Cryo-EM structure of the Shigella type III needle complex.

The Type III Secretion Systems (T3SS) needle complex is a conserved syringe-shaped protein translocation nanomachine with a mass of about 3.5 MDa essential for the survival and virulence of many Gram-negative bacterial pathogens. This system is composed of a membrane-embedded basal body and an extracellular needle that deliver effector proteins into host cells. High-resolution structures of the T3SS from different organisms and infection stages are needed to understand the underlying molecular mechanisms of effector translocation. Here, we present the cryo-electron microscopy structure of the isolated Shigella T3SS needle complex. The inner membrane (IM) region of the basal body adopts 24-fold rotational symmetry and forms a channel system that connects the bacterial periplasm with the export apparatus cage. The secretin oligomer adopts a heterogeneous architecture with 16- and 15-fold cyclic symmetry in the periplasmic N-terminal connector and C-terminal outer membrane ring, respectively. Two out of three IM subunits bind the secretin connector via a ß-sheet augmentation. The cryo-EM map also reveals the helical architecture of the export apparatus core, the inner rod, the needle and their intervening interfaces.

Role of flagellar hydrogen bonding in Salmonella motility and flagellar polymorphic transition.

Bacterial flagellar filaments are assembled by tens of thousands flagellin subunits, forming 11 helically arranged protofilaments. Each protofilament can take either of the two bistable forms L-type or R-type, having slightly different conformations and inter-protofilaments interactions. By mixing different ratios of L-type and R-type protofilaments, flagella adopt multiple filament polymorphs and promote bacterial motility. In this study, we investigated the hydrogen bonding networks at the flagellin crystal packing interface in Salmonella enterica serovar typhimurium (S. typhimurium) by site-directed mutagenesis of each hydrogen bonded residue. We identified three flagellin mutants D108A, N133A and D152A that were non-motile despite their fully assembled flagella. Mutants D108A and D152A trapped their flagellar filament into inflexible right-handed polymorphs, which resemble the previously predicted 3L/8R and 4L/7R helical forms in Calladine's model but have never been reported in vivo. Mutant N133A produces floppy flagella that transform flagellar polymorphs in a disordered manner, preventing the formation of flagellar bundles. Further, we found that the hydrogen bonding interactions around these residues are conserved and coupled to flagellin L/R transition. Therefore, we demonstrate that the hydrogen bonding networks formed around flagellin residues D108, N133 and D152 greatly contribute to flagellar bending, flexibility, polymorphisms and bacterial motility.

Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productive Salmonella gut colonisation.

The flagellum is a sophisticated nanomachine and an important virulence factor of many pathogenic bacteria. Flagellar motility enables directed movements towards host cells in a chemotactic process, and near-surface swimming on cell surfaces is crucial for selection of permissive entry sites. The long external flagellar filament is made of tens of thousands subunits of a single protein, flagellin, and many Salmonella serovars alternate expression of antigenically distinct flagellin proteins, FliC and FljB. However, the role of the different flagellin variants during gut colonisation and host cell invasion remains elusive. Here, we demonstrate that flagella made of different flagellin variants display structural differences and affect Salmonella's swimming behaviour on host cell surfaces. We observed a distinct advantage of bacteria expressing FliC-flagella to identify target sites on host cell surfaces and to invade epithelial cells. FliC-expressing bacteria outcompeted FljB-expressing bacteria for intestinal tissue colonisation in the gastroenteritis and typhoid murine infection models. Intracellular survival and responses of the host immune system were not altered. We conclude that structural properties of flagella modulate the swimming behaviour on host cell surfaces, which facilitates the search for invasion sites and might constitute a general mechanism for productive host cell invasion of flagellated bacteria.

Crystal structure of PrgI-SipD: insight into a secretion competent state of the type three secretion system needle tip and its interaction with host ligands.

Many infectious gram-negative bacteria, including Salmonella typhimurium, require a Type Three Secretion System (T3SS) to translocate virulence factors into host cells. The T3SS consists of a membrane protein complex and an extracellular needle together that form a continuous channel. Regulated secretion of virulence factors requires the presence of SipD at the T3SS needle tip in S. typhimurium. Here we report three-dimensional structures of individual SipD, SipD in fusion with the needle subunit PrgI, and of SipD:PrgI in complex with the bile salt, deoxycholate. Assembly of the complex involves major conformational changes in both SipD and PrgI. This rearrangement is mediated via a π bulge in the central SipD helix and is stabilized by conserved amino acids that may allow for specificity in the assembly and composition of the tip proteins. Five copies each of the needle subunit PrgI and SipD form the T3SS needle tip complex. Using surface plasmon resonance spectroscopy and crystal structure analysis we found that the T3SS needle tip complex binds deoxycholate with micromolar affinity via a cleft formed at the SipD:PrgI interface. In the structure-based three-dimensional model of the T3SS needle tip, the bound deoxycholate faces the host membrane. Recently, binding of SipD with bile salts present in the gut was shown to impede bacterial infection. Binding of bile salts to the SipD:PrgI interface in this particular arrangement may thus inhibit the T3SS function. The structures presented in this study provide insight into the open state of the T3SS needle tip. Our findings present the atomic details of the T3SS arrangement occurring at the pathogen-host interface.

Integrative structural analysis of the type III secretion system needle complex from Shigella flexneri.

The type III secretion system (T3SS) is a large, transmembrane protein machinery used by various pathogenic gram-negative bacteria to transport virulence factors into the host cell during infection. Understanding the structure of T3SSs is crucial for future developments of therapeutics that could target this system. However, much of the knowledge about the structure of T3SS is available only for Salmonella, and it is unclear how this large assembly is conserved across species. Here, we combined cryo-electron microscopy, cross-linking mass spectrometry, and integrative modeling to determine the structure of the T3SS needle complex from Shigella flexneri. We show that the Shigella T3SS exhibits unique features distinguishing it from other structurally characterized T3SSs. The secretin pore complex adopts a new fold of its C-terminal S domain and the pilotin MxiM[SctG] locates around the outer surface of the pore. The export apparatus structure exhibits a conserved pseudohelical arrangement but includes the N-terminal domain of the SpaS[SctU] subunit, which was not present in any of the previously published virulence-related T3SS structures. Similar to other T3SSs, however, the apparatus is anchored within the needle complex by a network of flexible linkers that either adjust conformation to connect to equivalent patches on the secretin oligomer or bind distinct surface patches at the same height of the export apparatus. The conserved and unique features delineated by our analysis highlight the necessity to analyze T3SS in a species-specific manner, in order to fully understand the underlying molecular mechanisms of these systems. The structure of the type III secretion system from Shigella flexneri delineates conserved and unique features, which could be used for the development of broad-range therapeutics.

Synthetic polyamines as potential amine oxidase inhibitors: a preliminary study.

In the last few decades, medicinal chemists have carried out extensive research on synthetic polyamines for use as anticancer drugs and multitarget-directed ligands in neurodegenerative diseases. The aim of this study was to evaluate the effect of some synthetic polyamines as inhibitors of two new potential targets, human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1) and monoamine oxidases B (MAO B), enzymes involved in various multi-factorial diseases such as Alzheimer's disease. N,N'-Dibenzyl-dodecane-1,12-diamine (Bis-Bza-Diado), a newly synthesised compound, and ELP 12, a muscarinic cholinergic M(2) receptor antagonist, were found to behave as reversible and mixed non-competitive inhibitors of both amine oxidases (dissociation constants of about 100 µM). ELP 12 was found to be more selective for SSAO/VAP-1. Combining kinetic and structural approaches, the binding mode of ELP 12 to SSAO/VAP-1 was investigated. ELP 12 may bind at the entrance of the active site channel by ionic interactions with ASP446 and/or ASP180; one end of the polyamine may be accommodated inside the channel, reaching the TPQ cofactor area. The binding of ELP 12 induces rearrangement of the secondary structure of the enzyme and impedes substrate entry and/or product release and catalysis. These structural data reveal that the entrance and the first part of the SSAO/VAP-1 channel may be considered as a new target area, or a "secondary binding site", for modulators of human SSAO/VAP-1 activity. These results indicate ELP 12 and Bis-Bza-Diado as new "skeletons" for the development of novel SSAO/VAP-1 and MAO B inhibitors.

IpaB-IpgC interaction defines binding motif for type III secretion translocator.

The delivery of virulence factors into host cells through type III secretion systems is essential for enterobacterial pathogenesis. Molecular chaperones bind specifically to virulence factors in the bacterial cytosol before secretion. Invasion plasmid gene C (IpgC) is a chaperone that binds 2 essential virulence factors of Shigella: invasion plasmid antigens (Ipa) B and C. Here, we report the crystal structure of IpgC alone and in complex with the chaperone binding domain (CBD) of IpaB. The chaperone captures the CBD in an extended conformation that is stabilized by conserved residues lining the cleft. Analysis of the cocrystal structure reveals a sequence motif that is functional in the IpaB translocator class from different bacteria as determined by isothermal titration calorimetry. Our results show how translocators are chaperoned and may allow the design of inhibitors of enterobacterial diseases.

Combination of two separate binding domains defines stoichiometry between type III secretion system chaperone IpgC and translocator protein IpaB.

Type III secretion systems (TTSSs) utilized by enteropathogenic bacteria require the presence of small, acidic virulence-associated chaperones for effective host cell infection. We adopted a combination of biochemical and cellular techniques to define the chaperone binding domains (CBDs) in the translocators IpaB and IpaC associated with the chaperone IpgC from Shigella flexneri. We identified a novel CBD in IpaB and furthermore precisely mapped the boundaries of the CBDs in both translocator proteins. In IpaC a single binding domain associates with IpgC. In IpaB, we show that the binding of the newly characterized CBD is essential in maintaining the ternary arrangement of chaperone-translocator complex. This hitherto unknown function is reflected in the co-crystal structure as well, with an IpgC dimer bound to an IpaB fragment comprising both CBDs. Moreover, in the absence of this novel CBD the IpaB/IpgC complex aggregates. This dual-recognition of a domain in the protein by the chaperone in facilitating the correct chaperone-substrate organization describes a new function for the TTSS associated chaperone-substrate complexes.

Helical reconstruction of Salmonella and Shigella needle filaments attached to type 3 basal bodies.

Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of Salmonella PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development.

Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

Structural analysis of ligand-bound states of the Salmonella type III secretion system ATPase InvC.

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram-negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone-effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N-terminal domains. Using size-exclusion chromatography coupled to multi-angle light scattering and native mass spectrometry, we show that in the absence of the N-terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone-effector recognition. Furthermore, we validate the InvC ATP-binding site by co-crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP-analogue recognition, these structures reveal remodeling of the ATP-binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo-oligomerization process and ATP-dependent conformational changes underlying the T3SS ATPase activity.

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates.

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37 degrees C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is -OH>-OCH(3)>-H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.

Crystal structure of amine oxidase from bovine serum.

Copper-containing amine oxidase extracted from bovine serum (BSAO) was crystallized and its three-dimensional structure at 2.37A resolution is described. The biological unit of BSAO is a homodimer, formed by two monomers related to each other by a non-crystallographic 2-fold axis. Each monomer is composed of three domains, similar to those of other amine oxidases from lower species. The two monomers are structurally equivalent, despite some minor differences at the two active sites. A large funnel allows access of substrates to the active-site; another cavity, accessible to the solvent, is also present between the two monomers; this second cavity could allow the entrance of molecular oxygen necessary for the oxidative reaction. Some sugar residues, bound to Asn, were still present and visible in the electron density map, in spite of the exhaustive deglycosylation necessary to grow the crystals. The comparison of the BSAO structure with those of other resolved AO structures shows strong dissimilarities in the architecture and charge distribution of the cavities leading to the active-site, possibly explaining the differences in substrate specificity.

Phosphonium compounds as new and specific inhibitors of bovine serum amine oxidase.

TPP+ (tetraphenylphosphonium ion) and its analogues were found to act as powerful competitive inhibitors of BSAO (bovine serum amine oxidase). The binding of this new class of inhibitors to BSAO was characterized by kinetic measurements. TPP+ can bind to the BSAO active site by hydrophobic and by coulombian interactions. The binding probably occurs in the region of the 'cation-binding site'[Di Paolo, Scarpa, Corazza, Stevanato and Rigo (2002) Biophys. J. 83, 2231-2239]. Under physiological conditions, the association constant of TPP+ for this site is higher than 10(6) M(-1), the change of enthalpy being the main free-energy term controlling binding. Analysis of the relationships between substrate structure and extent of inhibition by TPP+ reveals some new molecular features of the BSAO active site.

Active site residue involvement in monoamine or diamine oxidation catalysed by pea seedling amine oxidase.

The structures of copper amine oxidases from various sources show good similarity, suggesting similar catalytic mechanisms for all members of this enzyme family. However, the optimal substrates for each member differ, depending on the source of the enzyme and its location. The structural factors underlying substrate selectivity still remain to be discovered. With this in view, we examined the kinetic behaviour of pea seedling amine oxidase with cadaverine and hexylamine, the first bearing two, and the second only one, positively charged amino group. The dependence of K(m) and catalytic constant (k(c)) values on pH, ionic strength and temperature indicates that binding of the monoamine is driven by hydrophobic interactions. Instead, binding of the diamine is strongly facilitated by electrostatic factors, controlled by polar side-chains and two titratable residues present in the active site. The position of the docked substrate is also essential for the participation of titratable amino acid residues in the following catalytic steps. A new mechanistic model explaining the substrate-dependent kinetics of the reaction is discussed.

A structure-activity study to identify novel and efficient substrates of the human semicarbazide-sensitive amine oxidase/VAP-1 enzyme.

Kinetic studies were performed with various alkanamines as "substrate probes" of the properties of the active site of the human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1). We found that the enzyme-substrate recognition step is mainly controlled by apolar interactions and that a "good" substrate has a molecular structure containing a long aliphatic chain and a second positive charge at a distance greater than 12 A from the reactive amino group. In this context, we identified a novel substrate for the human SSAO/VAP-1, 1,12-diaminododecane (DIADO), which is characterised by the highest catalytic efficiency reported to date in comparison to the prototypic substrate benzylamine. Computational docking studies revealed the structural basis of this behaviour, highlighting the key role played by Lys393 in hindering substrate docking. Maximum SSAO/VAP-1 activity is reached at relatively low concentrations of DIADO (10-30 microM), and, in these conditions, it has good selectivity: it is a good substrate of SSAO/VAP-1 but not of human adipocyte monoamine oxidases or pig kidney diamine oxidase. From these findings, it appears that DIADO can be used as a new substrate for human SSAO/VAP-1 to elicit glucose transport into adipocytes, and may consequently have potential pharmacological applications in the design of anti-diabetic agents.

N-alkanamines as substrates to probe the hydrophobic region of bovine serum amine oxidase active site: a kinetic and spectroscopic study.

Kinetic and spectroscopic studies were carried out to study the role of hydrophobic effect on the activity of bovine serum amine oxidase (BSAO). Increasing the chain length of the substrates (linear aliphatic primary monoamines), the affinity for the active site increases while the catalytic constant decreases in accordance with a relative low value of dielectric constant (about 10) estimated for the microenvironment of BSAO active site using a fluorescent probe sensitive to solvent polarity. The aliphatic chain of 1-aminononane induces a shift in the pK(a) of the product Schiff base, the hydrolysis of which appears to be a rate-determining step of the reaction. Furthermore, circular dichroism studies highlighted the "flexibility" of BSAO secondary structure that can explain the wide substrate specificity of this enzyme. These results should be useful to elucidate the substrate/inhibitor preferences of CuAOs, in particular of the human enzyme.

Synergistic antioxidant effect of catechin and malvidin 3-glucoside on free radical-initiated peroxidation of linoleic acid in micelles.

The inhibitory effect of anthocyanins has been investigated in the peroxidation of linoleic acid in micelles in the presence and in the absence of (+)-catechin. The peroxidation was initiated by thermal decomposition of 2,2(')-azobis[2-(2-imidazolin-2-yl)propane], and the kinetics of peroxidation were followed by measuring the rate of oxygen consumption and the rate of disappearance of the antioxidant. The analysis of the antioxidant effect of various anthocyanins, alone or in the presence of catechin, demonstrates that catechin, which is relatively inefficient at inhibiting linoleic acid oxidation, regenerates the highly efficient antioxidant malvidin 3-glucoside and, at a lower extent, peonidin 3-glucoside. The malvidin 3-glucoside recycling by catechin strongly increases the antioxidant efficiency of these two antioxidants. This protective mechanism appears specific for malvidin and peonidin 3-glucosides. The high unpaired spin density of the phenolic O atoms in the radicals generated by these anthocyanins, calculated by the semiempirical quantum chemical AM1 method, may explain the observed behavior.