptide in HBMECs. The work indicated that PorA-Loop4 caused perturbations of Cyclin D1/CDK4 as cell cycle regulators at G1 phase. Transcriptome analysis using qRT-PCR showed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunoblotting confirmed these results as the 24 h-treatment of PorA-Loop4 peptide caused the downregulation of Cyclin D1 and the upregulation of pCDK4 (Thr172). PorA-Loop4 caused a blockade of PI3K/Akt signalling. Using ZSTK474 as a PI3K inhibitor, we observed that the effect of PorA-Loop4 on Cyclin D1 and pCDK4 (Thr172) was PI3K-dependent at both mRNA and protein levels. Exposure of HBMECs to PorA-Loop4 peptide caused a downregulation of pAkt (Ser473) and an upregulation of pGSK-3β (Ser9) at the protein level 24 h post treatment. The upregulation of pGSK-3β (Ser9) was dependent to Akt, and the downregulation of Cyclin D1 was independent of GSK-3β. Using the whole bacterial cells of N. meningitidis wild-type MC58 and its mutants lacking of PorA-Loop4, PorA, and/or PilQ, the role of PorA-Loop4, PorA and PilQ in influencing the PI3K/Akt pathway were investigated. Similar perturbations of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) occurred in PorA-Loop4 peptide treated cells and wild-type infected cells while the abrogation of the PorA-Loop4 erased the perturbations in HBMECs. Both PorA and PilQ have significant roles in the perturbation of pAkt (Ser473), pGSK-3β (Ser9), and Cyclin D1. However, the upregulation of pCDK4 (Thr172) was mediated by Loop4, not by other parts of the PorA, nor by PilQ. Confocal microscopy showed the localisation of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) in the uninfected, wild-type-infected, and PorA-Loop4 mutant-infected HBMECs. The pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1 were localised mainly in the cytoplasm, while the pCDK4 (Thr172) was localised in cytoplasmic and nuclear regions. Confocal microscopy confirmed the immunoblotting results, as the diffuse pattern of pAkt (Ser473), pGSK-3β (Ser9), Cyclin D1, and pCDK4 (Thr172) in the wild-type infected cells were in contrast with the uninfected and the PorA-Loop4 mutant infected cells. In summary, PorA-Loop4 causes the perturbation of PI3K/Akt pathway via Akt/GSK-3β/Cyclin D1/CDK4. The data presented in this thesis has extended knowledge of meningococcal-host pathogen interaction. A thorough understanding of PorA-Loop4 interaction with LAMR and its effect in the PI3K/Akt pathway may provide another strategy for the development of interventions against the meningococcus and perhaps other Gram-negative pathogens