Characterization of the interaction of IpaB and IpaD, proteins required for entry of Shigella flexneri into epithelial cells, with a lipid membrane

Abstract

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...

IcmR-regulated Membrane Insertion and Efflux by the Legionella pneumophila IcmQ Protein

“…IcmQ also has the unusual property of being soluble and being able to insert into membranes where it forms pores, similar to membrane-active toxins such as the S. aureus ␣-hemolysin (31) or Colicin E1 (55). A similar pore-forming activity is observed with the Shigella flexneri protein IpaB (44) and the A. tumefaciens VirE2 protein (30), which are translocated to host cells. Future research will focus on further defining the role of the pore-forming activity of IcmQ during infection and how it relates to the other Dot/Icm components.…”

“…The capacity of IcmQ to exist in a soluble form associated with a chaperone and to insert into lipid bilayers in the absence of its chaperone is reminiscent of proteins such as IpaB or VirE2 that have pore-forming activity (30,44). Therefore, we used the fluorescent dye calcein to examine whether IcmQ was able to form pores in a target membrane (39).…”

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

“…The high concentration of SRB in the liposomes results in autoquenching that is relieved when the dye is released. [24][25][26] The oligomeric OPOE-IpaB quickly and efficiently disrupts the phospholipid bilayers, thereby relieving SRB auto-quenching [ Fig. 4(A)].…”

“…The stock solutions were combined and dried under nitrogen followed by vacuum for 3 h to create lipid films of 66.8, 23.5, and 9.7 mol% DOPC, DOPG, and cholesterol, respectively, with 10 mol% DGS-NTA and/or 1 mol% TRITC DHPE added where appropriate. Lipid films were hydrated in PBS alone or in water containing 100 mM SRB for liposome flotation assays 36 and SRB release assays, 25,26 respectively. Liposomes used for dextran release assays were hydrated with 3 mM, 1 mM, 125 mM, or 71.5 mM solutions of fluorescein-labeled 3, 10, 40, or 70 kDa anionic dextrans, respectively.…”

“…Release of 3, 10, 40, and 70 kDa fluorescein-labeled dextrans from lipid vesicles was monitored using a modified protocol described previously 37 following the addition of 15 nM oligomeric IpaB. It is important to note that all of these release assays are strongly dependent on protein and liposome concentration 25 as well as the final concentration of detergent. Proper controls preceded each assay to ensure that the liposomes were not compromised and that minimal lysis was incurred in the absence of protein.…”