Study of host pathogen interactions using a model of Pseudomonas aeruginosa infection of lung epithelial cells / by Rebecca Barnes.
Barnes, Rebecca Jean
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Pseudomonas aeruginosa is an opportunistic pulmonary pathogen that can cause acute, life-threatening pneumonia in immunocompromised individuals, it is a common cause of high-mortality ventilator associated pneumonia, and frequently colonizes the lungs of cystic fibrosis patients. As an opportunist, P. aeruginosa uses multiple strategies to exploit host cells during pulmonary infections, and many of these mechanisms are not completely understood. The goal of this study was to develop and optimize a set of experimental methods that would allow us to explore the host-pathogen interactions between P. aeruginosa and lung epithelial cells, and to use these methods to specifically investigate the role of lung epithelial integrin receptors in P. aeruginosa pathogenesis. We labelled a strain of P. aeruginosa PAK with green fluorescent protein (gfp) gene and optimized conditions using this strain to measure its interaction with A549 lung epithelial cells using flow cytometry, a plate reader based assay and fluorescence microscopy. We then used these methods to investigate the importance of integrin inhibitory peptides or antibodies and integrin ECM ligands for PAKgfp interactions with A549 cells. In addition, we measured the effect of inhibiting the functional involvement of integrins using siRNA against integrin linked kinase (ILK) on A549 cellular responses (cytokine release) to PAK. Finally, isogenic PAK mutants and clinical isolates of P. aeruginosa were used to determine the importance of various bacterial virulence factors in activation A549 cells. We found that P. aeruginosa PAK preferentially binds the [alpha]5[beta]1 integrin ligand fibronectin which can increase PAKgfp association with A549 cells, and that ILK mediated signaling is necessary for the maximal cytokine response of A549 to P. aeruginosa infection. In addition, infection with PAK mutants lacking flagella or LPS, and all studied P. aeruginosa clinical isolates resulted in decreased cytokine release by A549 cells compared to PAK WT. We conclude that P. aeruginosa is able to exploit integrins, particularly aSpi, to activate cellular signaling events in host airway epithelial cells during infection, and that the ability of the bacterium to elicit such responses can vary depending on the phenotype and virulence of the particular P. aeruginosa strain.