Release of Toll-Like Receptor-2-Activating Bacterial Lipoproteins inShigella flexneriCulture Supernatants

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Abstract
Shigellaspp. cause dysentery, a severe form of bloody diarrhea. Apoptosis, or programmed cell death, is induced duringShigellainfections and has been proposed to be a key event in the pathogenesis of dysentery. Here, we describe a novel cytotoxic activity in the sterile-culture supernatants ofShigella flexneri. An identical activity was identified in purifiedS. flexneriendotoxin, defined here as a mixture of lipopolysaccharide (LPS) and endotoxin-associated proteins (EP). Separation of endotoxin into EP and LPS revealed the activity to partition exclusively to the EP fraction. Biochemical characterization ofS. flexneriEP and culture supernatants, including enzymatic deactivation, reverse-phase high-pressure liquid chromatography analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a Toll-like receptor-2 (TLR2) activation assay, indicates that the cytotoxic component is a mixture of bacterial lipoproteins (BLP). We show that biologically active BLP are liberated into culture supernatants of actively growingS. flexneri. In addition, our data indicate that BLP, and not LPS, are the component of endotoxin of gram-negative organisms responsible for activating TLR2. The activation of apoptosis by BLP shed fromS. flexneriis discussed as a novel aspect of the interaction of bacteria with the host.