Structure and Dynamic Studies of the Nuclear Pore Complex at the Single-Molecule Level

Nuclear pore complexes (NPCs) are large macromolecular structures forming the only known direct route across the double bilayer membrane of the nuclear envelope. The NPC structure has been extensively explored in an effort to elucidate the mechanisms by which they control transport. Many of these studies have found the presence of a central mass or plug within the central channel of NPCs, although neither the function nor identity of the central mass were clear. Here, several techniques including electron microscopy, Förster resonance energy transfer (FRET), and high-resolution near-field scanning optical microscopy (NSOM) are utilized to specifically locate vault ribonucleoproteins to NPCs. This interaction, along with several other results, strongly suggests that vaults represent the central mass of NPCs. A single-molecule transport assay was also developed in order to record the translocation of individual fluorescent dextrans through NPCs. Comparison of the single-molecule dwell times under various conditions led to a better understanding of the specific mechanism controlling the non signal-mediated transport of cargo through NPCs

Phosphomimetic Tyrosine Mutations in Spa47 Inhibit Type Three Secretion ATPase Activity and Shigella Virulence Phenotype

Shigella is a highly infectious human pathogen responsible for 269 million infections and 200,000 deaths per year. Shigella virulence is absolutely reliant on the injection of effector proteins into the host cell cytoplasm via its type three secretion system (T3SS). The protein Spa47 is a T3SS ATPase whose activity is essential for the proper function of the Shigella T3SS needle-like apparatus through which effectors are secreted. A phosphoproteomics study recently found several Shigella T3SS proteins, including Spa47, to be tyrosine phosphorylated, suggesting a means of regulating Spa47 enzymatic activity, T3SS function, and overall Shigella virulence. The work presented here employs phosphomimetic mutations in Spa47 to probe the effects of phosphorylation at these targeted tyrosines through in vitro radiometric ATPase assays and circular dichroism as well as in vivo characterization of T3SS secretion activity, erythrocyte hemolysis, and cellular invasion. Results presented here demonstrate a direct correlation between Spa47 tyrosine phosphorylation state, Spa47 ATPase activity, T3SS function, and Shigella virulence. Together, these findings provide a strong foundation that leads the way to uncovering the specific pathway(s) that Shigella employ to mitigate wasteful ATP hydrolysis and effector protein secretion when not required as well as T3SS activation in preparation for host infection and immune evasion

Kinetic Characterization of the Shigella Type Three Secretion System ATPase Spa47 Using α-32P ATP

ATPases represent a diverse class of enzymes that utilize ATP hydrolysis to support critical biological functions such as driving ion pumps, providing mechanical work, unfolding/folding proteins, and supporting otherwise thermodynamically unfavorable chemical reactions. We have recently shown that the Shigella protein Spa47 is an ATPase that supports protein secretion through its specialized type three secretion apparatus (T3SA), supporting infection of human host cells. Characterizing ATPases, such as Spa47, requires a means to accurately determine enzyme activity (ATP hydrolysis) as a function of time, reaction conditions, and potential cofactors, regulators, inhibitors, etc. Here, we describe a detailed protocol for characterizing the enzyme kinetics of Spa47 using a direct α−32P ATPase assay

Characterization of Power Induced Heating and Damage in Fiber Optic Probes for Near-Field Scanning Optical Microscopy

Tip-induced sample heating in near-field scanning optical microscopy (NSOM) is studied for fiber optic probes fabricated using the chemical etching technique. To characterize sample heating from etched NSOM probes, the spectra of a thermochromic polymer sample are measured as a function of probe output power, as was previously reported for pulled NSOM probes. The results reveal that sample heating increases rapidly to ~55–60°C as output powers reach ~50 nW. At higher output powers, the sample heating remains approximately constant up to the maximum power studied of ~450 nW. The sample heating profiles measured for etched NSOM probes are consistent with those previously measured for NSOM probes fabricated using the pulling method. At high powers, both pulled and etched NSOM probes fail as the aluminum coating is damaged. For probes fabricated in our laboratory we find failure occurring at input powers of 3.4 ± 1.7 and 20.7 ± 6.9 mW for pulled and etched probes, respectively. The larger half-cone angle for etched probes (∼15° for etched and ~6° for pulled probes) enables more light delivery and also apparently leads to a different failure mechanism. For pulled NSOM probes, high resolution images of NSOM probes as power is increased reveal the development of stress fractures in the coating at a taper diameter of ~6μm. These stress fractures, arising from the differential heating expansion of the dielectric and the metal coating, eventually lead to coating removal and probe failure. For etched tips, the absence of clear stress fractures and the pooled morphology of the damaged aluminum coating following failure suggest that thermal damage may cause coating failure, although other mechanisms cannot be ruled out

Characterization of power induced heating and damage in fiber optic probes for near-field scanning optical microscopy

This is the published version, also available here: http://dx.doi.org/10.1063/1.2740133.Tip-induced sample heating in near-field scanning optical microscopy (NSOM) is studied for fiber optic probes fabricated using the chemical etching technique. To characterize sample heating from etched NSOM probes, the spectra of a thermochromic polymer sample are measured as a function of probe output power, as was previously reported for pulled NSOM probes. The results reveal that sample heating increases rapidly to ∼55–60°C as output powers reach ∼50nW. At higher output powers, the sample heating remains approximately constant up to the maximum power studied of ∼450nW. The sample heating profiles measured for etched NSOM probes are consistent with those previously measured for NSOM probes fabricated using the pulling method. At high powers, both pulled and etched NSOM probes fail as the aluminumcoating is damaged. For probes fabricated in our laboratory we find failure occurring at input powers of 3.4±1.7 and 20.7±6.9mW for pulled and etched probes, respectively. The larger half-cone angle for etched probes (∼15° for etched and ∼6° for pulled probes) enables more light delivery and also apparently leads to a different failure mechanism. For pulled NSOM probes, high resolution images of NSOM probes as power is increased reveal the development of stress fractures in the coating at a taper diameter of ∼6μm. These stress fractures, arising from the differential heating expansion of the dielectric and the metalcoating, eventually lead to coating removal and probe failure. For etched tips, the absence of clear stress fractures and the pooled morphology of the damaged aluminumcoating following failure suggest that thermal damage may cause coating failure, although other mechanisms cannot be ruled out

Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from \u3cem\u3eShigella flexneri\u3c/em\u3e

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents

Dominant Negative Effects by Inactive Spa47 Mutants Inhibit T3SS Function and Shigella Virulence

Type three secretion systems (T3SS) are complex nano-machines that evolved to inject bacterial effector proteins directly into the cytoplasm of eukaryotic cells. Many high-priority human pathogens rely on one or more T3SSs to cause disease and evade host immune responses, underscoring the need to better understand the mechanisms through which T3SSs function and their role(s) in supporting pathogen virulence. We recently identified the Shigella protein Spa47 as an oligomerization-activated T3SS ATPase that fuels the T3SS and supports overall Shigella virulence. Here, we provide both in vitro and in vivo characterization of Spa47 oligomerization and activation in the presence and absence of engineered ATPase-inactive Spa47 mutants. The findings describe mechanistic details of Spa47-catalyzed ATP hydrolysis and uncover critical distinctions between oligomerization mechanisms capable of supporting ATP hydrolysis in vitro and those that support T3SS function in vivo. Concentration-dependent ATPase kinetics and experiments combining wild-type and engineered ATPase inactive Spa47 mutants found that monomeric Spa47 species isolated from recombinant preparations exhibit low-level ATPase activity by forming short-lived oligomers with active site contributions from at least two protomers. In contrast, isolated Spa47 oligomers exhibit enhanced ATP hydrolysis rates that likely result from multiple preformed active sites within the oligomeric complex, as is predicted to occur within the context of the type three secretion system injectisome. High-resolution fluorescence microscopy, T3SS activity, and virulence phenotype analyses of Shigella strains co-expressing wild-type Spa47 and the ATPase inactive Spa47 mutants demonstrate that the N-terminus of Spa47, not ATPase activity, is responsible for incorporation into the injectisome where the mutant strains exhibit a dominant negative effect on T3SS function and Shigella virulence. Together, the findings presented here help to close a significant gap in our understanding of how T3SS ATPases are activated and define restraints with respect to how ATP hydrolysis is ultimately coupled to T3SS function in vivo

Influence of Oligomerization State on the Structural Properties of Invasion Plasmid Antigen B (IpaB) from Shigella flexneri in the Presence and Absence of Phospholipid Membranes

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 (IpaD) lies at the nascent needle tip and IpaB is recruited into the needle tip complex upon 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

Interfacial Amino Acids Support Spa47 Oligomerization and Shigella Type Three Secretion System Activation

Like many Gram-negative pathogens, Shigella rely on a type three secretion system (T3SS) for injection of effector proteins directly into eukaryotic host cells to initiate and sustain infection. Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47, making it a likely target for in vivo regulation of T3SS activity and an attractive target for small molecule therapeutics against shigellosis. Here, we developed a model of an activated Spa47 homo-hexamer, identifying two distinct regions at each protomer interface that we hypothesized to provide intermolecular interactions supportingSpa47 oligomerization and enzymatic activation. Mutational analysis and a series of high-resolution crystal structures confirm the importance of these residues, as many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro. Furthermore, in vivo evaluation of Shigella virulence phenotype uncovered a strong correlation between T3SS effector protein secretion, host cell membrane disruption, and cellular invasion by the tested mutant strains, suggesting that perturbation of the identified interfacial residues/interactions influences Spa47 activity through preventing oligomer formation, which in turn regulates Shigella virulence. The most impactful mutations are observed within the conserved Site 2 interface where the native residues support oligomerization and likely contribute to a complex hydrogen bonding network that organizes the active site and supports catalysis. The critical reliance on these conserved residues suggests that aspects of T3SS regulation may also be conserved, providing promise for the development of a cross-species therapeutic that broadly targetsT3SS ATPase oligomerization and activation

Liposomes Recruit IpaC to the Shigella flexneri Type III Secretion Apparatus Needle as a Final Step in Secretion Induction

Shigella flexneri contact with enterocytes induces a burst of protein secretion via its type III secretion apparatus (TTSA) as an initial step in cellular invasion. We have previously reported that IpaD is positioned at the TTSA needle tip (M. Espina et al., Infect. Immuno. 74:4391-4400, 2006). From this position, IpaD senses small molecules in the environment to control the presentation of IpaB to the needle tip. This step occurs without type III secretion induction or IpaC recruitment to the S. flexneri surface. IpaC is then transported to the S. flexneri surface when target cell lipids are added, and this event presumably mimics host cell contact. Unlike IpaB mobilization, IpaC surface presentation is closely linked to secretion induction. This study demonstrates that sphingomyelin and cholesterol are key players in type III secretion induction and that they appear to interact with IpaB to elicit IpaC presentation at the TTSA needle tip. Furthermore, IpaB localization at the needle tip prior to membrane contact provides the optimal set of conditions for host cell invasion. Thus, the S. flexneri type III secretion system can be induced in a stepwise manner, with the first step being the stable association of IpaD with the needle tip, the second step being the sensing of small molecules by IpaD to mobilize IpaB to the tip, and the third step being the interaction of lipids with IpaB to induce IpaC localization at the needle tip concomitant with translocon insertion into the host membrane and type III secretion induction. Shigella flexneri, the causative agent of shigellosis, is responsible for more than 1 million deaths each year, especially among children in developing regions (www.who.int/vaccines-documents/DocsPDF99/www9947.pdf). Once ingested, S. flexneri crosses M cells and passes into the underlying gut-associated lymphoid tissues of the colon (20), where it kills macrophages (29) and then invades epithelial cells by macropinocytosis (17). The S. flexneri invasive phenotype localizes genetically to a 31-kb region of its large virulence plasmid and is absolutely tied to its type III secretion system (TTSS) (6, 23). TTSSs are used by numerous gram-negative bacteria to introduce bacterially derived effector proteins into the membrane and cytoplasm of a target cell, resulting in the subversion of normal cell functions (8). Linking the bacterium and host cell in this process is the type III secretion apparatus (TTSA), which structurally resembles a molecular needle and syringe. The system is controlled by a basal body (the syringe) that spans both bacterial membranes and an external needle that provides a conduit from the basal body to the sensory needle tip complex (8, 28). The needle in S. flexneri is comprised of a polymer of MxiH and is approximately 50 nm long and 7 nm in diameter, with a central channel that is about 2.5 nm in diameter (5). At the top of the MxiH needle resides the tip protein IpaD, most likely as a pentamer, which serves as an environmental sensor for the MxiH-IpaD tip complex (4, 7). When the presence of bile salts such as deoxycholate (DOC) is sensed by IpaD, the first translocator protein, IpaB, is mobilized to the TTSA needle tip to form an MxiH-IpaD-IpaB ternary complex. At this stage, the TTSA structure is primed for subsequent host cell contact (19, 24). In previous studies IpaC had not been found to localize to the S. flexneri surface of the log-phase bacterium (7, 19). As a next step in describing the process of type III secretion, we show here that liposomes trigger mobilization of IpaC to the needle tip complex, where it is immediately inserted into the host cell membrane, along with IpaB, to complete the TTSA conduit into the host cell just prior to initiating host cytoskeleton rearrangements. IpaC is most efficiently recruited to the S. flexneri surface with a defined liposome composition that includes phospholipids, sphingomyelin (SM), and cholesterol (Chol). Furthermore, IpaC recruitment occurs concomitantly with induction of type III secretion of IpaB, IpaC, and IpaD into the S. flexneri culture supernatant. It thus appears that IpaB mobilization to the S. flexneri TTSA needle tip represents a second discrete step in TTSA assembly, with the final third step being IpaC recruitment to the needle tip, which occurs after IpaB contacts and inserts into the host cell membrane