Pseudomonas aeruginosa causes aggressive infection in patients with pre-existing disorders and recurrent pulmonary infections in cystic fibrosis patients. Pathogenesis of P. aeruginosa infections is multifactorial owing to numerous virulence factors. The focus of this thesis research was to investigate whether P. aeruginosa elastase (PE) causes remodeling of the cytoskeleton by increasing the phosphorylation of RhoA GTPase proteins. In addressing our hypothesis, we utilized Small GTPase Immuno-sorbent Activation assays (G-LISA) and Enzyme linked Immuno-sorbent assay (ELISA) to quantitate changes in the total as well as phosphorylated RhoA protein in Calu3 cell lines. Fluorescence microscopy aided in understanding the changes in morphological organization of F-actin. Changes in expression of TJ protein, ZO1, due to PE induced RhoA GTPase activity, was analyzed with SDS PAGE and Western Blot Analysis. Our data from G-LISA and ELISA assays indicate that PE increases the amount of active RhoA protein by 50.8% in comparison to PBS treated control cells. RhoA inhibitor reduced the PE-induced phosphorylation of RhoA proteins by 30.35 % in PE treated cells. Images from fluorescence microscopy revealed that increase in RhoA GTPase activity causes formation of specific morphological protrusions such as stress fibers, lamellipodium and filopodium. Presence of RhoA inhibitor reverses the changes induced by PE. Results from immuno-sorbent assays and fluorescence microscopy indicate that inhibition of EGFR and MAPK significantly reduces the PE-induced GTPase activity of RhoA by 48.07 % and 42.2 % respectively. Data from G-LISA and ELISA assays correlate well with the morphology data obtained by fluorescence microscopy. Taken together, our data indicate that PE, at least in part, activates RhoA kinase via upstream EGFR and downstream MAPK signaling pathways, in vitro. The impact of these PE-induced alterations in the anatomy and physiology of the tight junctional complex, and their impact on the homeostasis of the lungs, demands further investigation.
Date of publication
Pal, Bidisha, "Pseudomonas Aeruginosa Elastase Induces Restructuring of Actin Cytoskeleton by Phosphorylation of RhoA Proteins" (2015). Biology Theses. Paper 24.