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1.
Protein Sci ; 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393998

RESUMO

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.

2.
Mol Microbiol ; 2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444817

RESUMO

Bacterial flagellar filaments are assembled by tens of thousands flagellin subunits, forming 11 helically arranged protofilaments. Each protofilament can take either of the two bi-stable forms L-type or R-type, having slightly different conformations and inter-protofilaments interactions. By mixing different ratios of L- 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 of 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 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. This article is protected by copyright. All rights reserved.

3.
J Mol Biol ; 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31288030

RESUMO

Many medically relevant Gram-negative bacteria use the type III secretion system (T3SS) to translocate effector proteins into the host for their invasion and intracellular survival. A multi-protein complex located at the cytosolic interface of the T3SS is proposed to act as a sorting platform by selecting and targeting substrates for secretion through the system. However, the precise stoichiometry and 3D organization of the sorting platform components are unknown. Here we reconstitute soluble complexes of the Salmonella Typhimurium sorting platform proteins including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified subunit SpaOC, which we show to be essential for the solubility of SpaO. We establish domain-domain interactions, determine for the first time the stoichiometry of each subunit within the complexes by native mass spectrometry and gain insight into their organization using small-angle X-ray scattering. Importantly, we find that in solution the assembly of SpaO/SpaOC/OrgB/InvC adopts an extended L-shaped conformation resembling the sorting platform pods seen in in situ cryo-electron tomography, proposing that this complex is the core building block that can be conceivably assembled into higher oligomers to form the T3SS sorting platform. The determined molecular arrangements of the soluble complexes of the sorting platform provide important insights into its architecture and assembly.

4.
EMBO Rep ; 20(4)2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30872316

RESUMO

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.

5.
J Synchrotron Radiat ; 26(Pt 2): 535-542, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30855265

RESUMO

At the Metrology Light Source, an electron storage ring dedicated to metrological applications, the U125 insertion device beamline utilizes undulator radiation for various applications over a broad spectral range. Using a hybrid normal-incidence and grazing-incidence in-vacuum switchable plane-grating monochromator, a spectral region ranging from the near-infrared to soft X-ray is covered. The beamline is dedicated to surface-analytical methods, e.g. ellipsometry, photoelectron spectroscopy or photoemission tomography. The traceability of radiometric quantities, i.e. quantitative determination of the available radiant power (or photon flux), is required for some of these applications to support the metrological aspect of the measurements. In particular, attention is paid to the suppression of unwanted spectral contributions from higher diffraction orders, and to the monitoring of the radiation intensity during the measurements. With the results from the beamline commissioning, an uncertainty budget for all relevant radiometric quantities was established.

6.
Cell Microbiol ; 19(8)2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28295924

RESUMO

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.


Assuntos
Células Epiteliais/microbiologia , Flagelina/metabolismo , Trato Gastrointestinal/microbiologia , Salmonella/fisiologia , Animais , Locomoção , Camundongos , Salmonelose Animal/microbiologia
7.
Nanotechnology ; 27(32): 324005, 2016 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-27363480

RESUMO

Monochromatic radiation with known absolute radiant power from an undulator at the electron storage ring Metrology Light Source (MLS) was used to irradiate PTB7 (a thieno[3, 4-b]thiophene-alt-benzodithiophene polymer) thin films at wavelengths (photon energies) of 185 nm (6.70 eV), 220 nm (5.64 eV), 300 nm (4.13 eV), 320 nm (3.88 eV), 356 nm (3.48 eV) and 675 nm (1.84 eV) under ultra-high vacuum conditions for the investigation of radiation-induced degradation effects. The characterization of the thin films is focused at ultraviolet photoelectron spectroscopy (UPS) of valence bands and is complemented by S 2p x-ray photoelectron spectroscopy (S 2p XPS) before and after the irradiation procedure. The radiant exposure was determined for each irradiation by means of photodiodes traceably calibrated to the international system of units SI. The valence band spectra show the strongest changes for the shortest wavelengths and no degradation effect at 356 nm and 675 nm even with the highest radiant exposure applied. In the spectral range where the Sun appears bright on the Earth's surface, no degradation effects are observed.

8.
Phys Rev Lett ; 113(16): 163001, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-25361254

RESUMO

An experimental method for the verification of the individually different energy dependencies of L(1)-, L(2)-, and L(3)- subshell photoionization cross sections is described. The results obtained for Pd and Mo are well in line with theory regarding both energy dependency and absolute values, and confirm the theoretically calculated cross sections by Scofield from the early 1970 s and, partially, more recent data by Trzhaskovskaya, Nefedov, and Yarzhemsky. The data also demonstrate the questionability of quantitative x-ray spectroscopical results based on the widely used fixed jump ratio approximated cross sections with energy independent ratios. The experiments are carried out by employing the radiometrically calibrated instrumentation of the Physikalisch-Technische Bundesanstalt at the electron storage ring BESSY II in Berlin; the obtained fluorescent intensities are thereby calibrated at an absolute level in reference to the International System of Units. Experimentally determined fixed fluorescence line ratios for each subshell are used for a reliable deconvolution of overlapping fluorescence lines. The relevant fundamental parameters of Mo and Pd are also determined experimentally in order to calculate the subshell photoionization cross sections independently of any database.

9.
Nature ; 512(7515): 387-92, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-25119038

RESUMO

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mycobacterium tuberculosis/imunologia , Pigmentos Biológicos/metabolismo , Pseudomonas aeruginosa/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Animais , Antibacterianos/metabolismo , Células da Medula Óssea/citologia , Citocinas/imunologia , Citocinas/metabolismo , Retroalimentação Fisiológica , Humanos , Ligantes , Ativação de Macrófagos , Camundongos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Fenazinas/metabolismo , Pigmentos Biológicos/química , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo
10.
PLoS Pathog ; 10(1): e1003881, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24453973

RESUMO

The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Citoplasma/metabolismo , Shigella flexneri/metabolismo , Proteínas de Bactérias/genética , Citoplasma/genética , Transporte Proteico/fisiologia , Shigella flexneri/genética , Shigella flexneri/ultraestrutura
11.
Cell Microbiol ; 15(11): 1809-17, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23927570

RESUMO

Many Gram-negative pathogens utilize type 3 secretion systems (T3SSs) for a successful infection. The T3SS is a large macromolecular complex which spans both bacterial membranes and delivers effector proteins into the host cell. The infection requires spatiotemporal control of diverse sets of secreted effectors and various mechanisms have evolved to regulate T3SS in response to external stimuli. This review will describe mechanisms that may control type 3 secretion, revealing a multi-step regulatory strategy. We then propose an updated model of T3SS that illustrates different stages of secretion and integrates the most recent structural and functional data.


Assuntos
Sistemas de Secreção Bacterianos , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Proteínas de Bactérias/metabolismo , Substâncias Macromoleculares/metabolismo , Fatores de Virulência/metabolismo
12.
PLoS Pathog ; 9(3): e1003245, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23555258

RESUMO

The Type Three Secretion System (T3SS), or injectisome, is a macromolecular infection machinery present in many pathogenic Gram-negative bacteria. It consists of a basal body, anchored in both bacterial membranes, and a hollow needle through which effector proteins are delivered into the target host cell. Two different architectures of the T3SS needle have been previously proposed. First, an atomic model of the Salmonella typhimurium needle was generated from solid-state NMR data. The needle subunit protein, PrgI, comprises a rigid-extended N-terminal segment and a helix-loop-helix motif with the N-terminus located on the outside face of the needle. Second, a model of the Shigella flexneri needle was generated from a high-resolution 7.7-Å cryo-electron microscopy density map. The subunit protein, MxiH, contains an N-terminal α-helix, a loop, another α-helix, a 14-residue-long ß-hairpin (Q51-Q64) and a C-terminal α-helix, with the N-terminus facing inward to the lumen of the needle. In the current study, we carried out solid-state NMR measurements of wild-type Shigella flexneri needles polymerized in vitro and identified the following secondary structure elements for MxiH: a rigid-extended N-terminal segment (S2-T11), an α-helix (L12-A38), a loop (E39-P44) and a C-terminal α-helix (Q45-R83). Using immunogold labeling in vitro and in vivo on functional needles, we located the N-terminus of MxiH subunits on the exterior of the assembly, consistent with evolutionary sequence conservation patterns and mutagenesis data. We generated a homology model of Shigella flexneri needles compatible with both experimental data: the MxiH solid-state NMR chemical shifts and the state-of-the-art cryoEM density map. These results corroborate the solid-state NMR structure previously solved for Salmonella typhimurium PrgI needles and establish that Shigella flexneri and Salmonella typhimurium subunit proteins adopt a conserved structure and orientation in their assembled state. Our study reveals a common structural architecture of T3SS needles, essential to understand T3SS-mediated infection and develop treatments.


Assuntos
Proteínas de Bactérias/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Salmonella typhimurium/química , Shigella flexneri/química , Sequência de Aminoácidos , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Salmonella typhimurium/ultraestrutura , Alinhamento de Sequência , Shigella flexneri/ultraestrutura
13.
Cell Host Microbe ; 12(3): 381-9, 2012 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-22980334

RESUMO

Shigella infection causes destruction of the human colonic epithelial barrier. The Golgi network and recycling endosomes are essential for maintaining epithelial barrier function. Here we show that Shigella epithelial invasion induces fragmentation of the Golgi complex with consequent inhibition of both secretion and retrograde transport in the infected host cell. Shigella induces tubulation of the Rab11-positive compartment, thereby affecting cell surface receptor recycling. The molecular process underlying the observed damage to the Golgi complex and receptor recycling is a massive redistribution of plasma membrane cholesterol to the sites of Shigella entry. IpaB, a virulence factor of Shigella that is known to bind cholesterol, is necessary and sufficient to induce Golgi fragmentation and reorganization of the recycling compartment. Shigella infection-induced Golgi disorganization was also observed in vivo, suggesting that this mechanism affecting the sorting of cell surface molecules likely contributes to host epithelial barrier disruption associated with Shigella pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Endossomos/ultraestrutura , Complexo de Golgi/ultraestrutura , Interações Hospedeiro-Patógeno , Proteínas/metabolismo , Shigella/patogenicidade , Linhagem Celular , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Microscopia de Fluorescência , Fatores de Virulência/metabolismo
14.
Nature ; 486(7402): 276-9, 2012 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-22699623

RESUMO

Pathogenic bacteria using a type III secretion system (T3SS) to manipulate host cells cause many different infections including Shigella dysentery, typhoid fever, enterohaemorrhagic colitis and bubonic plague. An essential part of the T3SS is a hollow needle-like protein filament through which effector proteins are injected into eukaryotic host cells. Currently, the three-dimensional structure of the needle is unknown because it is not amenable to X-ray crystallography and solution NMR, as a result of its inherent non-crystallinity and insolubility. Cryo-electron microscopy combined with crystal or solution NMR subunit structures has recently provided a powerful hybrid approach for studying supramolecular assemblies, resulting in low-resolution and medium-resolution models. However, such approaches cannot deliver atomic details, especially of the crucial subunit-subunit interfaces, because of the limited cryo-electron microscopic resolution obtained in these studies. Here we report an alternative approach combining recombinant wild-type needle production, solid-state NMR, electron microscopy and Rosetta modelling to reveal the supramolecular interfaces and ultimately the complete atomic structure of the Salmonella typhimurium T3SS needle. We show that the 80-residue subunits form a right-handed helical assembly with roughly 11 subunits per two turns, similar to that of the flagellar filament of S. typhimurium. In contrast to established models of the needle in which the amino terminus of the protein subunit was assumed to be α-helical and positioned inside the needle, our model reveals an extended amino-terminal domain that is positioned on the surface of the needle, while the highly conserved carboxy terminus points towards the lumen.


Assuntos
Sistemas de Secreção Bacterianos , Modelos Moleculares , Salmonella typhimurium/química , Células HeLa , Humanos , Microscopia Eletrônica , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína
15.
FASEB J ; 26(4): 1717-26, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22247334

RESUMO

Gram-negative bacteria use the type 3 secretion system (T3SS) to colonize host cells. T3SSs are ring-shaped macromolecular complexes specific for the transport of effector molecules into host cells. It was recently suggested that a cytosolic ring-shaped protein complex delivers effector molecules to the T3SS. However, how transport of effector proteins is regulated is not known. Here, we report the high-resolution X-ray crystal structure of the whole cytosolic domain of MxiG (MxiG(1-126)), a major component of the inner T3SS rings in Shigella flexneri. MxiG(1-126) folds as an FHA domain, which specifically binds phosphorylated threonines. Indeed, MxiG(1-126) binds to Spa33, a cytoplasmic-ring component of Shigella, as revealed in pulldown studies. Surface plasmon resonance analysis showed specific interaction of MxiG with a Spa33 peptide only if phosphorylated. In total, 24 copies of the MxiG(1-126) crystal structure were fitted into the cryo-EM map of the Shigella T3SS. The phosphoprotein binding site of each MxiG molecule faces the channel of the T3SS, allowing interaction with cytosolic binding partners. Secretion assays and host cell invasion studies of complemented Shigella knockout cells indicated that the phosphoprotein binding of MxiG is essential for bacterial virulence. Our findings suggest that MxiG is involved in T3SS regulation.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Citosol/metabolismo , Via Secretória/fisiologia , Shigella flexneri/patogenicidade , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Cristalografia por Raios X , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Alinhamento de Sequência , Shigella flexneri/genética , Shigella flexneri/metabolismo
16.
PLoS Pathog ; 7(8): e1002163, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21829362

RESUMO

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.


Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Proteínas de Membrana/química , Subunidades Proteicas/química , Salmonella typhimurium/fisiologia , Animais , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Ácido Desoxicólico/química , Proteínas de Membrana/metabolismo , Membranas/química , Membranas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão , Ressonância de Plasmônio de Superfície
17.
J Biol Chem ; 285(51): 39965-75, 2010 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-20937829

RESUMO

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.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Shigella flexneri/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Óperon/fisiologia , Mapeamento de Peptídeos/métodos , Ligação Proteica , Multimerização Proteica/fisiologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Shigella flexneri/química , Shigella flexneri/genética
18.
Nat Struct Mol Biol ; 17(7): 788-92, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20543831

RESUMO

Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from alpha-helix into beta-strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level.


Assuntos
Proteínas de Bactérias/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Salmonella typhimurium/química , Salmonella typhimurium/genética , Shigella flexneri/química , Shigella flexneri/genética
19.
Anal Bioanal Chem ; 396(8): 2825-32, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19941133

RESUMO

A need for analysis techniques, complementary to secondary ion mass spectrometry (SIMS), for depth profiling dopants in silicon for ultra shallow junction (USJ) applications in CMOS technologies has recently emerged following the difficulties SIMS is facing there. Grazing incidence X-ray fluorescence (GIXRF) analysis in the soft X-ray range is a high-potential tool for this purpose. It provides excellent conditions for the excitation of the B-K and the As-L(iii,ii) shells. The X-ray standing wave (XSW) field associated with GIXRF on flat samples is used here as a tunable sensor to obtain information about the implantation profile because the in-depth changes of the XSW intensity are dependent on the angle of incidence. This technique is very sensitive to near-surface layers and is therefore well suited for the analysis of USJ distributions. Si wafers implanted with either arsenic or boron at different fluences and implantation energies were used to compare SIMS with synchrotron radiation-induced GIXRF analysis. GIXRF measurements were carried out at the laboratory of the Physikalisch-Technische Bundesanstalt (PTB) at the electron storage ring BESSY II using monochromatized undulator radiation of well-known radiant power and spectral purity. The use of an absolutely calibrated energy-dispersive detector for the acquisition of the B-Kalpha and As-Lalpha fluorescence radiation enabled the absolute determination of the total retained dose. The concentration profile was obtained by ab initio calculation and comparison with the angular measurements of the X-ray fluorescence.

20.
Proc Natl Acad Sci U S A ; 106(24): 9661-6, 2009 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-19478065

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Chaperonas Moleculares/química , Conformação Proteica , Homologia de Sequência de Aminoácidos , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidade
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