NK cell killing of extracellular Pseudomonas aeruginosa

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dc.contributor.advisorMody, Christopher Hugh
dc.contributor.authorFeehan, David
dc.contributor.committeememberCobo, Eduardo R.
dc.contributor.committeememberHarrison, Joe J.
dc.date2020-11
dc.date.accessioned2020-07-06T13:56:48Z
dc.date.available2020-07-06T13:56:48Z
dc.date.issued2020-06-26
dc.description.abstractPseudomonas 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.en_US
dc.identifier.citationFeehan, D. (2020). NK cell killing of extracellular Pseudomonas aeruginosa (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.en_US
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/37973
dc.identifier.urihttp://hdl.handle.net/1880/112251
dc.language.isoengen_US
dc.publisher.facultyCumming School of Medicineen_US
dc.publisher.institutionUniversity of Calgaryen
dc.rightsUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.en_US
dc.subject.classificationMicrobiologyen_US
dc.subject.classificationImmunologyen_US
dc.titleNK cell killing of extracellular Pseudomonas aeruginosaen_US
dc.typemaster thesisen_US
thesis.degree.disciplineMedicine – Microbiology & Infectious Diseasesen_US
thesis.degree.grantorUniversity of Calgaryen_US
thesis.degree.nameMaster of Science (MSc)en_US
ucalgary.item.requestcopytrueen_US
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