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Title: Use of lux gene technology to investigate real-time in-situ interactions of bacterial pathogens with a model blood-brain barrier system
Author: Grimshaw, K. L.
ISNI:       0000 0004 2742 7848
Awarding Body: University of the West of England, Bristol
Current Institution: University of the West of England, Bristol
Date of Award: 2012
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Bacterial bioluminescence has been shown to be an accurate real-time reporter of bacterial internalisation and a valid alternative to viable counts. A self-bioluminescent strain of the bacterial pathogen Neisseria meningitidis was used to develop and optimise an internalisation assay with a range of cell lines including ECV-304, C6 and Caco-2 as an alternative to traditional indirect methods such as viable counting. The use of bioluminescence as a reporter of bacterial internalisation did not produce any robust evidence of internalisation of N. meningitidis C751 pGLITE within cell lines suggesting that internalisation is short lived, does not occur or is below the minimum level of detection. The traditional approach of using a gentamicin protection assay followed by viable counts to investigate internalisation of N. meningitidis suggested evidence of very low level internalisation, the reliability and reproducibility of which remains questionable due to methodological limitations. Although studying bacterial-cell interactions in internalisation assays using monolayers can provide useful insight into bacterial pathogenesis, when infecting the human host, bacteria are required to react with more than a monolayer of cells. The human blood-brain barrier (BBB) is considered to be the main physiological barrier controlling entry and exit from the brain parenchyma of molecules and bacteria. Knowledge of the exact mechanisms by which pathogens invade the brain remains incomplete for two main reasons; most of these pathogens are restricted to humans, limiting the relevance of any animal models and there are few good in vitro models of the BBB. It has been shown, that upon co-culture with C6 cells, ECV-304 cells demonstrate many of the key features of the BBB in vivo including an upregulation of endothelial tight junctions. This continuous co-culture model was further developed and adapted to enhance transendothelial electrical resistance (TEER), indicative of tight junction formation and adapted for use with bioluminescent strains of bacterial pathogens capable of crossing the BBB and entering the CNS to cause meningitis. Penetration of lux-expressing N. meningitidis, Pseudomonas aeruginosa, Staphylococcus aureus and pathogenic Escherichia coli across the BBB model was studied and subsequent effects on TEER and cell viability were also measured. Breaching of the BBB co-culture model by bioluminescent P. aeruginosa was observed after only 6 hours for 106 cfu whereas the other pathogens were not observed to cross the BBB model until in excess of 20 hours. For all pathogenic bacteria tested BBB penetration was associated with high bacterial numbers and all pathogens were observed to have a significant effect on TEER. The effect on cell viability was more variable. By combining the use of bioluminescence and a continuous cell culture model of the BBB, further investigation into breaching of the BBB by bacterial pathogens could be made that would be difficult or impossible to carry out in vivo. This study has resulted in the development of a robust system which has the potential to significantly enhance our understanding of bacterial translocation of the BBB and possible preventative measures.
Supervisor: Not available Sponsor: UWE
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available