Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H.
Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2022-02-01
|
| Series: | PLoS Pathogens |
| Online Access: | https://doi.org/10.1371/journal.ppat.1010325 |
| _version_ | 1811330043350089728 |
|---|---|
| author | David D Feehan Khusraw Jamil Maria J Polyak Henry Ogbomo Mark Hasell Shu Shun Li Richard F Xiang Michael Parkins Joseph A Trapani Joe J Harrison Christopher H Mody |
| author_facet | David D Feehan Khusraw Jamil Maria J Polyak Henry Ogbomo Mark Hasell Shu Shun Li Richard F Xiang Michael Parkins Joseph A Trapani Joe J Harrison Christopher H Mody |
| author_sort | David D Feehan |
| collection | DOAJ |
| description | Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (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, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show 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-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. 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 P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process. |
| first_indexed | 2024-04-13T15:54:29Z |
| format | Article |
| id | doaj.art-b778c7014af347808053473fb513adcf |
| institution | Directory Open Access Journal |
| issn | 1553-7366 1553-7374 |
| language | English |
| last_indexed | 2024-04-13T15:54:29Z |
| publishDate | 2022-02-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj.art-b778c7014af347808053473fb513adcf2022-12-22T02:40:44ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742022-02-01182e101032510.1371/journal.ppat.1010325Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H.David D FeehanKhusraw JamilMaria J PolyakHenry OgbomoMark HasellShu Shun LiRichard F XiangMichael ParkinsJoseph A TrapaniJoe J HarrisonChristopher H ModyPseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (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, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show 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-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. 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 P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.https://doi.org/10.1371/journal.ppat.1010325 |
| spellingShingle | David D Feehan Khusraw Jamil Maria J Polyak Henry Ogbomo Mark Hasell Shu Shun Li Richard F Xiang Michael Parkins Joseph A Trapani Joe J Harrison Christopher H Mody Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H. PLoS Pathogens |
| title | Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H. |
| title_full | Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H. |
| title_fullStr | Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H. |
| title_full_unstemmed | Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H. |
| title_short | Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H. |
| title_sort | natural killer cells kill extracellular pseudomonas aeruginosa using contact dependent release of granzymes b and h |
| url | https://doi.org/10.1371/journal.ppat.1010325 |
| work_keys_str_mv | AT daviddfeehan naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh AT khusrawjamil naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh AT mariajpolyak naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh AT henryogbomo naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh AT markhasell naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh AT shushunli naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh AT richardfxiang naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh AT michaelparkins naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh AT josephatrapani naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh AT joejharrison naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh AT christopherhmody naturalkillercellskillextracellularpseudomonasaeruginosausingcontactdependentreleaseofgranzymesbandh |