Lipopolysaccharide as a major virulence factor in the pathogenesis of Burkholderia cepacia syndrome

Bamford, Sarah 2008. Lipopolysaccharide as a major virulence factor in the pathogenesis of Burkholderia cepacia syndrome. PhD Thesis, Cardiff University.

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Abstract

In cystic fibrosis (CF), bacteria of the Burkholderia cepacia complex (Bcc) can induce a fulminant inflammation with pneumonitis and sepsis. Lipopolysaccharide (LPS) may be an important virulence factor associated with the increased morbidity and mortality seen in Bcc infection but little is known about the molecular pathogenesis of Bcc LPS. In this study, the inflammatory response to highly purified LPS from different Bcc clinical isolates and the cellular signalling pathways employed was investigated. It has been suggested that the large inflammatory response from BcLPS may be due to contaminants in the LPS preparation. Phenol-chloroform petroleum ether (PCP) purified BcLPS preparations were compared to more highly purified, enzyme treated preparations of BcLPS. There were no significant differences in the levels of IL-6 and TNF-a induced from monocytes (MM6) and in levels of IL-8 from epithelial cells (A549), which indicates that there were no contaminants present that could cause an inflammatory response from these cells. The inflammatory response elicited by LPS from different Bcc species that were tested was seen to be varied. LPS from different clinical isolates of the same clonal ET12 strain of Burkholderia cenocepacia were found to induce a varied inflammatory response. Some isolate's LPS produced as much cytokine as prototypical Escherichia coli LPS and all Bcc isolates tested with the exception of environmental samples produced higher levels of inflammatory cytokine than LPS from the CF pathogen Pseudomonas aeruginosa. It was shown that passaging over time under laboratory conditions was not responsible for this variation. Bcc LPS samples were tested in their ability to prime MM6 cells for respiratory burst, samples that had previously produced high levels of cytokine from direct stimulation of MM6 cells were found to not be the most efficient primers of respiratory burst. It was found that the inflammatory response from Bcc LPS stimulated monocytes was separate and in some cases opposite to the ability to prime for respiratory burst. Inhibition experiments were used to investigate the Toll-like receptors and associated adaptor molecules and pathways utilized when monocytes were stimulated by Bcc LPS. The use of anti-CD14, anti-TLR4 and anti-TLR2 antibodies in Bcc LPS stimulated monocytes showed that all Bcc LPS samples tested signalled via CD 14 and TLR4 and not via TLR2. Inhibition of MyD88 using an inhibitor peptide proved that all but one sample required MyD88 to signal. LPS from clinical isolate of Burkholderia multivorans was found to activate the inflammatory response via MyD88-independent pathways. Using MAP kinase inhibitors to test for reduction of cytokine response from stimulated MM6 cells and direct analysis of activation through western blotting, LPS from all clinical Bcc isolates were seen to activate all three major MAPKs p42/44, p38 and JNK. Degradation of the NF-kB inhibitory protein IicB-a was tested using anti-lKB-a antibodies and activation of the transcription factor NF-kB was tested using an electrophoretic mobility shift assay (EMSA). IicB-a was degraded and NF-kB was activated in all the BcLPS samples tested. This study suggests that LPS alone from clinical isolates of Bcc is major virulence factor in CF that it can cause a massive inflammatory response from cells, and that the LPS induced signalling cascade is via classical TLR4-mediated signalling pathways similar to highly inflammatory LPS purified from E.coli.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Dentistry
Subjects:	R Medicine > R Medicine (General)
ISBN:	9781303183379
Date of First Compliant Deposit:	30 March 2016
Last Modified:	09 Jan 2018 20:28
URI:	https://orca.cardiff.ac.uk/id/eprint/55761

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