Listeriolysin O-dependent activation of endothelial cells during infectionwith Listeria monocytogenes: activation of NF-kappa B and upregulation of adhesion molecules and chemokines

The facultative intracellular bacterium Listeria monocytogenes is an invasi ve pathogen that crosses the vascular endothelium and disseminates to the p lacenta and the central nervous system. Its interaction with endothelial ce lls is crucial for the pathogenesis of listeriosis. By infecting in vitro h uman umbilical vein endothelial cells (HUVEC) with L. monocytogenes, we fou nd that wild-type bacteria induced the expression of the adhesion molecules (ICAM-1 and E-selectin), chemokine secretion (IL-8 and monocyte chemotacti c protein-1) and NF-kappa B nuclear translocation. The activation of HUVEC required viable bacteria and was abolished in prfA-deficient mutants of L. monocytogenes, suggesting that virulence genes are associated with endothel ial cell activation. Using a genetic approach with mutants of virulence gen es, we found that listeriolysin O (LLO)-deficient mutants inactivated in th e hly gene did not induce HUVEC activation, as opposed to mutants inactivat ed in the other virulence genes. Adhesion molecule expression, chemokine se cretion and NF-kappa B activation were fully restored by a strain of Lister ia innocua transformed with the hly gene encoding LLO. The relevance in viv o of endothelial cell activation for listerial pathogenesis was investigate d in transgenic mice carrying an NF-kappa B-responsive lacZ reporter gene, NF-kappa B activation was visualized by a strong lacZ expression in endothe lial cells of capillaries of mice infected with a virulent haemolytic strai n, but was not seen in those infected with a non-haemolytic isogenic mutant . Direct evidence that LLO is involved in NF-kappa B activation in transgen ic mice was provided by injecting intravenously purified LLO, thus inducing stimulation of NF-kappa B in endothelial cells of blood capillaries. Our r esults demonstrate that functional listeriolysin O secreted by bacteria con tributes as a potent inflammatory stimulus to inducing endothelial cell act ivation during the infectious process.