Escherichia coli (E. coli) Kl is one of the commonest Gram negative bacteria causing neonatal bacterial meningitis in both developed and developing countries. Haematogenous spread is a key step in E. coli Kl meningitis; however, it is not clear how bacteria cross the brain endothelium to gain entry into the central nervous system. Previous studies have focussed mainly on the identification of bacterial virulence factors, as well as the signalling pathways that are activated for the recruitment of actin cytoskeleton to the bacterial adhesion site on the apical surface of human brain microvascular endothelial cells (HBMEC) and finally lead to bacterial uptake. However, the cellular requirements and mechanisms of post-invasion events are poorly understood. This study aims to further characterize E. coli KI entry, intracellular trafficking and the associated molecular mechanisms. To achieve this, a virulent fluorescent proteinexpressing E. coli K I strain was constructed. In a previous study, caveolin-l, a lipid raft marker associated with clathrin-independent endocytosis, was found associated with invading and intracellular bacteria in HBMEC. To further study the effect of caveolin-l on the bacterial entry, different caveolin-l mutants were applied here. Overexpression of caveolin-l Y 14A mutant and caveolin-l~, which is non-phosphorylatable, did not block E. coli Kl invasion of HBMEC. Furthermore, E. coli Kl invasion of caveolin-l knockout mouse lung endothelial cells (MLEC) was not blocked, which suggested that caveolin-l was not required for E. coli K 1 invasion of endothelial cells. The role of dynamin, a large GTPase that has been implicated in the membrane fission of caveolae buds, was also investigated. Based on quantitative microscopy scoring, no evidence of any inhibitory effect on the bacterial invasion was observed in cells overexpressing green fluorescent protein- (GFP) tagged dominant negative dynamin 2 [Dyn2(aa)K44A] and dominant negative dynamin 1 (DynlK44A). The experimental evidence from this study therefore suggests that E. coli Kl might invade HBMEC via a caveolae- and dynamin-independent endocytic pathway. To further explore the endocytosis pathway that the bacteria use to invade HBMEC, immunofluorescence staining of E. coli Kl infected HBMEC revealed colocalization of the bacteria with flotillin 1, another lipid raft marker associated with clathrin-independent endocytosis. However, E. coli K1 infection of flotillin 1 knockout MLEC demonstrated a significantly increased bacterial uptake. This observation suggests that E. coli K 1 uptake does not require flotillin 1. In parallel, the number of intracellular non-pathogenic E. coli K-12 recovered from the lysates of flotillin 1 knockout MLEC was also significantly higher than that recovered from the lysates of wild type MLEC. Further, overexpression of GFP-tagged flotillin 1 and flotillin 2 in HBMEC inhibited E. coli Kl invasion, which suggest flotillin might have a role as a regulatory cell barrier in host defence.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:557260 |
| Date | January 2011 |
| Creators | Loh, Lip Nam |
| Contributors | Ward, T. H. ; Khan, N. |
| Publisher | London School of Hygiene and Tropical Medicine (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://researchonline.lshtm.ac.uk/923208/ |
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