Determining the Role of Ca2+ and Ca2+- Binding Protein EfhP in Adherence of Pseudomonas Aeruginosa to Lung Epithelial Cells.

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen, responsible for severe acute and chronic infections. It is one of the primary organisms that form biofilms on airway mucosal epithelium in the lungs of patients with cystic fibrosis (CF). Ca2+ accumulates in pulmonary fluids of CF patients and regulates hyperinflammatory host response to bacterial infections. Studies show that Ca2+ binds directly to the Ca2+-binding protein EfhP of P. aeruginosa and elevated Ca2+ leads to increased virulence in P. aeruginosa, but little is known about how Ca2+ regulates P. aeruginosa virulence during infection of human cell lines. The goal of this study is to determine how Ca2+ affects initial adherence of P. aeruginosa and the role of Ca2+-binding protein, EfhP in this host-pathogen interaction. The adherence of P. aeruginosa was determined with the wild-type strain PAO1, PAO1043 (efhp deletion mutant) and PAO1043.pMF (complemented strain expressing EfhP) utilizing human lung epithelial cell lines (A549) in low and high Ca2+ conditions using RPMI. It was estimated that RPMI contains approximately 0.5-0.67 mM Ca2+, which is spiked to 5mM to obtain the high Ca2+ condition for the assays. Adherence studies show that there is no significant difference in percentage adherence of all the three strains when compared amongst low and high Ca2+ conditions. Thus, EfhP doesn’t seem to play a role in adherence of PAO1 with A549 cells.