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Staphylococcus aureus mediated disruption of blood-brain barrier phenotype in human brain microvascular endothelial cells

McLoughlin, Alisha (2016) Staphylococcus aureus mediated disruption of blood-brain barrier phenotype in human brain microvascular endothelial cells. PhD thesis, Dublin City University.

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Abstract

Background: Staphylococcus aureus (SA) is a gram-positive, cocci-forming pathogen capable of causing serious-life-threatening illnesses (e.g. meningitis). In 2010, it was the number one cause of bacterial deaths in the USA. Moreover, SA is one of just a few bacteria that are able to gain access to the central nervous system (CNS) via the blood-brain barrier (BBB). The BBB comprises a monolayer of unique brain microvascular endothelial cells (BMvEC), which act as a seal to separate the CNS from the main circulatory system. BMvEC are equipped with specialised interendothelial tight and adherens junction protein complexes that regulate the paracellular traffic of fluids and solutes into the neural microenvironment. However, when this barrier is compromised by injury and/or infection it can result in BBB dysfunction. The main objective of this project was to investigate the effects of SA infection on the human blood-brain barrier (BBB) microvascular endothelium in-vitro with respect to: (i) interendothelial tight/adherens junction (TJ/AJ) protein expression; (ii) endothelial permeability and; (iii) to pro-inflammatory signalling. Methods: Primary-derived human brain microvascular endothelial cells (HBMvEC) were infected with either “formaldehyde-fixed” or “live” SA Newman wild-type (NWT) or mutant staphylococcal protein A (ΔSpA) strains. Infection dose- (multiplicity of infection/MOI) and time-dependency studies were routinely performed. The impact of infection was monitored on: (i) cell viability (by flow cytometry);(ii) bacterial adherence to HBMvEC; (iii) interendothelial junction protein expression (by Western blotting); (iv) HBMvEC monolayer paracellular permeability (by transendothelial permeability assay using FITC-dextran); (v) activation of NF-κB (by Western blotting for NF-κB phospho-p65); (vi) inflammatory cytokine and chemokine release (by ELISA and cytokine array panel); (vii) reactive oxygen species production (by flow cytometry) and; (viii) endothelial microparticle release post-infection (by flow cytometry). Main Results: Following 48 hrs infection using “fixed” NWT-SA bacterium (MOI 0-250), the expression of HBMvEC junctional proteins (VE-Cadherin, ZO-1 and claudin-5) were dose- dependently downregulated, in parallel with increased monolayer permeability. Fixed NWT-SA infection significantly induced activation of NF-κB within just 1 hour of infection, whilst showing significant dose-dependent increases in release of IL-6, TNF-α and thrombomodulin. A cytokine array panel for fixed SA infections using MOI 0 and MOI 250 media samples showed six upregulated cytokines (RANTES, IP-10, G-CSF, GM-CSF, MCP-1 and IL-6). By contrast, minimal junctional protein decreases were recorded post-infection with “live” NWT-SA (MOI 0- 100, 3 hrs), in parallel with minimal barrier disruption, although NF-κB was activated in a time- dependent manner. There was no difference in cytokine release using live SA media samples after 3 and 12 hrs infection. Further infection studies using fixed NWT-SA showed dose-dependent increases in ROS generation and also release of annexin-V/VE-cadherin-positive endothelial microparticles. In a final series of experiments, the contribution of the virulence factor, SpA, to the effects of SA on HBMvEC barrier properties was investigated using the mutant bacterium (ΔSpA). The ΔSpA strain was attenuated in disrupting HBMvEC barrier properties as seen in the downregulation of adherens junction proteins (VE-cadherin), barrier permeabilization at low concentrations and in the activation of NF-κB in comparison to NWT-SA. Conclusion: Both SA strains (NWT-SA and ΔSpA) have shown decreased expression of interendothelial adherens/tight junction proteins, in parallel with elevated barrier permeabilization and pro-inflammatory index, albeit at different infection times and doses. Ultimately, the effects of SA infection result in BBB dysfunction and barrier compromisation indicative of a cerebral bacterial infection.

Item Type:Thesis (PhD)
Date of Award:March 2016
Refereed:No
Supervisor(s):Cummins, Philip
Uncontrolled Keywords:Endothelial cells
Subjects:Biological Sciences > Microbiology
Biological Sciences > Biochemistry
Biological Sciences > Neurochemistry
Biological Sciences > Cell biology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
Funders:Higher Education Authority
ID Code:21026
Deposited On:12 Apr 2016 14:16 by Philip Cummins. Last Modified 12 Apr 2016 14:16

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