Atomic model of the type III secretion system needle

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.     

Energy source of flagellar type III secretion

Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane. This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion. The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export. Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the non-flagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a proton-driven protein exporter and that ATP hydrolysis is not essential for type III secretion.     

Chaperone release and unfolding of substrates in type III secretion

Type III protein secretion systems are essential virulence factors of many bacteria pathogenic to humans, animals and plants. These systems mediate the transfer of bacterial virulence proteins directly into the host cell cytoplasm. Proteins are thought to travel this pathway in a largely unfolded manner, and a family of customized cytoplasmic chaperones, which specifically bind cognate secreted proteins, are essential for secretion. Here we show that InvC, an ATPase associated with a Salmonella enterica type III secretion system, has a critical function in substrate recognition. Furthermore, InvC induces chaperone release from and unfolding of the cognate secreted protein in an ATP-dependent manner. Our results show a similarity between the mechanisms of substrate recognition by type III protein secretion systems and AAA + ATPase disassembly machines.     

Ecosystem size determines food-chain length in lakes

Food-chain length is an important characteristic of ecological communities: it influences community structure, ecosystem functions and contaminant concentrations in top predators. Since Elton first noted that food-chain length was variable among natural systems, ecologists have considered many explanatory hypotheses, but few are supported by empirical evidence. Here we test three hypotheses that predict food-chain length to be determined by productivity alone (productivity hypothesis), ecosystem size alone (ecosystem-size hypothesis) or a combination of productivity and ecosystem size (productive-space hypothesis). The productivity and productive-space hypotheses propose that food-chain length should increase with increasing resource availability; however, the productivity hypothesis does not include ecosystem size as a determinant of resource availability. The ecosystem-size hypothesis is based on the relationship between ecosystem size and species diversity, habitat availability and habitat heterogeneity. We find that food-chain length increases with ecosystem size, but that the length of the food chain is not related to productivity. Our results support the hypothesis that ecosystem size, and not resource availability, determines food-chain length in these natural ecosystems.     

The effect of pmpR on the type III secretion system in Pseudomonas aeruginosa

The type III secretion system(T3SS) plays important roles in Pseudomonas aeruginosa pathogenicity.Previously,we reported that the uncharacterized protein PmpR could regulate pqsR,an important regulator in the quorum-sensing system,by directly binding to its promoter region.As the T3SS is controlled by the quorum-sensing system,here,we investigated the relationship between PmpR and the T3SS.Our data showed that expression of the T3SS genes exoS,exoY,exoT,and exsD was dramatically increased in a pmpR-deletion mutant compared with that in the wild-type P.aeruginosa strain PAO1.Data from DNA mobility assays indicated that PmpR affects the T3SS indirectly.It is unlikely that PmpR controls the T3SS via the Pseudomonas quinolone signal(PQS) because the PQS negatively regulates the T3SS,while pmpR negatively regulates the PQS.The effect of PmpR on the T3SS seems to be independent of the PQS;further investigation is required to uncover the underlying regulatory pathways.     

Structural characterization of the molecular platform for type III secretion system assembly

Type III secretion systems (TTSSs) are multi-protein macromolecular 'machines' that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross 'needle-shaped' morphology of the TTSS, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-A crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit 'ring' superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.     

Protein delivery into eukaryotic cells by type III secretion machines

Bacteria that have sustained long-standing close associations with eukaryotic hosts have evolved specific adaptations to survive and replicate in this environment. Perhaps one of the most remarkable of those adaptations is the type III secretion system (T3SS)--a bacterial organelle that has specifically evolved to deliver bacterial proteins into eukaryotic cells. Although originally identified in a handful of pathogenic bacteria, T3SSs are encoded by a large number of bacterial species that are symbiotic or pathogenic for humans, other animals including insects or nematodes, and plants. The study of these systems is leading to unique insights into not only organelle assembly and protein secretion but also mechanisms of symbiosis and pathogenesis.     

Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS

During infection by Gram-negative pathogenic bacteria, the type III secretion system (T3SS) is assembled to allow for the direct transmission of bacterial virulence effectors into the host cell. The T3SS system is characterized by a series of prominent multi-component rings in the inner and outer bacterial membranes, as well as a translocation pore in the host cell membrane. These are all connected by a series of polymerized tubes that act as the direct conduit for the T3SS proteins to pass through to the host cell. During assembly of the T3SS, as well as the evolutionarily related flagellar apparatus, a post-translational cleavage event within the inner membrane proteins EscU/FlhB is required to promote a secretion-competent state. These proteins have long been proposed to act as a part of a molecular switch, which would regulate the appropriate chronological secretion of the various T3SS apparatus components during assembly and subsequently the transported virulence effectors. Here we show that a surface type II beta-turn in the Escherichia coli protein EscU undergoes auto-cleavage by a mechanism involving cyclization of a strictly conserved asparagine residue. Structural and in vivo analysis of point and deletion mutations illustrates the subtle conformational effects of auto-cleavage in modulating the molecular features of a highly conserved surface region of EscU, a potential point of interaction with other T3SS components at the inner membrane. In addition, this work provides new structural insight into the distinct conformational requirements for a large class of self-cleaving reactions involving asparagine cyclization.     

Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion.

Many bacterial pathogens use a type III protein secretion system to deliver virulence effector proteins directly into the host cell cytosol, where they modulate cellular processes. A requirement for the effective translocation of several such effector proteins is the binding of specific cytosolic chaperones, which typically interact with discrete domains in the virulence factors. We report here the crystal structure at 1.9 A resolution of the chaperone-binding domain of the Salmonella effector protein SptP with its cognate chaperone SicP. The structure reveals that this domain is maintained in an extended, unfolded conformation that is wound around three successive chaperone molecules. Short segments from two different SptP molecules are juxtaposed by the chaperones, where they dimerize across a hydrophobic interface. These results imply that the chaperones associated with the type III secretion system maintain their substrates in a secretion-competent state that is capable of engaging the secretion machinery to travel through the type III apparatus in an unfolded or partially folded manner.     

The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus

Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death. The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component. How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages, is a universal component of the flagellin-NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5-NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity.     

Tyrosine-containing motif that transduces cell activation signals also determines internalization and antigen presentation via type III receptors for IgG.

Type III receptors for IgG (Fc gamma RII; ref. 1), high-affinity IgE receptors (Fc epsilon RI; ref. 2), as well as the T- and B-cell antigen receptors, consist of multiple components with specialized ligand-binding and signal transduction functions. Fc gamma RII alpha (ligand-binding) and gamma (signal-transducing) subunits are expressed in macrophages, a cell type involved in the uptake of antigen, its processing and the presentation of the resulting peptides to major histocompatibility complex class II-restricted T lymphocytes. Here we show that murine Fc gamma RIII, transfected into Fc gamma R-negative antigen-presenting B-lymphoma cells, mediate rapid ligand internalization and strongly increase the efficiency of antigen presentation when antigen is complexed to IgG. Efficient internalization and antigen presentation via Fc gamma RIII did not require the cytoplasmic domain of the ligand-binding alpha-chain, but did require the gamma-subunit. Using chimaeric molecules, we show that gamma-chain contains a signal for receptor internalization and that the mutation of either of the two tyrosine residues present in its cytoplasmic domain prevents efficient internalization and antigen presentation of immune complexes. Thus, associated chains and their tyrosine-containing motif are not exclusively involved in cell activation, but also determine multimeric receptor internalization.     

汽车第三代轮毂轴承全自动内组件装配机研制

按照汽车第三代圆锥轮毂轴承单元的装配工艺要求,通过对滚子上料机构、保持架上料机构、传输机械爪、分布盘、卸料机械爪和芯轴上料机构等的设计,配合PLC和触摸屏控制实现了轴承内组件的自动装配,解决了人工装配效率低、易出现倒装漏装以及对滚动体划伤等问题,达到了提高装配质量、装配效率,降低工人劳动强度的目的。     

端面研磨Nd:YAG晶体棒热透镜焦距测量

主要分析和测量端面研磨凹面的Nd:YAG激光晶体棒的热透镜焦距。实验通过He-Ne光测试法,分别测量了不同功率下的端面研磨和未研磨晶体棒的热透镜焦距,并通过作图比较二者的热透镜焦距随泵浦功率变化情况,得出随着泵浦功率的增大,热透镜焦距逐渐减小的结论。文中对未研磨晶体棒热透镜焦距进行了理论和实验值的比较,曲线情况基本相符,故根据理论计算出补偿热透镜效应所需要的晶体棒的研磨半径。由于通过理论计算得出的研磨半径在实验过程中出现了出光阈值高,输出功率降低等一些现象,不利于生产的需要,故在实际经验的指导下,增大了晶体棒的研磨半径。结论指出端面研磨晶体棒的方法在一定条件下能对激光晶体棒的热透镜效应进行补偿,对大功率固体激光器的研发有一定积极作用。     

晶体中化学键的研究Ⅲ具有NiAs结构的二元间隙化合物

本文用LCBO方法研究了NiA_B结构二元化合物的能带结构特点,从而探讨了这类化合物的导电性。     

Different receptors for distribution of peanut and ricin agglutinins between inner and outer segments of rod cells

Lectins can be used as probes for cell-surface oligosaccharides1-4. These proteins display high specificities for certain haptene sugars, although the details of the sugar linkages and the three-dimensional array of the oligosaccharide may all be involved in determining the affinity of a lectin for its receptor. We have now shown that peanut and ricin agglutinins bind differentially to the surfaces of rod inner and outer segments.     

基于SMA智能复合棒的新型驱动器研究

提出了一种新型的基于偏心内嵌SMA丝复合弹性棒的驱动器结构,推导了复合弹性棒的三维变形描述方程式,应用SMA丝和弹性棒的界面连续性原理,求解棒和丝的平衡方程式,得到了两者的合力和合力矩,将三维方程应用于SMA丝平行轴线嵌入圆直棒的特定情形,得到二维的简化式,设计和制作了基于新型驱动器的多指机械手,实验结果进一步验证了弹性棒变形理论。     

电脑刺绣机针杆及挑线机构设计

以电脑刺绣机原始针杆机构和挑线机构为研究对象,在保持原有针杆运动规律和挑线杆运动规律的前提下,运用机构创新设计理论,分析电脑刺绣机针杆机构和挑线机构的可能选型.为研制新型电脑刺绣机针杆机构和挑线机构奠定了理论基础.     

The secretion of lecithinase of Pseudomonas alcaligenes S2 was via type II secretion pathway

Phosphorus is one of the major essential macronutrients for virtually metabolic processes in plant growth and de-velopment[1]. This creates a paradox with major agro-nomic implications since the phosphate form of phospho-rus is one of the least soluble mineral nutrient ions in the soil. The concentration of soluble phosphorus in soil is usually very low, normally at levels of 1 ppm or less (10 mol/L H2PO4?). Mineral forms of phosphorus are repre-sented in soil by primary minerals, such as ap…     

On theories of solar type III radio bursts

In solar radiophysics,many theories for type Ⅲ bursts have been proposed during the past 60 years.Almost all these theories are based on the plasma hypothesis,which assumes that(i)the radiation is mainly generated by Langmuir waves via nonlinear processes and(ii)the radiation has frequencies close to the local plasma frequency and/or its second harmonic in the source region. We feel strongly that it is time to advocate an alternative approach without recourse to the plasma hypothesis.This brief discussion explains why.