Significance: Many infectious bacteria such as Shigella and Salmonella use type III secretion machines, also called injectisomes, to transfer virulence proteins into eukaryotic host cells. A cytoplasmic sorting platform is required for effector selection and assembly of the needle but has not been visualized in any bacteria. We combine advanced imaging and genetic techniques to visualize the frozen-hydrated diarrheal pathogen Shigella flexneri and reveal the intact type III secretion machine and its interaction with a host cell for the first time to our knowledge. The structures characterized herein provide new insights into the mechanisms underlying type III secretion and pathogenesis and also highlight the major distinctions from the evolutionarily related bacterial flagellum.

Shigella flexneri causes human dysentery after invading the cells of the colonic epithelium. The best-studied effectors of Shigella entry into colonocytes are the invasion plasmid antigens IpaC and IpaB. These proteins are exported via a type III secretion system (TTSS) to form a pore in the host membrane that may allow the translocation of other effectors into the host cytoplasm. TTSS-mediated secretion of IpaD is also required for translocation pore formation, bacterial invasion, and virulence, but the mechanistic role of this protein is unclear. IpaD is also known to be involved in controlling Ipa protein secretion, but here it is shown that this activity can be separated from its requirement for cellular invasion. Amino acids 40 to 120 of IpaD are not essential for IpaD-dependent invasion; however, deletions in this region still lead to constitutive IpaB/IpaC secretion. Meanwhile, a central deletion causes only a partial loss of control of Ipa secretion but completely eliminates IpaD's invasion function, indicating that IpaD's role in invasion is not a direct outcome of its ability to control Ipa secretion. As shigellae expressing ipaD N-terminal deletion mutations have reduced contact-mediated hemolysis activity and are less efficient at introducing IpaB and IpaC into erythrocyte membranes, it is possible that IpaD is responsible for insertion of IpaB/IpaC pores into target cell membranes. While efficient insertion of IpaB/IpaC pores is needed for optimal invasion efficiency, it may be especially important for Ipa-dependent membrane disruption and thus for efficient vacuolar escape and intercellular spread.

Shigella flexneri uses its type III secretion apparatus (TTSA) to deliver invasins into human cells. This TTSA possesses an external needle with IpaD at its tip. We now show that deoxycholate promotes the stable recruitment of IpaB to the needle tip without inducing a rapid burst of type III secretion. The maintenance of IpaB at the needle tip requires a stable association of IpaD with the Shigella surface. This is the first demonstration of a translocator protein being stably associated with the TTSA needle.

Type III secretion (TTS) is an essential virulence function for Shigella flexneri that delivers effector proteins that are responsible for bacterial invasion of intestinal epithelial cells. The Shigella TTS apparatus (TTSA) consists of a basal body that spans the bacterial inner and outer membranes and a needle exposed at the pathogen surface. At the distal end of the needle is a “tip complex” composed of invasion plasmid antigen D (IpaD). IpaD not only regulates TTS, but is required for the recruitment and stable association of the translocator protein IpaB at the TTSA needle tip in the presence of deoxycholate or other bile salts. This phenomenon is not accompanied by induction of TTS or the recruitment of IpaC to the Shigella surface. We now show that IpaD specifically binds fluorescein-labeled deoxycholate and, based on energy transfer measurements and docking simulations, this interaction appears to occur where the N-terminal domain of IpaD meets its central coiled-coil, a region that may also be involved in needle-tip interactions. TTS is initiated as a series of distinct steps and that small molecules present in the bacterial milieu are capable of inducing the first step of TSS through interactions with the needle tip protein IpaD. Furthermore, the amino acids proposed to be important for deoxycholate binding by IpaD appear to have significant roles in regulating tip complex composition and pathogen entry into host cells.

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 comprises four species of human-restricted pathogens causing bacillary dysentery. While Shigella possesses multiple genetic loci contributing to virulence, a type III secretion system (T3SS) is its primary virulence factor. The Shigella T3SS nanomachine consists of four major assemblies: the cytoplasmic sorting platform; the envelope-spanning core/basal body; an exposed needle; and a needle-associated tip complex with associated translocon that is inserted into host cell membranes. The initial subversion of host cell activities is carried out by the effector functions of the invasion plasmid antigen (Ipa) translocator proteins, with the cell ultimately being controlled by dedicated effector proteins that are injected into the host cytoplasm though the translocon. Much of the information now available on the T3SS injectisome has been accumulated through collective studies on the T3SS from three systems, those of Shigella flexneri, Salmonella typhimurium and Yersinia enterocolitica/Yersinia pestis. In this review, we will touch upon the important features of the T3SS injectisome that have come to light because of research in the Shigella and closely related systems. We will also briefly highlight some of the strategies being considered to target the Shigella T3SS for disease prevention.