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.     

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.     

Signal sequence directs localized secretion of bacterial surface proteins

All living cells require specific mechanisms that target proteins to the cell surface. In eukaryotes, the first part of this process involves recognition in the endoplasmic reticulum of amino-terminal signal sequences and translocation through Sec translocons, whereas subsequent targeting to different surface locations is promoted by internal sorting signals. In bacteria, N-terminal signal sequences promote translocation across the cytoplasmic membrane, which surrounds the entire cell, but some proteins are nevertheless secreted in one part of the cell by poorly understood mechanisms. Here we analyse localized secretion in the Gram-positive pathogen Streptococcus pyogenes, and show that the signal sequences of two surface proteins, M protein and protein F (PrtF), direct secretion to different subcellular regions. The signal sequence of M protein promotes secretion at the division septum, whereas that of PrtF preferentially promotes secretion at the old pole. Our work therefore shows that a signal sequence may contain information that directs the secretion of a protein to one subcellular region, in addition to its classical role in promoting secretion. This finding identifies a new level of complexity in protein translocation and emphasizes the potential of bacterial systems for the analysis of fundamental cell-biological problems.     

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.     

嗜水气单胞菌Ⅲ型分泌系统研究进展

主要对嗜水气单胞菌Ⅲ型分泌系统、效应蛋白及其致病机理等方面的研究进展进行了综述.细菌分泌系统的发现是近年来细菌致病机制研究的重要进展,许多革兰氏阴性细菌借助于细菌的分泌系统,分泌出毒性因子和效应蛋白,与寄主进行分子交流.     

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.     

Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein

The three-dimensional structure of the amino-terminal 44K ATPase fragment of the 70K bovine heat-shock cognate protein has been solved to a resolution of 2.2 A. The ATPase fragment has two structural lobes with a deep cleft between them; ATP binds at the base of the cleft. Surprisingly, the nucleotide-binding 'core' of the ATPase fragment has a tertiary structure similar to that of hexokinase, although the remainder of the structures of the two proteins are completely dissimilar, suggesting that both the phosphotransferase mechanism and the substrate-induced conformational change intrinsic to the hexokinases may be used by the 70K heat shock-related proteins.     

Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells

The regulation of the subclass of immunoglobulin secreted by B cells has been studied in vitro in polyclonal systems using mitogens, such as lipopolysaccharide (LPS), to bypass the requirement for cognate interaction between antigen-specific T and B cells. In these systems, interleukin-(IL)-4 induces the secretion of IgG1 (ref. 1) and IgE (ref. 2); IL-5 enhances the secretion of IgA, and interferon-gamma (IFN-gamma) enhances the secretion of IgG2a (ref. 5). Clones of murine TH cells can be divided into two subsets, TH1 and TH2 (ref. 6). Both subsets synthesize IL-3 and granulocyte-monocyte colony-stimulating factor (GM-CSF), but only TH1 clones produce IL-2, IFN-gamma, and lymphotoxin (LT) and TH2 clones produce IL-4 and IL-5 (ref. 7). We have examined the role of clones of antigen-specific TH1 and TH2 cells in the regulation of the subclasses of IgG antibody secreted by antigen-specific B cells. Our results show that both types of TH cells induce the secretion of IgM and IgG3, whereas clones of TH1 and TH2 cells specifically induce antigen-specific B cells to secrete IgG2a and IgG1, respectively. We also demonstrate that regulation of commitment to the secretion of a particular IgG isotype occurs in two distinct stages: cognate interaction between T and B cells and interaction between T-cell-derived lymphokines and B cells.     

Signal sequence mutations disrupt feedback between secretion of an exported protein and its synthesis in E. coli

Recent studies in a eukaryotic system indicate that a block in secretion can lead to a block in the translation of secretory proteins. This feedback on protein synthesis is thought to be a result of an interaction of the signal recognition particle with the signal sequences of nascent proteins. Genetic studies in the prokaryote Escherichia coli suggest that a complex secretion machinery and a similar feedback mechanism exist. In addition, mutations affecting two genes, secA and secC, thought to encode components of the bacterial secretion machinery, selectively interfere with the synthesis of exported proteins. This selective interference with translation may be a result of recognition by the secretion machinery of signal sequences. If so, alteration of the signal sequence of a particular protein by mutation should eliminate the block in synthesis for that protein. We show here that signal sequence mutants for an exported protein, maltose binding protein, prevent the block in synthesis of this protein in a secA mutant.     

A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by retro-translocation is an important physiological adaptation to ER stress. This process requires recognition of a substrate in the ER lumen and its subsequent movement through the membrane by the cytosolic p97 ATPase. Here we identify a p97-interacting membrane protein complex in the mammalian ER that links these two events. The central component of the complex, Derlin-1, is a homologue of Der1, a yeast protein whose inactivation prevents the elimination of misfolded luminal ER proteins. Derlin-1 associates with different substrates as they move through the membrane, and inactivation of Derlin-1 in C. elegans causes ER stress. Derlin-1 interacts with US11, a virally encoded ER protein that specifically targets MHC class I heavy chains for export from the ER, as well as with VIMP, a novel membrane protein that recruits the p97 ATPase and its cofactor.     

Global unfolding of a substrate protein by the Hsp100 chaperone ClpA.

The bacterial protein CIpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP. ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences, much as the 19S ATPase 'cap' of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation. Fluorescence studies both with and without a 'trap' version of the chaperonin GroEL, which binds non-native forms of GFP, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.     

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.     

Xenopus oocytes can secrete bacterial beta-lactamase

Most secretory proteins are synthesized as precursor polypeptides carrying N-terminal, hydrophobic sequences which, by means of a signal recognition particle (SRP), trigger the membrane transfer of the polypeptide and are subsequently cleaved off. The signal sequences appear to be interchangeable between prokaryotes and eukaryotes. In bacteria, secretion only involves the crossing of a membrane, whereas in eukaryotes the secretory process can be separated into two distinct phases: translocation across the membrane of the rough endoplasmic reticulum and subsequent intraluminal transport by processes involving vesicle budding and fusion. Since secretory proteins must be distinguished from other soluble proteins destined for various sites in the reticular system, it is conceivable that eukaryotic secretory proteins possess additional markers distinct from the signal peptide to guide the polypeptide after its transfer through the membrane. Proteins are secreted at different rates from a eukaryotic cell, suggesting a role in intracellular transport for receptors with differing affinities for some topogenic features in secretory proteins. We have tested this possibility by introducing into the lumen of eukaryotic rough endoplasmic reticulum a prokaryotic protein which, by virtue of its origin, had not been adapted to the eukaryotic secretory pathway. We reasoned that secretion of the bacterial protein would indicate that after membrane transfer no topogenic signal(s) and corresponding recognition system(s) are required. We report here that this is indeed the case.     

Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export

Translocation of many soluble proteins across cell membranes occurs in an ATPase-driven manner. For construction of the bacterial flagellum responsible for motility, most of the components are exported by the flagellar protein export apparatus. The FliI ATPase is required for this export, and its ATPase activity is regulated by FliH; however, it is unclear how the chemical energy derived from ATP hydrolysis is used for the export process. Here we report that flagellar proteins of Salmonella enterica serovar Typhimurium are exported even in the absence of FliI. A fliH fliI double null mutant was weakly motile. Certain mutations in FlhA or FlhB, which form the core of the export gate, substantially improved protein export and motility of the double null mutant. Furthermore, proton motive force was essential for the export process. These results suggest that the FliH-FliI complex facilitates only the initial entry of export substrates into the gate, with the energy of ATP hydrolysis being used to disassemble and release the FliH-FliI complex from the protein about to be exported. The rest of the successive unfolding/translocation process of the substrates is driven by proton motive force.     

Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium

Genetic transformation of plant cells by Agrobacterium represents a unique case of trans-kingdom DNA transfer. During this process, Agrobacterium exports its transferred (T) DNA and several virulence (Vir) proteins into the host cell, within which T-DNA nuclear import is mediated by VirD2 (ref. 3) and VirE2 (ref. 4) and their host cell interactors AtKAP-alpha and VIP1 (ref. 6), whereas its integration is mediated mainly by host cell proteins. The factors involved in the uncoating of T-DNA from its cognate proteins, which occurs before integration into the host genome, are still unknown. Here, we report that VirF-one of the few known exported Vir proteins whose function in the host cell remains unknown-is involved in targeted proteolysis of VIP1 and VirE2. We show that VirF localizes to the plant cell nucleus and interacts with VIP1, a nuclear protein. VirF, which contains an F-box motif, significantly destabilizes both VIP1 and VirE2 in yeast cells. Destabilization of VIP1 in the presence of VirF was then confirmed in planta. These results suggest that VIP1 and its cognate VirE2 are specifically targeted by the VirF-containing Skp1-Cdc53-cullin-F-box complex for proteolysis. The critical role of proteasomal degradation in Agrobacterium-mediated genetic transformation was also evident from inhibition of T-DNA expression by a proteasomal inhibitor.     

The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-II reverse turn

Many short synthetic peptides have now been shown to induce antibodies reactive with their cognate sequences in the intact folded protein. Aside from the usefulness of such antibodies as site-specific reagents, the frequency with which this recognition occurs has raised several theoretical issues, the central one being that of how an antibody to a short synthetic peptide, which represents one of the most disordered states of a site in a protein, can react with the more ordered version of the same sequence in the folded protein. This apparent paradox can be resolved if the target site on the protein approaches disorder or if the peptide in solution or on a carrier adopts, with significant frequency, a conformation compatible with that of the cognate site in the protein. Various studies already suggest that antigenic sites in proteins correspond to regions of high atomic mobility. We now show, using high-field nuclear magnetic resonance (NMR) spectroscopy, that a nonapeptide selected by several monoclonal antibodies as the immunodominant site of a 36-amino-acid immunogen (residues 75-110 of influenza virus haemagglutinin) adopts a highly populated type-II reverse-turn conformation in water. This suggests that in this case the antibodies have selected a sequence possessing a conformational preference. Apart from helping us to understand immunological recognition, anti-peptide antibodies may provide reagents of sufficient precision for an immunological approach to the problem of protein folding.     

Metabolic priming by a secreted fungal effector

Maize smut caused by the fungus Ustilago maydis is a widespread disease characterized by the development of large plant tumours. U. maydis is a biotrophic pathogen that requires living plant tissue for its development and establishes an intimate interaction zone between fungal hyphae and the plant plasma membrane. U. maydis actively suppresses plant defence responses by secreted protein effectors. Its effector repertoire comprises at least 386 genes mostly encoding proteins of unknown function and expressed exclusively during the biotrophic stage. The U. maydis secretome also contains about 150 proteins with probable roles in fungal nutrition, fungal cell wall modification and host penetration as well as proteins unlikely to act in the fungal-host interface like a chorismate mutase. Chorismate mutases are key enzymes of the shikimate pathway and catalyse the conversion of chorismate to prephenate, the precursor for tyrosine and phenylalanine synthesis. Root-knot nematodes inject a secreted chorismate mutase into plant cells likely to affect development. Here we show that the chorismate mutase Cmu1 secreted by U. maydis is a virulence factor. The enzyme is taken up by plant cells, can spread to neighbouring cells and changes the metabolic status of these cells through metabolic priming. Secreted chorismate mutases are found in many plant-associated microbes and might serve as general tools for host manipulation.