Abstract
Purpose :
There are 71,000 cases of bacterial keratitis in the United States every year, and the identity of certain pathological bacteria remains poorly characterized. Corynebacterium macginleyi (C. mac) is a bacterium linked with ocular surface disease; however, mechanisms of disease related to Corynebacterium spp. at the ocular surface are poorly defined. Here, we aimed to better understand how C. mac affects the host at the ocular surface by showing that C. mac can cause rapid cell death of ocular cells.
Methods :
Human Corneal Epithelial Cells (HCLEs) were plated at 10,000 cells per well. Bacteria were grown in Luria-Bertani broth supplemented with Tween 80. We added whole bacteria or supernatants from C. mac or C. mastitidis (C. mast). Controls received media. We used size exclusion columns (100 kDa) from Millipore to help identify the secreted toxin. After 40 minutes of incubation with bacterial supernatant, the levels of active caspases 3 and 7 were measured in HCLEs. Microscopic images of HCLEs were collected 24-hours after inoculation. Cytokines were monitored by ELISA. To identify the C. mac gene responsible, we are creating a TN5 transposon mutant library to screen for candidates. Finally, mice were colonized with C. mac or C. mast during steady state or after HSV-1-induced keratitis.
Results :
HCLEs inoculated with C. mac supernatant (>100 kDa) had significantly more PI uptake as compared to the media control (p = 0.0005) and a trend of more activated Caspase 3 and 7. Supernatant from HCLEs inoculated with C. mac supernatant had higher levels of IL-1β and IL-8. C. mac was unable to colonize the eyes of mice with no disease; however, it was able to colonize the mice with severe keratitis. Conversely, C. mast could only colonize the eyes of mice with no disease and was unable to colonize eyes with keratitis.
Conclusions :
C. mac uniquely induces robust cell death in HCLEs, which is concurrent with the release of IL-1β and IL-8. These data suggest that C. mac potentially causes disease at the ocular surface. Since the C. mac supernatant retains the ability to kill HCLEs, we have concluded that the lethal factor may be a secreted endotoxin larger than 100 kDa. Furthermore, C. mac lacks an ability to colonize the eye; however, once a niche is opened after ocular perturbation, C. mac can remain at the ocular surface long-term increasing the likelihood that C. mac can worsen corneal inflammation and disease.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.