Release of toll-like receptor-2-activating bacterial lipoproteins in Shigella flexneri culture supernatants

Shigella spp. cause dysentery, a severe form of bloody diarrhea. Apoptosis, or programmed cell death, is induced during Shigella infections and has be en proposed to be a key event in the pathogenesis of dysentery. Here, we de scribe a novel cytotoxic activity in the sterile-culture supernatants of Sh igella flexneri. An identical activity was identified in purified S. flexne ri endotoxin, defined here as a mixture of lipopolysaccharide (LPS) and end otoxin-associated proteins (EP). Separation of endotoxin into EP and LPS re vealed the activity to partition exclusively to the EP fraction. Biochemica l characterization of S. flexneri EP and culture supernatants, including en zymatic deactivation, reverse-phase high-pressure liquid chromatography ana lysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a Tol l-like receptor-2 (TLR2) activation assay, indicates that the cytotoxic com ponent is a mixture of bacterial lipoproteins (BLP). We show that biologica lly active BLP are liberated into culture supernatants of actively growing S. flexneri. In addition, our data indicate that BLP, and not LPS, are the component of endotoxin of gram-negative organisms responsible for activatin g TLR2. The activation of apoptosis by BLP shed from S. flexneri is discuss ed as a novel aspect of the interaction of bacteria with the host.