Abstract
Purpose: :
Bacillus endophthalmitis causes permeability of the blood-retinal barrier (BRB). We sought to determine whether permeability was caused by a secreted bacterial factor and characterize the factor.
Methods: :
An in vitro BRB consisting of polarized RPE monolayers was cultivated on collagen-, fibronectin-, and protein-coated glass coverslips and incubated with 102 CFU/ml of wild type or plcR-deficient B. cereus, sterile B. cereus supernatants, heat inactivated supernatants, purified cell walls, or 102 CFU/ml B. subtilis. Permeability was measured by dextran conjugate transmigration assay, transepithelial resistance, and ZO-1 immunocytochemistry. Supernatants prepared in BHI and RPE media were analyzed by silver stain, zymogram, and hide azure blue assay to analyze for the presence of proteases.
Results: :
Incubation of polarized RPE with supernatants of wild typeor plcR-deficient B. cereus resulted in similar degrees of permeability by 8 h, with wild type Bacillus supernatant causing more rapid permeability than the plcR-deficient supernatant. PlcR-deficient supernatant caused alterations in ZO-1 as early as 6 h, while wild type supernatant caused ZO-1 alterations after 6 h. Neither heat inactivated supernatants, cell wall preparations, nor the growth of B. subtilus caused RPE monolayer permeability. The plcR-deficient strain retained significant protease activity in both BHI and RPE media, compared to wild type controls.
Conclusions: :
B. cereus infection of an in vitro BRB resulted in permeability that was independent of plcR-regulation and caused by a secreted factor. Future studies will focus on confirming the identity of the permeability factor and identifying the mechanism of BRB breakdown during B. cereus endophthalmitis.
Keywords: endophthalmitis • bacterial disease • retinal pigment epithelium