Entry of

Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the IpaB, IpaC, and IpaD proteins, which are secreted by type III secretion machinery. We report here the purification of IpaB and IpaD and the characterization of their lipid-binding properties as a function of pH. The interaction of IpaB with the membrane was quite independent of the pH whereas that of IpaD took place only at low pH. To support the data obtained with the purified proteins, we designed a system in which protein secretion by live bacteria was induced in the presence of liposomes, thereby allowing interaction of proteins with lipids directly after secretion and bypassing any purification step. In these conditions, both IpaB and IpaC, as well as minor amounts of IpaA and IpgD, were associated with the membrane and the ratio of IpaB to IpaC was modulated by the pH. The relevance of these results with respect to the dual roles of IpaB, IpaC and IpaD in induction of membrane ruffles and lysis of the endosomal membrane is discussed.Keywords: membrane; protein±lipid interaction; Shigella flexneri; type III secretion.Members of Shigella spp., including S. flexneri, are the etiological agents of shigellosis. In this disease, destruction of the epithelium of the colonic mucosa is provoked by the strong inflammatory response that is induced by invasion of the epithelium by bacteria. Entry of bacteria into epithelial cells involves actin polymerization, generating local membrane ruffling at the site of contact between the bacterium and the host cell. Bacteria then lyse the membrane of the endocytic vacuole, multiply in the cytoplasm, and move by inducing actin polymerization at one of their poles. The movement of intracellular bacteria generates the formation of cell protrusions that contain a bacterium at their tip, which are internalized by neighbouring cells. Lysis of the two cell membranes that surround bacteria in internalized protrusions completes the process of intercellular dissemination, which allows bacteria to spread from cell to cell without being exposed to the outside medium (reviewed in [1]).All bacterial genes necessary for entry into epithelial cells are clustered within a 30-kb region of a 220-kb virulence plasmid [2]. This region encodes the secreted IpaA-D and IpgD proteins, their cytoplasmic IpgC and IpgE chaperones, and the Mxi-Spa type III secretion machinery [3±9]. Genetic analysis indicated that IpaB, IpaC, and IpaD are essential for entry into epithelial cells [10]. IpaB and IpaC are stored in the bacterial cytoplasm in association with IpgC, which is necessary for the stability of IpaB and the partitioning of IpaB and IpaC [11]. Once secreted, IpaB and IpaC associate in a complex in the extracellular medium. This complex is necessary and sufficient to induce membrane ruffling and entry of latex beads into epithelial cells [12]. When bacteria are endocytosed by macrophages, the same proteins are responsible for the lysis of the membrane vacuole [13,14]. Both proteins have several hydrophobic regions...