Listeriolysin O mediates cytotoxicity against human brain microvascular endothelial cells

Penetration of the brain microvascular endothelial layer is one of the routes Listeria monocytogenes use to breach the blood-brain barrier. Because host factors in the blood severely limit direct invasion of human brain microvascular endothelial cells (HBMECs) by L. monocytogenes, alternative mechanisms might be used by this bacterium to penetrate the endothelial cell layer. In this study, we evaluated the cytotoxicity of proteins secreted by L. monocytogenes against HBEMCs using a live/dead staining method. Interestingly, the integrity of the plasma membrane of HBMECs was impaired by proteins secreted by the EGD wild-type strain but not proteins secreted by the isogenic ΔprfA strain. Therefore, we investigated the cytotoxicity of proteins secreted by several isogenic mutant strains (ΔplcA, Δmpl and Δhly) incapable of producing the prfA-regulated bacterial products PlcA, Mpl and LLO, respectively. Results from both fluorescent microscopy and flow cytometry analyses showed that proteins secreted by the Δhly strain were not cytotoxic to HBMECs, whereas those secreted by the ΔplcA and Δmpl strains were cytotoxic. These results suggest that LLO-mediated cytotoxicity against brain microvascular endothelial cells enables L. monocytogenes to effectively penetrate the brain microvascular endothelial layer.