Structure and biochemical analysis of a secretin pilot protein

The ability to translocate virulence proteins into host cells through a type III secretion apparatus (TTSS) is a hallmark of several Gram-negative pathogens including Shigella, Salmonella, Yersinia, Pseudomonas, and enteropathogenic Escherichia coli. In common with other types of bacterial secretion apparatus, the assembly of the TTSS complex requires the preceding formation of its integral outer membrane secretin ring component. We have determined at 1.5 A the structure of MxiM28-142, the Shigella pilot protein that is essential for the assembly and membrane association of the Shigella secretin, MxiD. This represents the first atomic structure of a secretin pilot protein from the several bacterial secretion systems containing an orthologous secretin component. A deep hydrophobic cavity is observed in the novel 'cracked barrel' structure of MxiM, providing a specific binding domain for the acyl chains of bacterial lipids, a proposal that is supported by our various lipid/MxiM complex structures. Isothermal titration analysis shows that the C-terminal domain of the secretin, MxiD525-570, hinders lipid binding to MxiM.     

Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton

Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram-negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the "needle complex" (NC), produced an average image at 17 A resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2-3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N-terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.     

Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors

We investigated the supramolecular structure of the SHIGELLA: type III secretion machinery including its major components. Our results indicated that the machinery was composed of needle and basal parts with respective lengths of 45.4 +/- 3.3 and 31.6 +/- 0.3 nm, and contained MxiD, MxiG, MxiJ and MxiH. spa47, encoding a putative F(1)-type ATPase, was required for the secretion of effector proteins via the type III system and was involved in the formation of the needle. The spa47 mutant produced a defective, needle-less type III structure, which contained MxiD, MxiG and MxiJ but not MxiH. The mxiH mutant produced a defective type III structure lacking the needle and failed to secrete effector proteins. Upon overexpression of MxiH in the mxiH mutant, the bacteria produced type III structures with protruding dramatically long needles, and showed a remarkable increase in invasiveness. Our results suggest that MxiH is the major needle component of the type III machinery and is essential for delivery of the effector proteins, and that the level of MxiH affects the length of the needle.     

Structural characterization of the type-III pilot-secretin complex from Shigella flexneri

Assembly of the type-III secretion apparatus, which translocates proteins through both membranes of Gram-negative bacterial pathogens into host cells, requires the formation of an integral outer-membrane secretin ring. Typically, a small lipidated pilot protein is necessary for the stabilization and localization of this ring. Using NMR spectroscopy, we demonstrate that the C-terminal residues 553-570 of the Shigella flexneri secretin MxiD encompass the minimal binding domain for its cognate pilot MxiM. Although unstructured in isolation, upon complex formation with MxiM, these residues fold into an amphipathic turn-helix motif that caps the elongated hydrophobic cavity of the cracked beta-barrel pilot. Along with a rearrangement of core aromatic residues, this prevents the binding of lipids within the cavity. The mutually exclusive association of lipids and MxiD with MxiM establishes a framework for understanding the role of a pilot in the outer-membrane insertion and multimerization of the secretin ring.     

MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins.

Shigella flexneri causes bacillary dysentery by invading epithelial cells of the colonic mucosa. The invasion process requires the synthesis and secretion of the virulence plasmid-encoded Ipa proteins. Using TnphoA mutagenesis, we have identified two virulence plasmid genes, mxiJ and mxiM, that encode proteins exported by the general export pathway. Analysis of the MxiJ and MxiM deduced amino acid sequences suggested that mxiJ and mxiM might encode lipoproteins, which was confirmed by [3H]palmitate labeling of MxiJ:PhoA and MxiM:PhoA fusion proteins. A mxiJ mutant was unable to invade HeLa cells, to induce the formation of plaques on confluent monolayers of HeLa cells, and to provoke keratoconjunctivitis in guinea pigs. In addition, secretion of seven polypeptides, including IpaA, IpaB, and IpaC, was abolished in the mxiJ mutant. Sequence comparisons indicated that MxiJ and MxiH, which is encoded by a gene located upstream from mxiJ, are homologous to the Yersinia enterocolitica YscJ and YscF proteins, respectively.     

Role of EscF, a putative needle complex protein, in the type III protein translocation system of enteropathogenic Escherichia coli

Type III secretion systems, designed to deliver effector proteins across the bacterial cell envelope and the plasma membrane of the target eukaryotic cell, are involved in subversion of eukaryotic cell functions in a variety of human, animal and plant pathogens. In enteropathogenic Escherichia coli (EPEC), several protein substrates for the secretion apparatus were identified, including EspA, EspB and EspD. EspA is a structural protein and the major component of a large transiently expressed filamentous surface organelle that forms a direct link between the bacterium and the host cell, whereas EspD and EspB seem to form the mature translocation pore. Recent studies of the type III secretion systems of Shigella and Salmonella pathogenicity island (SPI)-1 revealed the existence of a macromolecular complex that spans both bacterial membranes and consists of a basal structure with two upper and two lower rings and a needle-like projection that extends outwards from the bacterial surface. MxiH ( Shigella ) and PrgI ( Salmonella ) are the main components of the needle of the type III secretion complex. A needle-like complex has not yet been reported in EPEC. In this study, we investigated EscF, a protein sharing sequence similarity with MxiH and PrgI. We report that EscF is required for type III protein secretion and EspA filament assembly. Moreover, we show that EscF binds EspA, suggesting that EspA filaments are an extension of the type III secretion needle complexes in EPEC.     

Shigella Spa32 is an essential secretory protein for functional type III secretion machinery and uniformity of its needle length

The Shigella type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion. The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ. In S. flexneri, the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated. Here we show that Spa32, encoded by one of the spa genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity. When the spa32 gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm. Upon introduction of a cloned spa32 into the spa32 mutant, the bacteria produced needles of wild-type length. The spa32 mutant overexpressing MxiH produced extremely long (>5 microm) needles. Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system. The spa32 mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells. Upon introduction of Salmonella invJ, encoding InvJ, which has 15.4% amino acid identity with Spa32, into the spa32 mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells. These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in Shigella spp. and is involved in the control of needle length. Furthermore, its function is interchangeable with that of Salmonella InvJ.     

Genetic analysis of assembly of the Salmonella enterica serovar Typhimurium type III secretion-associated needle complex

Several pathogenic bacteria have evolved a specialized protein secretion system termed type III to secrete and deliver effector proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium uses one such system to mediate entry into nonphagocytic cells. This system is composed of more than 20 proteins which are encoded within a pathogenicity island (SPI-1) located at centisome 63 of its chromosome. A subset of these components form a supramolecular structure, termed the needle complex, that resembles the flagellar hook-basal body complex. The needle complex is composed of a multiple-ring cylindrical base that spans the bacterial envelope and a needle-like extension that protrudes from the bacterial outer surface. Although the components of this structure have been identified, little is known about its assembly. In this study we examined the effect of loss-of-function mutations in each of the type III secretion-associated genes encoded within SPI-1 on the assembly of the needle complex. This analysis indicates that the assembly of this organelle occurs in discrete, genetically separable steps. A model for the assembly pathway of this important organelle is proposed that involves a sec-dependent step leading to the assembly of the base substructure followed by a sec-independent process resulting in the assembly of the needle portion.     

Protein-peptidoglycan interactions modulate the assembly of the needle complex in the Salmonella invasion-associated type III secretion system

The invasion-associated type III secretion system of Salmonella enterica assembles as a supra-molecular structure, termed needle complex, which spans the bacterial envelope. Here, we present evidence for protein-peptidoglycan interactions that modulate the assembly of this organelle. The presence of major membrane components of the needle complex (PrgH, PrgK and InvG) and InvH, required for efficient assembly of the organelle, was examined in peptidoglycan purified by extensive boiling of bacteria in 4% SDS. InvH, PrgH and PrgK, but not InvG, were detected in this purified material. InvH was present in the peptidoglycan in higher relative amounts than PrgH or PrgK, and was the only protein efficiently bound to peptidoglycan in cross-linking experiments. Analysis in mutants defective for needle complex proteins showed that the needle proteins PrgI and PrgJ and, to a lesser extent, InvH, sustain the association of PrgH and PrgK with peptidoglycan. In contrast, the association of InvH with peptidoglycan did not necessitate other needle complex proteins. Functional analysis showed that the association of InvH, PrgH and PrgK with peptidoglycan is abolished in live bacteria carrying structural modifications in the peptidoglycan. The loss of these interactions caused a marked reduction in the number of needle complexes and, concomitantly, in protein secretion and bacterial invasion of cultured eukaryotic cells. Altogether, these data provide the first evidence for an association between proteins of the Salmonella needle complex and the peptidoglycan. In addition, we demonstrate that these protein-peptidoglycan interactions are critical for an efficient and correct assembly of this specialized organelle.     

MxiK and MxiN interact with the Spa47 ATPase and are required for transit of the needle components MxiH and MxiI,but not of Ipa proteins,through the type III secretion apparatus of Shigella flexneri

The type III secretion (TTS) pathway is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. The Shigella flexneri TTS apparatus (TTSA) spans the bacterial envelope and its assembly requires the products of approximately 20 mxi and spa genes. We present a functional analysis of the mxiK, mxiN and mxiL genes. Inactivation of mxiK and mxiN, but not mxiL, resulted in the assembly of a non-functional TTSA that lacked the outer needle. The amounts of needle components MxiH and MxiI were drastically reduced in mxiK and mxiN mutants and in the secretion defective spa47 mutant, indicating that MxiH and MxiI are degraded if they do not transit through the TTSA. Remarkably, expression of MxiH-His in the mxiN mutant and MxiI-His in the mxiK mutant restored assembly of a functional TTSA, as shown by the ability of these strains to enter into epithelial cells and to secrete Ipa proteins in response to activation by Congo red. Using a two-hybrid screen in yeast and immunoprecipitation assays from S. flexneri extracts, we identified interactions between MxiK and Spa33 and Spa47 and between MxiN and Spa33 and Spa47. These results suggest that transit of the needle components MxiH and MxiI through the TTSA involves the concerted action of the cytoplasmic proteins Spa47, Spa33, MxiK and MxiN. They also show that neither MxiK nor MxiN are absolutely required for secretion of Ipa proteins, provided that the TTSA is correctly assembled.     

The type III secretion system needle tip complex mediates host cell sensing and translocon insertion

Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow 'needle' protruding from the bacterial surface. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which proteins that facilitate host cell invasion are translocated. As the needle is implicated in host cell sensing and secretion regulation, its tip should contain components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we reveal tip complexes of differing compositions and functional states, which appear to represent the molecular events surrounding host cell sensing and pore formation. Our studies indicate that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.     

Binding affects the tertiary and quaternary structures of the Shigella translocator protein IpaB and its chaperone IpgC

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 determined, and we recently reported the 2.1 ? crystal structure of the N-terminal domain (IpaB(74.224)) 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 copurified after they had been co-expressed in Escherichia 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 N-terminus of IpaB adopts an alternative conformation as a result of chaperone binding. The importance of these findings within the functional context of these proteins is discussed.     

The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus

Gram-negative bacteria commonly interact with eukaryotic host cells by using type III secretion systems (TTSSs or secretons). TTSSs serve to transfer bacterial proteins into host cells. Two translocators, IpaB and IpaC, are first inserted with the aid of IpaD by Shigella into the host cell membrane. Then at least two supplementary effectors of cell invasion, IpaA and IpgD, are transferred into the host cytoplasm. How TTSSs are induced to secrete is unknown, but their activation appears to require direct contact of the external distal tip of the apparatus with the host cell. The extracellular domain of the TTSS is a hollow needle protruding 60 nm beyond the bacterial surface. The monomeric unit of the Shigella flexneri needle, MxiH, forms a superhelical assembly. To probe the role of the needle in the activation of the TTSS for secretion, we examined the structure-function relationship of MxiH by mutagenesis. Most point mutations led to normal needle assembly, but some led to polymerization or possible length control defects. In other mutants, secretion was constitutively turned "on." In a further set, it was "constitutively on" but experimentally "uninducible." Finally, upon induction of secretion, some mutants released only the translocators and not the effectors. Most types of mutants were defective in interactions with host cells. Together, these data indicate that the needle directly controls the activity of the TTSS and suggest that it may be used to "sense" host cells.     

PscI is a type III secretion needle anchoring protein with in vitro polymerization capacities

The export of bacterial toxins across the bacterial envelope requires the assembly of complex, membrane‐embedded protein architectures. Pseudomonas aeruginosa employs type III secretion (T3S) injectisome to translocate exotoxins directly into the cytoplasm of a target eukaryotic cell. This multi‐protein channel crosses two bacterial membranes and extends further as a needle through which the proteins travel. We show in this work that PscI, proposed to form the T3S system (T3SS) inner rod, possesses intrinsic properties to polymerize into flexible and regularly twisted fibrils and activates IL‐1β production in mouse bone marrow macrophages in vitro. We also found that point mutations within C‐terminal amphipathic helix of PscI alter needle assembly in vitro and T3SS function in cell infection assays, suggesting that this region is essential for an efficient needle assembly. The overexpression of PscF partially compensates for the absence of the inner rod in PscI‐deficient mutant by forming a secretion‐proficient injectisome. All together, we propose that the polymerized PscI in P. aeruginosa optimizes the injectisome function by anchoring the needle within the envelope‐embedded complex of the T3S secretome and – contrary to its counterpart in Salmonella – is not involved in substrate switching.     

The response of type three secretion system needle proteins MxiHDelta5, BsaLDelta5, and PrgIDelta5 to temperature and pH

The type III secretion system (TTSS) is a specialized supramolecular injectisome composed of 25 or more proteins which form basal and extracellular domains and share gross architectural similarities with bacterial flagella. The extracellular component of the "needle complex" is primarily composed of a single monomeric subunit organized in a helical array surrounding a hollow pore and protrudes from the bacterial membrane. It is through this surface appendage that virulence factors are translocated to the host cell cytoplasm and thereby subvert normal host cell functions. We present here a comprehensive biophysical analysis of the dynamic conformational behavior of the truncated monomeric needle subunit proteins MxiH(Delta5) (Shigella flexneri), BsaL(Delta5) (Burkholderia pseudomallei), and PrgI(Delta5) (Salmonella typhimurium) as well as their thermal stability over a pH range of 3-8. Circular dichroism spectroscopy indicates the secondary structure is largely alpha helical in all three proteins, and surprisingly thermally labile with transition midpoints in the range of 35-50 degrees C over the pH range of 3-8. Additionally, at the concentrations examined, the very broad thermal transitions were >90% reversible. Second derivative UV absorbance spectroscopy data indicates some disruption of the protein's tertiary structure occurs at temperatures in the range of 29-46 degrees C. The difference in the pH of maximal stability for each of the proteins and the variation for each protein with respect to both secondary and tertiary structural elements is striking. It appears, that at physiological temperatures all three proteins experience intermediate non-native molten globule like states in which they display significant secondary structure in the absence of extensive tertiary interactions. Because of the size difference between the inner pore of the needle and the fully folded needle proteins, it seems clear that the needle subunits must be secreted in a partially or completely unfolded state to reach the distal tip of the needle for assembly. It is proposed that the formation of these intermediate states in the physiological temperature range may play a role in passage through the pore and needle assembly.     

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular "needle" that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37 degrees C and MxiH having a value near 42 degrees C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (DeltaG degrees 0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein-protein interactions.     

The Shigella T3SS needle transmits a signal for MxiC release, which controls secretion of effectors

Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacteria, including animal and plant pathogens. They inject 'effector' proteins through a 'needle' protruding from the bacterial surface directly into eukaryotic cells after assembly of a 'translocator' pore in the host plasma membrane. Secretion is a tightly regulated process, which is blocked until physical contact with a host cell takes place. Host cell sensing occurs through a distal needle 'tip complex' and translocators are secreted before effectors. MxiC, a Shigella T3SS substrate, prevents premature effector secretion. Here, we examine how the different parts of T3SSs work together to allow orderly secretion. We show that T3SS assembly and needle tip composition are not altered in an mxiC mutant. We find that MxiC not only represses effector secretion but that it is also required for translocator release. We provide genetic evidence that MxiC acts downstream of the tip complex and then the needle during secretion activation. Finally, we show that the needle controls MxiC release. Therefore, for the first time, our data allow us to propose a model of secretion activation that goes from the tip complex to cytoplasmic MxiC via the needle.     

Deoxycholate interacts with IpaD of Shigella flexneri in inducing the recruitment of IpaB to the type III secretion apparatus needle tip

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 Spa33 is an essential C-ring component of type III secretion machinery

Type III secretion machinery (TTSM), composed of a needle, a basal body, and a C-ring compartment, delivers a subset of effectors into host cells. Here, we show that Shigella Spa33 is an essential component of the C-ring compartment involved in mediating the transit of various TTSM-associated translocated proteins. Electron microscopic analysis and pull-down assay revealed Spa33 to be localized beneath the TTSM via interaction with MxiG and MxiJ (basal body components). Spa33 is also capable of interacting with Spa47 (TTSM ATPase), MxiK, MxiN (required for the transit of MxiH, the needle component), Spa32 (required for determining needle length), and several effectors. Genetic and functional analyses of the Spa33 C-terminal region, which is highly conserved in the SpaO-YscQ-HrcQ(B)-FliN family, indicate that some of the conserved residues are crucial for needle formation via interactions with MxiN. Thus, Spa33 plays a central role as the C-ring component in recruiting/exporting TTSM-associated proteins.     

Ultrastructural analysis of IpaD at the tip of the nascent MxiH type III secretion apparatus of Shigella flexneri

Shigella flexneri is a Gram-negative enteric pathogen that is the predominant cause of bacillary dysentery. Shigella uses a type III secretion system to deliver effector proteins that alter normal target cell functions to promote pathogen invasion. The type III secretion apparatus (T3SA) consists of a basal body, an extracellular needle, and a tip complex that is responsible for delivering effectors into the host cell cytoplasm. IpaD [Ipa (invasion plasmid antigen)] is the first protein to localize to the T3SA needle tip, where it prevents premature effector secretion and serves as an environmental sensor for triggering recruitment of the translocator protein IpaB to the needle tip. Thus, IpaD would be expected to form a stable structure whose overall architecture supports its functions. It is not immediately obvious from the published IpaD crystal structure (Protein Data Bank ID 2j0o) how a multimer of IpaD would be incorporated at the tip of the first static T3SA intermediate, nor what its functional role would be in building a mature T3SA. Here, we produce three-dimensional reconstructions from transmission electron microscopy images of IpaD localized at the Shigella T3SA needle tip for comparison to needle tips from a Shigella ipaD-null mutant. The results demonstrate that IpaD resides as a homopentamer at the needle tip of the T3SA. Furthermore, comparison to tips assembled from the distal domain IpaD(Δ192-267) mutation shows that IpaD adopts an elongated conformation that facilitates its ability to control type III secretion and stepwise assembly of the T3SA needle tip complex.