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Infection and Immunity

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Shigella flexneri contact with enterocytes induces a burst of protein secretion via its type III secretion apparatus (TTSA) as an initial step in cellular invasion. We have previously reported that IpaD is positioned at the TTSA needle tip (M. Espina et al., Infect. Immuno. 74:4391-4400, 2006). From this position, IpaD senses small molecules in the environment to control the presentation of IpaB to the needle tip. This step occurs without type III secretion induction or IpaC recruitment to the S. flexneri surface. IpaC is then transported to the S. flexneri surface when target cell lipids are added, and this event presumably mimics host cell contact. Unlike IpaB mobilization, IpaC surface presentation is closely linked to secretion induction. This study demonstrates that sphingomyelin and cholesterol are key players in type III secretion induction and that they appear to interact with IpaB to elicit IpaC presentation at the TTSA needle tip. Furthermore, IpaB localization at the needle tip prior to membrane contact provides the optimal set of conditions for host cell invasion. Thus, the S. flexneri type III secretion system can be induced in a stepwise manner, with the first step being the stable association of IpaD with the needle tip, the second step being the sensing of small molecules by IpaD to mobilize IpaB to the tip, and the third step being the interaction of lipids with IpaB to induce IpaC localization at the needle tip concomitant with translocon insertion into the host membrane and type III secretion induction.

Shigella flexneri, the causative agent of shigellosis, is responsible for more than 1 million deaths each year, especially among children in developing regions ( Once ingested, S. flexneri crosses M cells and passes into the underlying gut-associated lymphoid tissues of the colon (20), where it kills macrophages (29) and then invades epithelial cells by macropinocytosis (17). The S. flexneri invasive phenotype localizes genetically to a 31-kb region of its large virulence plasmid and is absolutely tied to its type III secretion system (TTSS) (6, 23).

TTSSs are used by numerous gram-negative bacteria to introduce bacterially derived effector proteins into the membrane and cytoplasm of a target cell, resulting in the subversion of normal cell functions (8). Linking the bacterium and host cell in this process is the type III secretion apparatus (TTSA), which structurally resembles a molecular needle and syringe. The system is controlled by a basal body (the syringe) that spans both bacterial membranes and an external needle that provides a conduit from the basal body to the sensory needle tip complex (8, 28). The needle in S. flexneri is comprised of a polymer of MxiH and is approximately 50 nm long and 7 nm in diameter, with a central channel that is about 2.5 nm in diameter (5). At the top of the MxiH needle resides the tip protein IpaD, most likely as a pentamer, which serves as an environmental sensor for the MxiH-IpaD tip complex (4, 7). When the presence of bile salts such as deoxycholate (DOC) is sensed by IpaD, the first translocator protein, IpaB, is mobilized to the TTSA needle tip to form an MxiH-IpaD-IpaB ternary complex. At this stage, the TTSA structure is primed for subsequent host cell contact (19, 24). In previous studies IpaC had not been found to localize to the S. flexneri surface of the log-phase bacterium (7, 19).

As a next step in describing the process of type III secretion, we show here that liposomes trigger mobilization of IpaC to the needle tip complex, where it is immediately inserted into the host cell membrane, along with IpaB, to complete the TTSA conduit into the host cell just prior to initiating host cytoskeleton rearrangements. IpaC is most efficiently recruited to the S. flexneri surface with a defined liposome composition that includes phospholipids, sphingomyelin (SM), and cholesterol (Chol). Furthermore, IpaC recruitment occurs concomitantly with induction of type III secretion of IpaB, IpaC, and IpaD into the S. flexneri culture supernatant. It thus appears that IpaB mobilization to the S. flexneri TTSA needle tip represents a second discrete step in TTSA assembly, with the final third step being IpaC recruitment to the needle tip, which occurs after IpaB contacts and inserts into the host cell membrane.



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