Browsing by Author "Feehan, David"

Now showing 1 - 1 of 1
  • Thumbnail Image
    ItemOpen Access
    NK cell killing of extracellular Pseudomonas aeruginosa
    (2020-06-26) Feehan, David; Mody, Christopher Hugh; Cobo, Eduardo R.; Harrison, Joe J.
    Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects individuals with the respiratory 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. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes. The hypothesis of this project is that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co¬-culture of YT cells, a human NK cell line, and GFP P. aeruginosa stained with the membrane 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 effect on YT cell killing of P. aeruginosa, as determined by CFU count. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of PAO1 in comparison to wild type controls. This demonstrates that granzymes are required for killing, but exhibit redundancy in their function. In summary, these results suggest that NK cell kill P. aeruginosa through a membrane damaging, contact-dependent process, requiring granzymes.