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.     

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.     

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 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.     

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 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.     

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.     

Assembly of the inner rod determines needle length in the type III secretion injectisome

Assembly of multi-component supramolecular machines is fundamental to biology, yet in most cases, assembly pathways and their control are poorly understood. An example is the type III secretion machine, which mediates the transfer of bacterial virulence proteins into host cells. A central component of this nanomachine is the needle complex or injectisome, an organelle associated with the bacterial envelope that is composed of a multi-ring base, an inner rod, and a protruding needle. Assembly of this organelle proceeds in sequential steps that require the reprogramming of the secretion machine. Here we provide evidence that, in Salmonella typhimurium, completion of the assembly of the inner rod determines the size of the needle substructure. Assembly of the inner rod, which is regulated by the InvJ protein, triggers conformational changes on the cytoplasmic side of the injectisome, reprogramming the secretion apparatus to stop secretion of the needle protein.     

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.     

新型烃源岩——沉凝灰岩

通过对准东石炭系沉凝灰岩地化特征分析,确定沉凝灰岩为一种与火山活动有关的新型烃源岩,并对取自不同井的沉凝灰岩的生物标志化合物和饱和烃总离子流图进行比较,认为沉凝灰岩具有3种不同的成烃特征.本文研究成果为沉凝灰岩这种新型烃源岩的研究奠定了基础.     

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.     

一种新型的三相电流型多电平整流器

传统的多电平技术研究主要集中在电压型变流器,对电流型多电平的研究还不多,对电流型多电平整流器的研究更为少见。将对偶思想应用于电压型多电平的调控策略对偶到电流型多电平,并采用模块并联和载波相移技术,提出并实现了以三相电流型7电平整流器为代表的全新的三相电流型多电平整流器。着重分析了其工作原理和调制策略,在搭建DSP+CPLD数字平台的基础上实现了三逻辑载波相移SPWM信号的全数字化以及多路PWM驱动信号的产生,最终通过建立样机验证了控制策略和调制方法运用于电流型多电平整流器的正确性和可行性。     

Helicobacter exploits integrin for type IV secretion and kinase activation

Integrins are important mammalian receptors involved in normal cellular functions as well as pathogenesis of chronic inflammation and cancer. We propose that integrins are exploited by the gastric pathogen and type-1 carcinogen Helicobacter pylori for injection of the bacterial oncoprotein cytotoxin-associated gene A (CagA) into gastric epithelial cells. Virulent H. pylori express a type-IV secretion pilus that injects CagA into the host cell; CagA then becomes tyrosine-phosphorylated by Src family kinases. However, the identity of the host cell receptor involved in this process has remained unknown. Here we show that the H. pylori CagL protein is a specialized adhesin that is targeted to the pilus surface, where it binds to and activates integrin alpha5beta1 receptor on gastric epithelial cells through an arginine-glycine-aspartate motif. This interaction triggers CagA delivery into target cells as well as activation of focal adhesion kinase and Src. Our findings provide insights into the role of integrins in H.-pylori-induced pathogenesis. CagL may be exploited as a new molecular tool for our further understanding of integrin signalling.     

应变式传感器恒压源间歇供电

分析了恒压源供电下的桥路输出特性以及影响电桥电压灵敏度的各种因素.供电电压的增加会受到桥臂允许功耗的限制,从而制约了电桥电压灵敏度的提高,并产生电阻"温升"和热噪声.针对电桥存在的上述问题提出的恒压源间歇供电方式,不仅可以提高传感器的灵敏度,还有效地降低了应变片电阻的"温升"和热噪声,提高了传感器的精度.     

水源侧大温差热泵系统运行能耗分析

水源热泵技术是一种主要回收低温余热的节能技术。利用水源热泵系统试验台研究了大温差(温差为6~10℃)运行热泵的特性,比较了各温差段热泵的主机和给水泵单位供热能耗的关系。实验结果表明:随着温差增大,虽然主机单位供热能耗不断增大,但是热泵系统总体的单位供热能耗呈现下降趋势。通过对热泵系统制冷系数(coefficient to performance,COP)分析可知,在温差为7℃时,热泵系统COP最大,因此热泵大温差运行时循环水温差控制在7~9℃比较合适,机组整体能耗低,节能效果明显。     

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。