To characterize the antimicrobial function of an airway specific gene, spurt, which is induced by retinoic acid and to examine the levels of expression and secretion of spurt in Cystic Fibrosis patients.
Antibacterial activity was evaluated by colony formation units (CFU) against gram negative P. aeruginosa (PAO1). Cell culture supernatant from CMV-spurt stably transfected airway cells, BAL from CCSP-spurttg transgenic mice, and specific peptides from SPURT were used to compare with their respective controls for CFU counts. Western blotting analysis, immunohistological staining, real time PCR, and ELISA were used to examine levels of secretion, gene and protein expression.
Higher antibacterial activity against P. aeruginosa using cell culture supernatant from CMV-spurt stably transfected spurt-overexpressing cells than those from wildtype and CMV vector control cells. BAL fluid from unchallenged CCSP-spurttg mice that overexpress SPURT (spurt protein) exhibited enhanced antibacterial activity than control group using BAL from wildtype mice. Lower numbers of CFUs in CCSP-spurttg transgenic mice than wildtype littermates were also observed in both 4h and 24h after both groups of mice were challenged with aerosolized P. aeruginosa infection. We also identified a region within SPURT peptide sequence that effectively killed P. aeruginosa at ng concentration range. Furthermore, we observed significant higher gene and protein expression levels of spurt in tissues and epithelial cell cultures originated from Cystic Fibrosis (CF) patients. The secretion of spurt was also consistently higher in BAL samples from CF patients and in apical cell culture supernatants.
SPURT is a novel BPI-like antibacterial protein that may play a critical role in airway specific innate immunity and the significant higher expression and secretion in CF epithelium may represent a response of airway epithelial cells to colonized and increased bacteria exist in CF patients.
Functional role of spurt may associate with pathogenesis of lung diseases such as cystic fibrosis. Aerosolized SPURT may be used to enhance airway specific antibacterial activity.
Y.P. Peter Di, None.