Type III secretion (TTS) is an essential virulence function forShigella 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 is the etiologic agent of shigellosis, a potentially life-threatening bacillary dysentery in humans. Although shigellosis is typically considered a disease of the developing world, it is an underreported problem in industrialized nations where it is an infectious agent in child daycare centers, nursing homes, and in any situation where sanitation procedures become compromised (1). Shigellosis is spread by the fecal-oral route with larger outbreaks linked to contaminated water, which makes it a serious public health problem anywhere treatment regimens are inadequate. Following ingestion, Shigella travels to the colon where it crosses M cells and kills macrophages to gain access to the basal side of the colonic epithelium. S. flexneri then promotes its own uptake into epithelial cells by inducing membrane ruffling at the site of pathogen contact.Shigella invasiveness is the product of a 31-kb segment on its large virulence plasmid, which encodes the components of a type III secretion system (TTSS). 4 The Shigella TTSS is used to subvert the normal host cell mechanisms that control the actin cytoskeleton to promote invasion (2, 3). Within this genetic region, the mxi/spa operons encode the type III secretion apparatus (TTSA) and the ipa/ipg operon encodes the type III secreted protein effectors/translocators, IpaA-D, and IpgC, the cytoplasmic chaperone for IpaB and IpaC (4). The TTSA is a nanomachine that provides a conduit for secretion from the bacterial cytoplasm to the membrane and cytoplasm of target cells. It is composed of two main parts: an external needle that allow...

Shigella flexneri uses its type III secretion apparatus (TTSA) to inject host-altering proteins into targeted eukaryotic cells. The TTSA is composed of a basal body and an exposed needle with invasion plasmid antigen D (IpaD) forming a tip complex that controls secretion. The bile salt deoxycholate (DOC) stimulates recruitment of the translocator protein IpaB into the maturing TTSA needle tip complex. This process appears to be triggered by a direct interaction between DOC and IpaD. Fluorescence spectroscopy and NMR spectroscopy are used here to confirm the DOC-IpaD interaction and to reveal that IpaD conformational changes upon DOC binding trigger the appearance of IpaB at the needle tip. Förster resonance energy transfer between specific sites on IpaD was used here to identify changes in distances between IpaD domains as a result of DOC binding. To further explore the effects of DOC binding on IpaD structure, NMR chemical shift mapping was employed. The environments of residues within the proposed DOC binding site and additional residues within the “distal” globular domain were perturbed upon DOC binding, further indicating that conformational changes occur within IpaD upon DOC binding. These events are proposed to be responsible for the recruitment of IpaB at the TTSA needle tip. Mutation analyses combined with additional spectroscopic analyses confirms that conformational changes in IpaD induced by DOC binding contribute to the recruitment of IpaB to the S. flexneri TTSA needle tip. These findings lay the foundation for determining how environmental factors promote TTSA needle tip maturation prior to host cell contact.

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein.We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n-octyl-oligooxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,Ndimethyldodecylamine N-oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size-dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose Abbreviations: AUC, analytical ultracentrifugation; CD, circular dichroism; CMC, critical micelle concentration; DGS-NTA, 1,2-dioleoylsn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid)succinyl]; DLS, dynamic light scattering; DMSO, dimethyl sulfoxide; DOPC, dioleoylphosphatidylcholine; DOPG, dioleoylphosphatidylglycerol; DSP, dithiobis[succinimidyl proprionate; FM, fluorescein maleimide; FRET, F-rster resonance energy transfer; Ipa, invasion plasmid antigen; Ipg, invasion plasmid gene; LDAO, N,N-dimethyldodecylamine N-oxide; Mxi, major exporter of Ipas; OPOE, n-Octyl-oligo-oxyethylene; SATA, N-succinimidyl-S-acetylthioacetate; SEC, size exclusion chromatography; SRB, sulforhodamine B; T m , thermal unfolding midpoint; TCEP, (tris (2-carboxyethyl)phosphine; TRITC DHPE, (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine); T3SS, type-III secretion system; T3SA, type III secretion apparatus.Additional Supporting Information may be found in the online version of this article. that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.

Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nano-machine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices α3 and α7, with concomitant movement in the orientation of helix α7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC-bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation.

Shigella flexneri uses its type III secretion system (T3SS) to promote invasion of human intestinal epithelial cells as the first step in causing shigellosis, a life threatening form of dysentery. The Shigella type III secretion apparatus (T3SA) consists of a basal body that spans the bacterial envelope and an exposed needle that injects effector proteins into target cells. The nascent Shigella T3SA needle is topped with a pentamer of the needle tip protein invasion plasmid antigen D (IpaD). Bile salts trigger recruitment of the first hydrophobic translocator protein, IpaB, to the tip complex where it senses contact with a host membrane. In the bacterial cytoplasm, IpaB exists in a complex with its chaperone IpgC. Several structures of IpgC have been solved and we recently reported the 2.1-Å crystal structure of the N-terminal domain (IpaB74.224) of IpaB. Like IpgC, the IpaB N-terminal domain exists as a homodimer in solution. We now report that when the two are mixed, these homodimers dissociate and form heterodimers having a nanomolar dissociation constant. This is consistent with the equivalent complexes co-purified after being co-expressed in E. coli. Fluorescence data presented here also indicate that the N-terminal domain of IpaB possesses two regions that appear to contribute additively to chaperone binding. It is also likely that the IpaB N terminus adopts an alternative conformation as a result of chaperone binding. The importance of these findings within the functional context of these proteins is discussed.