NATURAL KILLER CELL KILLING OF EXTRACELLULAR PSEUDOMONAS AERUGINOSA

Abstract

Abstract Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects individuals with the genetic illness, Cystic Fibrosis and contributes to airway blockage and loss of lung function. NK cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. NK cells, in addition to their cytotoxic effects on cancer and virally infected cells have been shown to play a role in an innate defense against microbes. Hypothesis NK cells will kill P. aeruginosa using cytolytic effector protein(perforin, granulysin or granzymes) alone or working synergistically. Results Live-cell imaging of a co-culture of YT cells, a human NK cell line, incubated with GFP P. aeruginosa in the presence of the viability dye, propidium iodide, demonstrated that YT cell killing of P. aeruginosa is contact-mediated. CRISPR knockout of granulysin or perforin in YT cells had no significant affect on NK cell killing of P. aeruginosa, as determined by CFU counts. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-Dichloroisocoumarin (DCI) to inhibit granzymes, resulted in an inhibition of killing. CRISPR knockout of granzyme B in YT cells, in addition to treatment with the granzyme A inhibitor Futhan, significantly inhibited killing of P. aeruginosa, as determined by CFU counts. These results suggest that NK cells induce membrane damage in P. aeruginosa through a contact dependent process, and that killing requires granzymes.