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Robin Kelleher Davis, David A Sullivan, Yang Liu; Human meibomian gland epithelial cell membrane lysate inhibits the growth rate of Pseudomonas aeruginosa.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4917. doi: https://doi.org/.
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We have previously determined that human meibomian gland epithelial cells (HMGECs) are enriched with genes encoding antimicrobial peptides, including leukocyte-associated immunoglobulin-like receptor, S100 calcium binding proteins, elafin, and secretory leukocyte peptidase inhibitor. Such peptides, if translated, could account for HMG resistance to bacterial infection in obstructive MG dysfunction, the leading cause of dry eye disease. We had previously shown that cell lysates from Immortalized (I) HMGECs inhibited the growth of the gram-negative bacteria, Pseudomonas (P.) aeruginosa. We hypothesize that this antimicrobial activity is conferred by one or more peptides or proteins residing either in the cell membrane or cytosol of HMGECs. This study was undertaken to begin to determine in which cellular fraction(s) the activity resides.
IHMGECs cultured in serum-containing or serum-free media for 7 days, were harvested, suspended in 1 ml of low osmolarity solution, and incubated at 20oC. Lysed cells were centrifuged to separate cellular membrane from cytosol, then lyophilized. Reconstituted lysate, was dialyzed to remove salts, and added (60 ul of 1 mg/ml protein) in sextuplicate to 96-well plates, followed by addition of 120 ul of P. aeruginosa 6294 (gift of Dr. Mihaela Gadjeva, Brigham & Women's Hospital, Boston, MA), which had been grown in Luria-Bertani media at 37oC, and diluted to an optical density at 600 nm (OD600) of 0.2. Plates were incubated at 37oC, and bacterial growth was monitored by a turbidity assay with OD600 readings in a spectrophotometer. Experiments were performed three times and included positive (growth inhibition with gentamycin) and negative (vehicle) controls.
Our results demonstrate that whole cell and membrane IHMGEC lysates inhibit the growth rate of P. aeruginosa in vitro. After 21 hours of incubation at 37 oC, IHMGEC whole cell and membrane lysates had significantly (p<0.002) reduced the P. aeruginosa growth rate by 18% and 30% respectively, whereas cytosol appeared to have no effect. This ability of IHMGEC whole cell and membrane lysates to suppress bacterial growth was expressed by cells cultured in serum-containing (i.e. differentiating), but not serum-free (i.e. proliferating), media.
Our findings support our hypothesis that the antimicrobial activity of HMGECs is compartmentalized and may reside in the cellular membrane.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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