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Shigella ssp cause bacillary dysentery (shigellosis) which has high global morbidity in young children and the elderly. The virulence of Shigella relies upon a type III secretion system (T3SS) which injects host altering effector proteins into targeted intestinal cells. The Shigella T3SS contains two components, invasion plasmid antigen D (IpaD) and invasion plasmid antigen B (IpaB), that were previously identified as broadly protective antigens. When IpaD and IpaB were co-expressed to give the DB fusion (DBF) protein, vaccine efficacy was further improved. Biophysical characterization under various pH conditions showed that DBF is most stable at pH 7 and 8 and loses its conformational integrity at 48 and 50 °C respectively. Forced degradation studies revealed significant effects on the secondary structure, tertiary structure and conformational stability of DBF. In the presence of phosphate buffers as well as other anionic excipients, DBF demonstrated a concentration dependent conformational stabilization. Molecular docking revealed potential polyanion binding sites in DBF that may interact with phytic acid. These sites can be exploited to stabilize the DBF protein. This work highlights potential destabilizing and stabilizing factors, which not only improves our understanding of the DBF protein but helps in future development of a stable Shigella vaccine.

Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisome’s cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 “pods” that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.

Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject effector proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common overall architecture in all systems studied thus far, comprising three major components – the cytoplasmic sorting platform, the envelope-spanning basal body and the external needle with controlling tip complex. The sorting platform consists of an ATPase (SctN) connected to “pods” (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa, as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two-hybrid analysis. The functional need for Arg96 in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome.HighlightsThe structures of Pseudomonas aeruginosa PscD (SctD) and PscK (SctK) were solvedThe interface between the T3SS basal body and sorting platform was modeledThe crystal structure of PscK is the first for any SctK family memberPscK represents a novel protein foldSite-directed mutagenesis supports a computational model of the PscD-PscK interfaceGraphical abstract.The first reported structure for a T3SS SctK protein family member was solved for PscK from Pseudomonas aeruginosa and this allowed for modeling of the interface between this sorting platform protein and the cytoplasmic domain of PscD (a SctD protein family member). This allowed for identification of amino acid residues that may play a role in the interaction between these proteins. The interface appears to be dominated by electrostatic interactions and mutagenesis confirmed the importance of key residues in driving their interaction based on two-hybrid analysis.

Numerous Gram-negative pathogens infect eukaryotes and use the type III secretion system (T3SS) to deliver effector proteins into host cells. One important T3SS feature is an extracellular needle with an associated tip complex responsible for assembly of a pore-forming translocon in the host cell membrane. Shigella spp. cause shigellosis, also called bacillary dysentery, and invade colonic epithelial cells via the T3SS. The tip complex of Shigella flexneri contains invasion plasmid antigen D (IpaD), which initially regulates secretion and provides a physical platform for the translocon pore. The tip complex represents a promising therapeutic target for many important T3SS-containing pathogens. Here, in an effort to further elucidate its function, we created a panel of single-VH domain antibodies (VHHs) that recognize distinct epitopes within IpaD. These VHHs recognized the in situ tip complex and modulated the infectious properties of Shigella. Moreover, structural elucidation of several IpaD–VHH complexes provided critical insights into tip complex formation and function. Of note, one VHH heterodimer could reduce Shigella hemolytic activity by >80%. Our observations along with previous findings support the hypothesis that the hydrophobic translocator (IpaB in Shigella) likely binds to a region within the tip protein that is structurally conserved across all T3SS-possessing pathogens, suggesting potential therapeutic avenues for managing infections by these pathogens.

Type III secretion systems are used by some Gram-negative bacteria to inject effector proteins into targeted eukaryotic cells for the benefit of the bacterium. The type III secretion injectisome is a complex nanomachine comprised of four main substructures including a cytoplasmic sorting platform, an envelope-spanning basal body, an extracellular needle and an exposed needle tip complex. Upon contact with a host cell, secretion is induced, resulting in the formation of a translocon pore in the host membrane. Translocon formation completes the conduit needed for effector secretion into the host cell. Control of type III secretion occurs in response to environmental signals, with the final signal being host cell contact. Secretion control occurs primarily at two sites-the cytoplasmic sorting platform, which determines secretion hierarchy, and the needle tip complex, which is critical for sensing and responding to environmental signals. The best-characterized injectisomes are those from Yersinia, Shigella and Salmonella species where there is a wealth of information on the tip complex and the two translocator proteins. Of these systems, the best characterized from a secretion regulation standpoint is Shigella. In the Shigella system, the tip complex and the first secreted translocon both contribute to secretion control and, thus, both are considered components of the tip complex. In this review, all three of these type III secretion systems are described with discussion focused on the structure and formation of the injectisome tip complex and what is known of the transition from nascent tip complex to assembled translocon pore.

Many Gram-negative bacteria use type III secretion systems (T3SSs) to inject virulence effector proteins into eukaryotic cells. The T3SS apparatus (T3SA) is structurally conserved among diverse bacterial pathogens and consists of a cytoplasmic sorting platform, an envelope-spanning basal body, and an extracellular needle with tip complex. The sorting platform is essential for effector recognition and powering secretion. Studies using bacterial “minicells” have revealed an unprecedented level of structural detail of the sorting platform; however, many of the structure-function relationships within this complex remain enigmatic. Here, we report on improved cryo-electron tomographic approaches to enhance the resolution of the Shigella T3SA sorting platform (at ≤2 nm resolution) done in concert with biochemical and genetic methods to define the sorting platform interactome and interactions with the T3SA inner membrane ring (IR). We observed that the sorting platform consists of “pods” with 6-fold symmetry that interact with the Spa47 ATPase via radial extensions comprising MxiN. Most importantly, MxiK maintained an interaction with the IR via specific interactions with the cytoplasmic domain of the IR protein MxiG (MxiG(C)), which is a noncanonical forkhead-associated domain, and MxiK has an elongated structure that interacts with the IR via MxiG(C). T4 lysozyme-mediated insertional mutagenesis of MxiK revealed its orientation within the sorting platform and enabled disruption of interactions with its binding partners, which abolished sorting platform assembly. Finally, a comparison with the homologous interactions in the Salmonella T3SS sorting platform revealed clear differences in their IR-sorting platform interfaces that have possible mechanistic implications.

Diarrheal diseases are a major cause of morbidity and mortality worldwide. They are most prevalent in settings with inadequate sanitation, poor hygiene and contaminated water. An important diarrheal pathogen in such settings is Shigella. No commercially available vaccine exists against shigellosis and immunity to the pathogen is serotype-restricted. We have previously shown that a polypeptide fusion of the Type Three Secretion Apparatus (T3SA) proteins IpaB and IpaD (named DBF) was efficacious as a vaccine against Shigella. Vaccination using different administration routes indicated that protection conferred by DBF did not fully correlate with antibodies. To define the immune responses involved in protection, we studied cellular responses to intranasal immunization with the DBF and the adjuvant dmLT. We found dendritic cell (DC) activation at the nasal associated lymphoid tissue (NALT). Activation markers CD86 and MHCII significantly increase in cells from immunized mice. Antigen exposure in vitro further confirmed the upregulation of CD80 and CD40 in primary dendritic cells. Animals immunized with antigen-primed dendritic cells were protected against Shigella infection, at levels comparable to the efficacy of immunization with the protein vaccine formulation. Therefore, we show that antigen-primed DCs are enough to provide immunity, and propose a mechanism of protection against Shigella spp. based on DC-mediated antigen presentation to T cells.

Nontyphoidal Salmonella enterica serotypes (NTS) are the leading cause of hospitalization and death due to foodborne illnesses. NTS are the costliest of the foodborne pathogens and cause ∼$4 billion annually in health care costs. In Africa, new invasive NTS are the leading cause of bacteremia, especially in HIV-positive children and adults. Current vaccines against S. enterica are not broadly protective and most are directed at the typhoid-causing serotypes, not the NTS. All S. enterica strains require two type III secretion systems (T3SS) for virulence. The T3SS needle tip protein and the first translocator are localized to the T3SS needle tip and are required for pathogenesis of S. enterica . Collectively they are 95 to 98% conserved at the amino acid sequence level among all S. enterica strains. The Salmonella pathogenicity island 1 or 2 tip and first translocator proteins were genetically fused to produce the S1 and S2 fusion proteins, respectively, as potential vaccine candidates. S1 and S2 were then characterized using spectroscopic techniques to understand their structural and biophysical properties. Formulated at the proper pH, S1, S2, or S1 plus S2 (S1S2), admixed with adjuvant, was used to immunize mice followed by a lethal challenge with S. enterica serotype Typhimurium or S. enterica serotype Enteritidis. The S1S2 formulation provided the highest protective efficacy, thus demonstrating that an S1S2 subunit vaccine can provide broad, serotype-independent protection, possibly against all S. enterica serotypes. Such a finding would be transformative in improving human health.

Diarrhea caused by Shigella, Salmonella, and Yersinia is an important public health problem, but development of safe and effective vaccines against such diseases is challenging. A new antigen delivery platform called bacterium-like particles (BLPs) was explored as a means for delivering protective antigens from the type III secretion systems (T3SS) of these pathogens. BLPs are peptidoglycan skeletons derived from Lactococcus lactis that are safe for newborns and can carry multiple antigens. Hydrophobic T3SS translocator proteins were fused to a peptidoglycan anchor (PA) for BLP attachment. The proteins and protein-BLP complexes associated with BLPs were characterized and the resulting data used to create three-index empirical phase diagrams (EPDs). On the basis of these EPDs, IpaB (Shigella) and SipB (Salmonella) behave distinctly from YopB (Yersinia) under different environmental stresses. Adding the PA domain appears to enhance the stability of both the PA and translocator proteins, which was confirmed using differential scanning calorimetry, and although the particles dominated the spectroscopic signals in the protein-loaded BLPs, structural changes in the proteins were still detected. The protein-BLPs were most stable near neutral pH, but these proteins' hydrophobicity made them sensitive to environmental stresses.

Bacterium-like particles (BLPs), derived from Lactococcus lactis , offer a self-adjuvanting delivery vehicle for subunit protein vaccines. Proteins can be specifically loaded onto the BLPs via a peptidoglycan anchoring (PA) domain. In this study, the tip proteins IpaD, SipD, and LcrV belonging to type III secretion systems of Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica , respectively, were fused to the PA and loaded onto the BLPs. Herein, we biophysically characterized these nine samples and condensed the spectroscopic results into three-index empirical phase diagrams (EPDs). The EPDs show distinctions between the IpaD/SipD and LcrV subfamilies of tip proteins, based on their physical stability, even upon addition of the PA. Upon attachment to the BLPs, the BLPs become defining moiety in the spectroscopic measurements, leaving the tip proteins to have a subtle yet modulating effect on the structural integrity of the tip proteins-BLPs binding. In summary, this work provides a comprehensive view of physical stability of the tip proteins and tip protein-BLPs and serves as a baseline for screening of excipients to increase the stability of the tip protein-BLPs for future vaccine formulation. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:424-432, 2015

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of the typhoid fever, is a pathogen of great public health importance. Typhoid vaccines have the potential to be cost-effective measures towards combating this disease, yet the antigens triggering host protective immune responses are largely unknown. Given the key role of cellular-mediated immunity in S. Typhi protection, it is crucial to identify S. Typhi proteins involved in T-cell responses. Here, cells from individuals immunized with Ty21a typhoid vaccine were collected before and after immunization and used as effectors. We also used an innovative antigen expressing system based on the infection of B-cells with recombinant Escherichia coli (E. coli) expressing one of four S. Typhi gene products (i.e., SifA, OmpC, FliC, GroEL) as targets. Using flow cytometry, we found that the pattern of response to specific S. Typhi proteins was variable. Some individuals responded to all four proteins while others responded to only one or two proteins. We next evaluated whether T-cells responding to recombinant E. coli also possess the ability to respond to purified proteins. We observed that CD4+ cell responses, but not CD8+ cell responses, to recombinant E. coli were significantly associated with the responses to purified proteins. Thus, our results demonstrate the feasibility of using an E. coli expressing system to uncover the antigen specificity of T-cells and highlight its applicability to vaccine studies. These results also emphasize the importance of selecting the stimuli appropriately when evaluating CD4+ and CD8+ cell responses., Author summary Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of the life-threatening typhoid fever that affects 11.9–20.6 million individuals annually in low-income and middle-income countries. The T-cells, CD4+ and CD8+ T cells, play a significant role in protection against S. Typhi infection. Yet, the antigens triggering host protective immune responses recognized by these cells are largely unknown. To address this shortcoming, in this study we used an E. coli expression system methodology for identifying immunogenic proteins of S. Typhi. We found that although the pattern of response to individual S. Typhi proteins was variable among the typhoid vaccinees, the E. coli expressing system uncovered the antigen specificity of T-cells, and highlight its applicability to vaccine studies.

The type III secretion system (T3SS) is Shigella’s most important virulence factor. The T3SS apparatus (T3SA) is comprised of an envelope-spanning basal body and an external needle topped by a tip complex protein called IpaD. This nanomachine is used to deliver effector proteins into host cells to promote pathogen entry. A key component of the matured T3SS needle tip complex is the translocator protein IpaB. IpaB can exist in multiple states when prepared as a recombinant protein, however, it has also been described as having additional roles in Shigella pathogenesis. This mini-review will briefly describe some of the features of IpaB as a T3SS needle tip protein, as a pore-forming translocator protein and as an effector protein. Reflection on the potential importance of the different in vitro states of IpaB on its function and importance in serotype-independent vaccines is also provided.

Forster resonance energy transfer (FRET) provides a powerful tool for monitoring intermolecular interactions and a sensitive technique for studying A-level protein conformational changes. One system that has particularly benefited from the sensitivity and diversity of FRET measurements is the maturation of the Shigella type III secretion apparatus (T3SA) needle tip complex. The Shigella T3SA delivers effector proteins into intestinal cells to promote bacterial invasion and spread. The T3SA is comprised of a basal body that spans the bacterial envelope and a needle with an exposed tip complex that matures in response to environmental stimuli. FRET measurements demonstrated bile salt binding by the nascent needle tip protein IpaD and also mapped resulting structural changes which led to the recruitment of the translocator IpaB. At the needle tip IpaB acts as a sensor for host cell contact but prior to secretion, it is stored as a heterodimeric complex with the chaperone IpgC. FRET analyses showed that chaperone binding to IpaB’s N-terminal domain causes a conformational change in the latter. These FRET analyses, with other biophysical methods, have been central to understanding T3SA maturation and will be highlighted, focusing on the details of the FRET measurements and the relevance to this particular system.

Bacterial infections caused by Shigella flexneri, Salmonella typhimurium, and Burkholderia pseudomallei are currently difficult to prevent due to the lack of a licensed vaccine. Here we present formulation and immunogenicity studies for the three type III secretion system (TTSS) needle proteins MxiH ∆5 , PrgI ∆5 , and BsaL ∆5 (each truncated by five residues at its C terminus) as potential candidates for vaccine development. These antigens are found to be thermally stabilized by the presence of carbohydrates and polyols. Additionally, all adsorb readily to aluminum hydroxide apparently through a combination of hydrogen bonds and/or Van der Waals forces. The interaction of these proteins with the aluminum-based adjuvant changes with time resulting in varying degrees of irreversible binding. Peptide maps of desorbed protein, however, suggest that chemical changes are not responsible for this irreversible association. The ability of MxiH ∆5 and PrgI ∆5 to elicit strong humoral immune responses was tested in a murine model. When administered intramuscularly as monomers, the needle components exhibited dose dependent immunogenic behavior. The polymerized version of MxiH was exceptionally immunogenic even at low doses. The responses of both monomeric and polymerized forms were boosted by adsorption to an aluminum salt adjuvant.

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.

Many pathogenic gram-negative bacteria employ type III secretion systems to transport proteins into the host cell membrane and cytoplasm to subvert normal cellular functions. The type III secretion apparatus consists of a basal body spanning the inner and outer bacterial membranes and a needle which extends away from the bacterium. Recent work has found that a special class of proteins localizes to the tip of the needle to control secretion of effector proteins. Five of these tip proteins are IpaD (Shigella flexneri), BipD (Burkholderia pseudomallei), SipD (Salmonella spp.), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa). In this study, the conformational stability of these proteins was characterized as a function of pH and temperature. Understanding the stability of the proteins in different pH environments is particularly important since they are expected to encounter different pH environments in their passage through the gastrointestinal tract and are exposed to low pH microenvironments near the surface of target cell membranes. Secondary and tertiary structural changes were monitored using the spectroscopic techniques of far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and ultraviolet absorption spectroscopy. Optical density and right angle scattering measurements were also used to evaluate protein association/dissociation. Empirical phase diagrams were then applied to mathematically combine data from the various spectroscopic techniques to provide a global picture of the proteins' structural behavior in solution. The responses of the proteins to changes in temperature and pH conditions reveal two distinct subfamilies in terms of stability. The first is that of IpaD, BipD, and SipD whose corresponding phase diagrams show conformational differences at pH 5–6. The conserved pH dependence in this subfamily suggests possible common mechanistic function. In the second subfamily (LcrV and PcrV), conformational stability is directly related to pH, also indicating mechanistic similarities. Proteins 2008. © 2008 Wiley-Liss, Inc.

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.

The pathogenesis of Shigella flexneri requires a functional type III secretion apparatus to serve as a conduit for injecting host-altering effector proteins into the membrane and cytoplasm of the targeted cell. The type III secretion apparatus is composed of a basal body and an exposed needle that is an extended polymer of MxiH with a 2.0-nm inner channel. Invasion plasmid antigen D (IpaD) resides at the tip of the needle to control type III secretion. The atomic structures of MxiH and IpaD have been solved. MxiH (8.3 kDa) is a helix-turn-helix, whereas IpaD (36.6 kDa) has a dumbbell shape with two globular domains flanking a central coiled-coil that stabilizes the protein. These structures alone, however, have not been sufficient to produce a workable in silico model by which IpaD docks at the needle tip. Thus, the work presented here provides an initial step in understanding this important protein-protein interaction. We have identified key MxiH residues located in its PSNP loop and the contiguous surface that uniquely contribute to the formation of the IpaD-needle interface as determined by NMR chemical shift mapping. Mutation of Asn-43, Leu-47, and Tyr-50 residues severely affects the stable maintenance of IpaD at the Shigella surface and thus compromises the invasive phenotype of S. flexneri. Other residues could be mutated to give rise to intermediate phenotypes, suggesting they have a role in tip complex stabilization while not being essential for tip complex formation. Initial in vitro fluorescence polarization studies confirmed that specific amino acid changes adversely affect the MxiH-IpaD interaction. Meanwhile, none of the mutations appeared to have a negative effect on the MxiH-MxiH interactions required for efficient needle assembly.

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.

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence; IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intra-molecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.

A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases.

Shigella flexneri is a facultative intracellular pathogen that causes severe gastroenteritis in humans. Invasion plasmid antigen D (IpaD) is an essential participant in Shigella invasion of intestinal cells, but no detailed structural information is available to help understand the proposed role of IpaD in invasion or its interaction with other invasion proteins. Therefore, the secondary and tertiary structure and thermal stability of IpaD as well as selected IpaD deletion mutants were investigated using Fourier transform infrared (FTIR), circular dichroism (CD), and both intrinsic and extrinsic fluorescence spectroscopies. The energetics of thermal unfolding were also evaluated by differential scanning calorimetry (DSC). Secondary-structure analysis by CD and FTIR suggests that that IpaD is primarily alpha-helical with characteristics of a intramolecular coiled coil. Thermal studies revealed that the unfolding of IpaD is a complex process consisting of two transitions centered near 59 and 80 degrees C. A comparison of the data obtained with the intact protein and selected deletion mutants indicated that the lower temperature transition is a reversible event attributable to the unfolding of a small domain located at the N terminus of IpaD. In contrast, the thermal unfolding of the proposed major and highly stable C-terminal domain was irreversible and led to protein aggregation. When the results are taken together, they strongly support the idea that IpaD has two independent folding domains.

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.

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.

Background We created a live, attenuated, oral Shigella vaccine by constructing a lineage of guanine auxotrophs and conducted a double-blind, placebo-controlled trial to ascertain (1) the attenuation profile of Delta guaBA Shigella flexneri 2a, which harbors deletions in the guanine nucleotide synthesis pathway (CVD 1204); (2) additional attenuation conferred by deletions in set and sen genes encoding Shigella enterotoxins (ShETs) 1 and 2, respectively (CVD 1208); and (3) the relative immunogenicity of these constructs. Methods Inpatient volunteers received a single oral dose of CVD 1204, CVD 1208 (10(7), 10(8), or 10(9) cfu), or placebo. Clinical, immunologic, and microbiologic responses were evaluated. Results Reactogenicity occurred in 8 of 23 recipients of CVD 1204, characterized by diarrhea (30%), fever (22%), and/or dysentery (17%), but in only 1 (5%) of 21 recipients of CVD 1208 (brief fever) (P=.02, Fisher's exact test). Antilipopolysaccharide responses, as measured by antibody-secreting cell, serum, or fecal antibody levels, occurred in 67%, 71%, and 100% of recipients of CVD 1204 and in 86%, 43%, and 100% of recipients of CVD 1208 at doses of 10(7), 10(8), and 10(9) cfu, respectively. Conclusions We conclude that 1 or both ShETs are virulence determinants in humans; their inactivation, in combination with Delta guaBA, leads to a well-tolerated and immunogenic Shigella vaccine candidate.

The virulence-associated phospholipase of Yersinia enterocolitica (YplA), which is secreted by a flagellar type III secretion system, was cloned and purified for structure-function analysis using a His(6)-tag expression system. Two versions of YplA have been proposed on the basis of two potential initiating methionine residues. The longer derivative possesses 59 additional amino acids at its N-terminus and appears to represent the native form of YplA; however, the shorter recombinant protein possesses enhanced activity in vitro. Both recombinant YplA derivatives are highly active as type-A(2) phospholipases and possess similar physical properties. Based on type III secretion substrates from other gram-negative bacteria, the N-terminus of YplA is probably required as a secretion signal; however, differences in the time-based activity of these two recombinant enzymes, the N-terminus of YplA may also have a regulatory function.

Shigella flexneri causes bacillary dysentery, an important cause of mortality among children in the developing world. Shigella secretes effector proteins via its type III secretion system (T3SS) to promote bacterial uptake into human colonic epithelial cells. The T3SS basal body spans the bacterial cell envelope anchoring a surface-exposed needle. A pentamer of invasion plasmid antigen D lies at the nascent needle tip and invasion plasmid antigen B (IpaB) is recruited into the needle tip complex on exposure to bile salts. From here, IpaB forms a translocon pore in the host cell membrane. Although the mechanism by which IpaB inserts into the membrane is unknown, it was recently shown that recombinant IpaB can exist as either a monomer or tetramer. Both of these forms of IpaB associate with membranes, however, only the tetramer forms pores in liposomes. To reveal differences between these membrane-binding events, Cys mutations were introduced throughout IpaB, allowing site-specific fluorescence labeling. Fluorescence quenching was used to determine the influence of oligomerization and/or membrane association on the accessibility of different IpaB regions to small solutes. The data show that the hydrophobic region of tetrameric IpaB is more accessible to solvent relative to the monomer. The hydrophobic region appears to promote membrane interaction for both forms of IpaB, however, more of the hydrophobic region is protected from solvent for the tetramer after membrane association. Limited proteolysis demonstrated that changes in IpaB's oligomeric state may determine the manner by which it associates with phospholipid membranes and the subsequent outcome of this association.

Shigella flexneri causes bacillary dysentery with symptoms resulting from the inflammation that accompanies bacterial entry into the cells of the colonic epithelium. The effectors of S. flexneri invasion are the Ipa proteins, particularly IpaB and IpaC, which are secreted at the host-pathogen interface following bacterial contact with a host cell. Of the purified Ipa proteins, only IpaC has been shown to possess quantifiable in vitro activities that are related to cellular invasion. In this study, ipaC deletion mutants were generated to identify functional regions within the IpaC protein. From these data, we now know that the N-terminus and an immunogenic central region are not required for IpaC-dependent enhancement of cellular invasion by S. flexneri. However, to restore invasiveness to an ipaC null mutant of S. flexneri, the N-terminus is essential, because IpaC mutants lacking the N-terminus are not secreted by the bacterium. Deletion of the central hydrophobic region eliminates IpaC's ability to interact with phospholipid membranes, and fusion of this region to a modified form of green fluorescent protein converts it into an efficient membrane-associating protein. Meanwhile, deletion of the C-terminus eliminates the mutant protein's ability to establish protein-protein contacts with full-length IpaC. Interestingly, the mutant form of ipaC that restores partial invasiveness to the S. flexneri ipaC null mutant also restores full contact-mediated haemolysis activity to this bacterium. These data support a model in which IpaC possesses a distinct functional organization that is important for bacterial invasion. This information will be important in defining the precise role of IpaC in S. flexneri pathogenesis and in exploring the potential effects of purified IpaC at mucosal surfaces.

Volunteers were orally administered invasive, non-Shiga toxin-producing Shigella dysenteriae 1 to establish a challenge model to assess vaccine efficacy. In stepwise fashion, four separate groups were given 3 × 102, 7 × 103, 5 × 104, or 7 × 105 CFU. Using PBMC, proliferative responses and cytokine production were measured to S. dysenteriae whole-cell preparations and to purified recombinant invasion plasmid Ags (Ipa) C and IpaD. Anti-LPS and anti-Ipa Abs and Ab-secreting cells were also evaluated. Preinoculation PBMC produced considerable quantities of IL-10 and IFN-γ, probably secreted by monocytes and NK cells, respectively, of the innate immune system. Following inoculation, PBMC from 95 and 87% of volunteers exhibited an increased production of IFN-γ and IL-10, respectively, in response to Shigella Ags. These increases included responses to IpaC and IpaD among those volunteers receiving the lowest inoculum. No IL-4 or IL-5 responses were detected. Whereas there were no Ab or Ab-secreting cell responses in volunteers receiving the lowest inoculum, other dose groups had moderate to strong anti-LPS and anti-Ipa responses. These results suggest that in humans, type 1 responses play an important role in mucosal and systemic immunity to S. dysentariae 1.

Shigellosis is an important disease in the developing world, where about 90 million people become infected with Shigella spp. each year. We previously demonstrated that the type three secretion apparatus (T3SA) proteins IpaB and IpaD are protective antigens in the mouse lethal pulmonary model. In order to simplify vaccine formulation and process development, we have evaluated a vaccine design that incorporates both of these previously tested Shigella antigens into a single polypeptide chain. To determine if this fusion protein (DB fusion) retains the antigenic and protective capacities of IpaB and IpaD, we immunized mice with the DB fusion and compared the immune response to that elicited by the IpaB/IpaD combination vaccine. Purification of the DB fusion required coexpression with IpgC, the IpaB chaperone, and after purification it maintained the highly α-helical characteristics of IpaB and IpaD. The DB fusion also induced comparable immune responses and retained the ability to protect mice against Shigella flexneri and S. sonnei in the lethal pulmonary challenge. It also offered limited protection against S. dysenteriae challenge. Our results show the feasibility of generating a protective Shigella vaccine comprised of the DB fusion.

The type III secretion system (T3SS) is an essential virulence factor for Shigella flexneri , providing a conduit through which host-altering effectors are injected directly into a host cell to promote uptake. The type III secretion apparatus (T3SA) is composed of a basal body, external needle, and regulatory tip complex. The nascent needle is a polymer of MxiH capped by a pentamer of invasion plasmid antigen D (IpaD). Exposure to bile salts (e.g., deoxycholate) causes a conformational change in IpaD and promotes recruitment of IpaB to the needle tip. It has been proposed that IpaB senses contact with host cell membranes, recruiting IpaC and inducing full secretion of T3SS effectors. Although the steps of T3SA maturation and their external triggers have been identified, details of specific protein interactions and mechanisms have remained difficult to study because of the hydrophobic nature of the IpaB and IpaC translocator proteins. Here, we explored the ability for a series of soluble N-terminal IpaB peptides to interact with IpaD. We found that DOC is required for the interaction and that a region of IpaB between residues 11-27 is required for maximum binding, which was confirmed in vivo. Furthermore, intramolecular FRET measurements indicated that movement of the IpaD distal domain away from the protein core accompanied the binding of IpaB11-226. Together, these new findings provide important new insight into the interactions and potential mechanisms that define the maturation of the Shigella T3SA needle tip complex and provide a foundation for further studies probing T3SS activation.

An important consideration in the development of subunit vaccines is the loss of activity caused by physical instability of the protein. Such instability often results from suboptimal solution conditions related to pH and temperature. Excipients can help to stabilize vaccines, but it is important to screen and identify excipients that adequately contribute to stabilization of a given formulation. CagL is a protein present in strains of Helicobacter pylori (H. pylori) that possess type IV secretion systems. It contributes to bacterial adherence via a5$1 integrin, thereby making it an attractive subunit vaccine candidate. We characterized the stability of CagL in different pH and temperature conditions using a variety of spectroscopic techniques. Stability was assessed in terms of transition temperature with the accumulated data, and then incorporated into an empirical phase diagram (EPD) that provided an overview of CagL physical stability. These analyses indicated maximum CagL stability at pH4-6 up to 40°C in the absence of excipient. Using this EPD analysis, aggregation assays were developed to screen a panel of excipients with some found to inhibit CagL aggregation. Candidate stabilizers were selected to confirm their enhanced stabilizing effect. These analyses will help in the formulation of a stable vaccine against H. pylori. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2508-2519, 2013

Shigella spp. are the causative agent of shigellosis, the second leading cause of diarrhea in children of ages 2–5. Despite many years of research, a protective vaccine has been elusive. We recently demonstrated that invasion plasmid antigens B and D (IpaB and IpaD) provide protection against S. flexneri and S. sonnei. These proteins, however, have very different properties which must be recognized and then managed during vaccine formulation. Herein, we employ spectroscopy to assess the stability of IpaB as well as IpgC (invasion protein gene), IpaB's cognate chaperone, and the IpaB/IpgC complex. The resulting data are mathematically summarized into a visual map illustrating the stability of the proteins and their complex as a function of pH and temperature. The IpaB/IpgC complex exhibits thermal stability at higher pH values but, though initially stable, quickly unfolds with increasing temperature when maintained at lower pH. In contrast, IpaB is a much more complex protein exhibiting increased stability at higher pH, but shows initial instability at lower pH values with pH 5 showing a distinct transition. IpgC precipitates at and below pH 5 and is stable above pH 7. Most strikingly, it is clear that complex formation results in stabilization of the two components. This work serves as a basis for the further development of IpaB as a vaccine candidate as well as extends our understanding of the structural stability of the Shigella type III secretion system.

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.

Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SS) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) which is responsible for over one million deaths per year. The Shigella type III secretion apparatus (T3SA) is comprised of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 Å and 2.8 Å limiting resolution, respectively. These newly identified domains are comprised of extended length (114 Å in IpaB and 71 Å in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably colicin Ia. This suggests that these mechanistically-separate and functionally-distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.

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. Forster 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 virulence of many pathogenic Gram-negative bacteria is dependent upon their type III secretion (TTS) systems. Here, we discuss initial formulation studies of five TTS needle tip proteins IpaD (Shigella flexneri), BipD (Burkholderia pseudomallei), SipD (Salmonella spp.), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) as targets for subunit vaccines. Excipient screening and subsequent assays lead to the selection of 10% sucrose and 5% dextrose as an optimal stabilizer combination for all five proteins. All of the proteins adsorb to aluminum hydroxide adjuvant, although the mechanisms of adsorption may vary. The proteins are physically stable when adsorbed to the adjuvant for at least 3 months at room temperature and chemical stability is enhanced in the presence of excipients. The ability of the IpaD and SipD proteins to elicit strong humoral immune responses was also tested in a murine model in the presence and absence of their needle counterparts MxiH and PrgI (see previous paper in this issue). Both proteins produce high antibody titers regardless of dose. While the IpaD titer is boosted slightly in the presence of its needle protein, MxiH, SipD titers appear to be reduced when administered in the presence of its needle counterpart, PrgI.

Many Gram-negative bacterial pathogens use type III secretion systems (TTSSs) for subverting the normal cellular functions of their target eukaryotic cells. The type III secretion apparatus (TTSA) functions like a syringe to inject proteins through an external needle and into a target cell's membrane and cytosol. The TTSA basal body spans the bacterial inner and outer membranes, and the external needle is topped with a tip complex that controls the secretion and delivery of translocator and effector proteins. Recently solved structures of TTSA proteins have greatly advanced our understanding of shared themes in apparatus assembly and function. In this highlight, the structure-function of TTSA needle and tip complex proteins are described and common themes discussed.

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 were90% 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.

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.

The results from experiments involving nonradiative energy transfer indicate that a fluorescent probe on the 5'-end of tRNA(Phe) moves more than 20 A towards probes on ribosomal protein L1 as a peptide bond is formed during the peptidyl transferase reaction on Escherichia coli ribosomes. The peptide itself moves no more than a few angstroms during peptide bond formation, as judged by the movement of fluorescent probes attached to the phenylalanine amino group of phenylalanyl-tRNA. Other results demonstrate that an analogue of peptidyl-tRNA, deacylated tRNA, and puromycin can be bound simultaneously to the same ribosome, indicating that there are three physically distinct sites to which tRNA is bound during the reaction steps by which peptides are elongated. The results appear to be consistent with the displacement model of peptide elongation.

Shigella flexneri causes a severe form of bacillary dysentery also known as shigellosis. Onset of shigellosis requires bacterial invasion of colonic epithelial cells which is initiated by the delivery of translocator and effector proteins to the host cell membrane and cytoplasm, respectively, by the Shigella type III secretion system (TTSS). The Shigella translocator proteins, IpaB and IpaC, form a pore complex in the host cell membrane to facilitate effector delivery; however, prior to their secretion IpaB and IpaC are partitioned in the bacterial cytoplasm by association with the cytoplasmic chaperone IpgC. To determine their structural and biophysical properties, recombinant IpaB/IpgC and IpaC/IpgC complexes were prepared for their first detailed in vitro analysis. Both IpaB/IpgC and IpaC/IpgC complexes are highly stable and soluble heterodimers whose formation prevents IpaB-IpaC interaction as well as Ipa-dependent disruption of phospholipid membranes. Circular dichroism spectroscopy shows that IpgC binding has a detectable influence on IpaC secondary/tertiary structure and stability. In contrast, IpaB structure is not as dramatically affected by chaperone binding. To more precisely ascertain the influence of chaperone binding on IpaC structure and stability, single tryptophan mutants were generated for detailed fluorescence spectroscopy analysis. These mutants provide a low-resolution picture of how IpaC exists in the Shigella cytoplasm with chaperone binding possibly involving distinct regions within the N- and C-terminal halves of IpaC. This preliminary assessment of the IpaC-IpgC interaction is supported by initial deletion mutagenesis studies. The data provide the first structural analysis of IpgC association with IpaB and IpaC.

Diverse Gram-negative bacteria use type III secretion systems (T3SS) to translocate effector proteins into the cytoplasm of eukaryotic cells. The type III secretion apparatus (T3SA) consists of a basal body spanning both bacterial membranes and an external needle. A sensor protein lies at the needle tip to detect environmental signals that trigger type III secretion. The Shigella flexneri T3SA needle tip protein, invasion plasmid antigen D (IpaD), possesses two independently folding domains in vitro. In this study, the solution behavior and thermal unfolding properties of IpaD's functional homologs SipD (Salmonella spp.), BipD (Burkholderia pseudomallei), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) were examined to identify common features within this protein family. CD and FTIR data indicate that all members within this group are alpha-helical with properties consistent with an intramolecular coiled-coil. SipD showed the most complex unfolding profile consisting of two thermal transitions, suggesting the presence of two independently folding domains. No evidence of multiple folding domains was seen, however, for BipD, LcrV, or PcrV. Thermal studies, including DSC, revealed significant destabilization of LcrV, PcrV, and BipD after N-terminal deletions. This contrasted with SipD and IpaD, which behaved like two-domain proteins. The results suggest that needle tip proteins share significant core structural similarity and thermal stability that may be the basis for their common function. Moreover, IpaD and SipD possess properties that distinguish them from the other tip proteins.

IpaD, the putative needle-tip protein of the S. flexneri type III secretion system, has been crystallized in a variety of crystal forms using in-drop proteolysis. Native and selenomethionine-labelled data collection and preliminary analyses are reported., IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P212121, with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 Å, and data were collected to 2.9 Å resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 Å resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 Å, β = 107.9°. An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit.

Type III secretion systems are essential virulence determinants for many Gram-negative bacterial pathogens. The type III secretion system consists of cytoplasmic, transmembrane, and extracellular domains. The extracellular domain is a hollow needle protruding above the bacterial surface and is held within a basal body that traverses both bacterial membranes. Effector proteins are translocated, via this external needle, directly into host cells, where they subvert normal cell functions to aid infection. Physical contact with host cells initiates secretion and leads to formation of a pore, thought to be contiguous with the needle channel, in the host-cell membrane. Here, we report the crystal structure of the Shigella flexneri needle subunit MxiH and a complete model for the needle assembly built into our three-dimensional EM reconstruction. The model, combined with mutagenesis data, reveals that signaling of host-cell contact is relayed through the needle via intersubunit contacts and suggests a mode of binding for a tip complex.

Shigella flexneri , the causative agent of shigellosis, is a gram-negative bacterial pathogen that initiates infection by invading cells within the colonic epithelium. Contact with host cell surfaces induces a rapid burst of protein secretion via the Shigella type III secretion system (TTSS). The first proteins secreted are IpaD, IpaB, and IpaC, with IpaB and IpaC being inserted into the host cell membrane to form a pore for translocating late effectors into the target cell cytoplasm. The resulting pathogen-host cross talk results in localized actin polymerization, membrane ruffling, and, ultimately, pathogen entry. IpaD is essential for host cell invasion, but its role in this process is just now coming to light. IpaD is a multifunctional protein that controls the secretion and presentation of IpaB and IpaC at the pathogen-host interface. We show here that antibodies recognizing the surface-exposed N terminus of IpaD neutralize Shigella 's ability to promote pore formation in erythrocyte membranes. We further show that MxiH and IpaD colocalize on the bacterial surface. When TTSS needles were sheared from the Shigella surface, IpaD was found at only the needle tips. Consistent with this, IpaD localized to the exposed tips of needles that were still attached to the bacterium. Molecular analyses then showed that the IpaD C terminus is required for this surface localization and function. Furthermore, mutations that prevent IpaD surface localization also eliminate all IpaD-related functions. Thus, this study demonstrates that IpaD localizes to the TTSA needle tip, where it functions to control the secretion and proper insertion of translocators into host cell membranes.

A monodisperse truncation mutant of MxiH, the subunit of the S. flexneri type III secretion system needle, has been crystallized. SeMet derivatives and a uranyl derivative have undergone preliminary crystallographic analysis., A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiHCΔ5 and diffraction data were collected to 1.9 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 Å, β = 96.5°. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 Å3 Da−1 for two molecules per asymmetric unit, corresponding to a solvent content of 33%.

Gram-negative bacteria commonly interact with eukaryotic host cells using type III secretion systems (TTSSs or secretons), which comprise cytoplasmic, transmembrane and extracellular domains. The extracellular domain is a hollow needle-like structure protruding 60 nm beyond the bacterial surface. The TTSS is activated to transfer bacterial proteins directly into a host cell only upon physical contact with the target cell. We showed previously that the monomer of the Shigella flexneri needle, MxiH, assembles into a helical structure with parameters similar to those defining the architecture of the extracellular components of bacterial flagella. By analogy with flagella, which are known to exist in different helical states, we proposed that changes in the helical packing of the needle might be used to sense host cell contact. Here, we show that, on the contrary, mutations within MxiH that lock the TTSS into altered secretion states do not detectably alter the helical packing of needles. This implies that either: (1) host cell contact is signalled through the TTSS via helical changes in the needle that are significantly smaller than those linked to structural changes in the flagellar filament and therefore too small to be detected by our analysis methods or (2) that signal transduction in this system occurs via a novel molecular mechanism.

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.

The primary effector for Shigella invasion of epithelial cells is IpaC, which is secreted via a type III secretion system. We recently reported that the IpaC N terminus is required for type III secretion and possibly other functions. In this study, mutagenesis was used to identify an N-terminal secretion signal and to determine the functional importance of the rest of the IpaC N terminus. The 15 N-terminal amino acids target IpaC for secretion by Shigella flexneri, and placing additional amino acids at the N terminus does not interfere with IpaC secretion. Furthermore, amino acid sequences with no relationship to the native IpaC secretion signal can also direct its secretion. Deletions introduced beyond amino acid 20 have no effect on secretion and do not adversely affect IpaC function in vivo until they extend beyond residue 50, at which point invasion function is completely eliminated. Deletions introduced at amino acid 100 and extending toward the N terminus reduce IpaC's invasion function but do not eliminate it until they extend to the N-terminal side of residue 80, indicating that a region from amino acid 50 to 80 is critical for IpaC invasion function. To explore this further, the ability of an IpaC N-terminal peptide to associate in vitro with its translocon partner IpaB and its chaperone IpgC was studied. The N-terminal peptide binds tightly to IpaB, but the IpaC central hydrophobic region also appears to participate in this binding. The N-terminal peptide also associates with the chaperone IpgC and IpaB is competitive for this interaction. Based on additional biophysical data, we propose that a region between amino acids 50 and 80 is required for chaperone binding, and that the IpaB binding domain is located downstream from, and possibly overlapping, this region. From these data, we propose that the secretion signal, chaperone binding region, and IpaB binding domain are located at the IpaC N terminus and are essential for presentation of IpaC to host cells during bacterial entry; however, IpaC effector activity may be located elsewhere.