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A. L. Moyer, J. Thurman, M. C. Callegan; A Bacillus cereus Secreted Factor InducesPermeability of an in vitro Blood Retinal Barrier. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2481. doi: https://doi.org/.
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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.
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.
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.
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.
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