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PubMed2002

Mechanism of action of EPEC type III effector molecules.

Enteropathogenic E. coli (EPEC) is a prototypic member of the family of related 'attaching and effacing (A/E)' pathogens that induce diarrhoeal disease, especially to the young that can be fatal, of a wide range of mammalian species. Disease is correlated with the loss of absorptive gut epithelial microvilli and the reorganisation of host cytoskeletal proteins into pedestal-like structures beneath the adherent bacteria. These phenotypes are dependent on a pathogenicity island (LEE; Locus of Enterocyte Effacement) encoding a type III secretion system, secreted proteins, chaperone molecules, regulatory proteins and the bacterial outer membrane protein intimin. The type III secretion apparatus directs the transfer of specific proteins across the bacterial envelope, with a subset (EPEC secreted proteins - EspA, EspB and EspD) functioning to transfer effector proteins into host cells. These effector molecules subvert cellular processes that undoubtedly benefit the pathogen and contribute to disease. Three LEE-encoded EPEC effector molecules have so far been identified with one, Tir (Translocated intimin receptor), being transferred into host cells where it is modified by host kinases and becomes inserted into the plasma membrane to orchestrate cytoskeletal rearrangements linked to disease. This activity is dependent on its interaction with intimin and on tyrosine phosphorylation, with Tir-intimin interaction essential for virulence. A second effector Map, Mitochondrial-associated protein, is targeted to mitochondria where it has membrane-potential disrupting activity. The third, EspF disrupts intestinal barrier function and can induce host cell death by unknown mechanisms. Recent data relating to the mechanism by which Tir and Map function within host cells is discussed.

Animals↗

PubMed1995

Study of stimulation-secretion coupling in a flow culture system: periodic secretion of hepatocyte growth factor by interleukin-1 alpha-stimulated human embryonic lung fibroblasts.

In order to closely examine stimulation-secretion coupling, the authors developed a 'flow culture system' in which it is possible to continuously replace the culture medium. This lessens the effects of cell-derived products observed in the conventional culture system. We compared our flow culture system and the conventional culture system based on the secretion patterns and concentrations of human hepatocyte growth factor (HGF) produced by interleukin 1 alpha (IL-1 alpha)-stimulated human embryonic lung fibroblasts (MRC-5). While the cells in the conventional culture system secreted HGF at a nearly constant rate, the cells in the flow culture system secreted HGF periodically. Even short-term stimulation with IL-1 alpha for 4 h resulted in significant HGF secretion continuing for at least 46 h. Thus the inflammatory cytokine IL-1 alpha was shown to modulate fibroblast secretion of HGF. The periodic secretion of HGF may play an important role in tissue repair and regeneration. Based on the results of actually applying it, we conclude that our flow culture system is an efficient and accurate model for the detailed examination of stimulation-secretion coupling.

Cell Line↗

PubMed2006

Identification of genistein-inducible and type III-secreted proteins of Bradyrhizobium japonicum.

Flagellin is the bulk protein secreted by Bradyrhizobium japonicum. For easier identification of minor protein fractions, the flagellin genes bll6865 and bll6866 were deleted. Extracellular proteins of the corresponding mutant were purified and separated by 2D gel electrophoresis. Several of the protein spots were detectable only after addition of genistein to the growth medium-genistein is an isoflavone secreted by soybean that activates the expression of genes encoding a type III secretion system. These secreted proteins were not present in supernatants of mutants in which conserved genes of the type III secretion system or the regulatory gene ttsI, which is essential for activation of the type III secretion system, are deleted. Out of 22 genistein-inducible protein spots 8 different proteins could be identified by mass spectrometry. One of the proteins, Blr1752, has similarity to NopP of Rhizobium sp. strain NGR234 that is known to be secreted. Another protein is Blr1656 (GunA2) that was shown previously to have endoglucanase activity. Three proteins have similarity to subunits of the flagellar apparatus. Some proteins appeared in several separate spots indicating posttranslational modification. A conserved tts box motif was found in the putative promoter region of six genes encoding secreted proteins.

Bacterial Outer Membrane Proteins↗

PubMed2001

How and when are substrates selected for type III secretion?

Many Gram-negative bacteria use type III secretion systems to secrete virulence factors as well as the structural components of the flagellum. Some bacterial secretion systems use a secretion signal contained in the amino acid sequence of the secreted substrate. However, substrates of type III systems lack a single, defined secretion signal. There is evidence for the existence of three independent secretion signals - the 5' region of the mRNA, the amino terminus of the substrate and the ability of a secretion chaperone to bind the substrate before secretion - that direct substrates for secretion through the type III pathways. One or more of these signals might be used for a given substrate. A recent study of flagellar assembly presented evidence for a role of translation in the type III secretion mechanism. We present a unifying model for type III secretion that can be applied to flagellar assembly, needle assembly and the secretion of virulence factors. The potential role of translation in regulating the timing of substrate secretion is also discussed.

5' Untranslated Regions↗

PubMed1992

Secretion of genetically-engineered dihydrofolate reductase from Escherichia coli using an E. coli alpha-hemolysin membrane translocation system.

Secretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli alpha-haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E. coli. DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured. The secretion was found to be inversely correlated with the intracellular DHFR activity. Moreover, when one amino acid (Ile155) in a beta-sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium. We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system.

Amino Acid Sequence↗

PubMed1984

A Bacillus subtilis secretion vector system derived from the B. subtilis alpha-amylase promoter and signal sequence region, and secretion of Escherichia coli beta-lactamase by the vector system.

A secretion vector system in Bacillus subtilis was constructed from the alpha-amylase promoter and signal sequence coding region of an alpha-amylase hyperproducing strain, B. subtilis NA64, and the major part of the plasmid pTUB4 which was derived from pUB110. When an Escherichia coli beta-lactamase gene, lacking its own promoter and signal sequence coding region, was introduced into the secretion vector system, beta-lactamase was expressed in B. subtilis. In addition, more than 95% of the enzyme synthesized was secreted into the culture medium via the secretion vector system. Secreted beta-lactamase crossreacted with rabbit antiserum raised against the E. coli enzyme.

Bacillus subtilis↗

PubMed2000

Assembly and function of type III secretory systems.

Type III secretion systems allow Yersinia spp., Salmonella spp., Shigella spp., Bordetella spp., and Pseudomonas aeruginosa and enteropathogenic Escherichia coli adhering at the surface of a eukaryotic cell to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane to destroy or subvert the target cell. These systems consist of a secretion apparatus, made of approximately 25 proteins, and an array of proteins released by this apparatus. Some of these released proteins are "effectors," which are delivered into the cytosol of the target cell, whereas the others are "translocators," which help the effectors to cross the membrane of the eukaryotic cell. Most of the effectors act on the cytoskeleton or on intracellular-signaling cascades. A protein injected by the enteropathogenic E. coli serves as a membrane receptor for the docking of the bacterium itself at the surface of the cell. Type III secretion systems also occur in plant pathogens where they are involved both in causing disease in susceptible hosts and in eliciting the so-called hypersensitive response in resistant or nonhost plants. They consist of 15-20 Hrp proteins building a secretion apparatus and two groups of effectors: harpins and avirulence proteins. Harpins are presumably secreted in the extracellular compartment, whereas avirulence proteins are thought to be targeted into plant cells. Although a coherent picture is clearly emerging, basic questions remain to be answered. In particular, little is known about how the type III apparatus fits together to deliver proteins in animal cells. It is even more mysterious for plant cells where a thick wall has to be crossed. In spite of these haunting questions, type III secretion appears as a fascinating trans-kingdom communication device.

Bacterial Outer Membrane Proteins↗

PubMed2001

Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis.

Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

Amino Acid Sequence↗

PubMedDate not supplied

Secretion of virulence factors by Escherichia coli.

In order to interact with their host, pathogenic strains of E. coli need to secrete some virulence factors which can modify the metabolism of host cells, contributing to disease. Since E. coli is a Gram-negative bacteria, this secretion process involves the crossing of both the inner and the outer membranes. E. coli uses mainly four secretion mechanisms called type I, type II, type III and type IV secretion systems. In the type I secretion system, the secretion machinery is composed of three proteins forming a channel through the inner and outer membranes. It is a one-step mechanism. The secretion signal is present in the carboxyterminal region of the secreted protein but without proteolytic cleavage. In E. coli, the best studied type I secreted protein is haemolysin. In type II and type IV secretion systems, the crossing of the inner membrane involves the sec machinery with the cleavage of an aminoterminal signal sequence. The crossing of the outer membrane involves the formation of a pore either by other proteins (type II) or by the carboxyterminal region of the protein (type IV). The A-B toxins, such as heat labile enterotoxin, are secreted by the first mechanism and members of the IgA proteases are secreted by the second. The type III secretion system involves at least 20 proteins including cytoplasmic, inner membrane and outer membrane proteins. The originality of this system is the ability to inject secreted bacteria into the cytosol of the host cells. Such a system is found in attaching and effacing E. coli and in diffusely adhering E. coli.

Animals↗

PubMed2006

Type IV pili-mediated secretion modulates Francisella virulence.

Francisella tularensis are the causative agent of the zoonotic disease, tularaemia. Among four F. tularensis subspecies, ssp. novicida (F. novicida) is pathogenic only for immunocompromised individuals, while all four subspecies are pathogenic for mice. This study utilized proteomic and bioinformatic approaches to identify seven F. novicida secreted proteins and the corresponding Type IV pilus (T4P) secretion system. The secreted proteins were predicted to encode two chitinases, a chitin binding protein, a protease (PepO), and a beta-glucosidase (BglX). The transcription of F. novicida pepO and bglX was regulated by the virulence regulator MglA. Intradermal infection of mice with F. novicida mutants defective in T4P secretion system or PepO resulted in enhanced F. novicida spread to systemic sites. Infection with F. novicida pepO mutants also resulted in increased neutrophil infiltration into the mouse airways. PepO is a zinc protease that is homologous to mammalian endothelin-converting enzyme ECE-1. Therefore, secretion of PepO likely results in increased production of endothelin and increased vasoconstriction at the infection site in skin that limits the F. novicida spread. Francisella human pathogenic strains contain a mutation in pepO predicted to abolish its secretion. Loss of PepO function may have contributed to evolution of highly virulent Francisellae.

Animals↗

PubMed1998

Initial binding of Shiga toxin-producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA-containing surface appendages.

Shiga toxin-producing Escherichia coli (STEC) induce so-called attaching and effacing lesions that enable the tight adherence of these pathogens to the gut epithelium. All of the genes necessary for this process are present in the locus of enterocyte effacement, which encodes a type III secretion system, the secreted Esp proteins and the surface protein intimin. In this study we sequenced the espA gene of STEC, generated and characterized a corresponding deletion mutant and raised EspA-specific monoclonal antibodies to analyse the functional role of this protein during infection. EspA was detected in often filament-like structures decorating all bacteria that had attached to HeLa cells. These appendages were especially prominent on bacteria that had not yet induced the formation of actin pedestals, indicating that they mediate the initial contact of STEC to their target cells. Consistently, a deletion of the espA gene completely abolished the capacity of such STEC mutants to bind to HeLa cells and to induce actin rearrangements. Surface appendages similar to those described in this study are also formed by Pseudomonas syringae and may represent a structural element common to many bacterial pathogens that deliver proteins into their target cells via a type III secretion system.

Actins↗

PubMed2002

Enzyme kinetics and glycan structural characterization of secreted alkaline phosphatase prepared using the baculovirus expression vector system.

Secreted human alkaline phosphatase (SEAP, a model protein containing a single N-glycan chain) was expressed in Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines infected with recombinant Autographa californica multiple nuclear polyhedrovirus expressing SEAP under control of the polyhedrin promoter. SDS-PAGE showed that both systems expressed fairly pure rSEAP products. The rSEAP expression level was 7.0 U/mL in Tn-5B1-4, higher than the 4.1 U/mL produced by Sf-9. Kinetic analysis showed that Vmax and Km of human placental SEAP were approx 10-fold higher than that of rSEAP, whereas the Vmax and Km of rSEAP prepared using both insect cell lines were comparable. To characterize the recombinant SEAP (rSEAP) glycosylation, the purified rSEAP was digested with PNGase F to release the N-glycan chains. Glycan analysis showed the presence of oligomannose-type N-linked glycans (i.e., Man(2-8)GlcNAc2 and FucMan(3 or 4)GlcNAc2) in rSEAP from Sf9 and Tn-5B1-4 cell lines. The proportions of these oligosaccharide structures were different in the two cell lines. Man4GlcNAc2 and FucMan4GlcNAc2 were the major rSEAP N-glycans produced in Sf-9 cells, while Man2GlcNAc2 was the major rSEAP N-glycan produced in Tn-5B1-4 cells.

Alkaline Phosphatase↗

PubMed2002

Buffered non-fermenter system for lab-scale production of secreted recombinant His-tagged proteins in Saccharomyces cerevisiae.

Expression of recombinant proteins using a secretion system can minimize co-purification of contaminating host proteins. Production of His-tagged recombinant proteins in the yeast alpha-factor secretion system has previously required a fermenter system to control the growth conditions such as pH of the yeast culture. We describe an inexpensive non-fermenter system for the production of secreted recombinant His-tagged proteins in Saccharomyces cerevisiae that uses a buffered low peptone YP glycerol medium, which does not interfere with immobilized metal affinity chromatography. Maspin, a tumor suppressor serpin, was expressed as a secreted N-terminal His/FLAG-tagged protein. Purification of the soluble active recombinant protein only requires centrifugation, concentration by ultrafiltration, and Ni2+ affinity chromatography. Purified protein yields of this system are 3-5 mg/L culture medium.

Buffers↗

PubMedDate not supplied

Secreted peptides as regulators of neuron-glia and glia-glia interactions in the developing nervous system.

Secreted peptides of the nervous system help to regulate neuron-glia and glia-glia interactions during development. These regulatory factors, referred to as glia-promoting factors (GPFs), act on specific classes of glia and include oligodendroglia-stimulating peptides, interleukin-1 (IL-1), colony-stimulating factors (CSF), and fibroblast growth factor (FGF). The maturity of secretory and target cells determines, in part, the ability of a factor to influence glial proliferation, activation, or differentiation. During neural development, GPFs help to control such fundamentally important events as cell movement, neurite outgrowth, and myelination.

Animals↗

PubMed2004

Analysis of the type IV secretion system-dependent cell motility of Helicobacter pylori-infected epithelial cells.

The pathogenesis of Helicobacter pylori-associated disorders is strongly dependent on a specialized type IV secretion system (T4SS) encoded by the cag pathogenicity island (PAI). Cytotoxin-associated gene A (CagA) is the only known H. pylori protein translocated into the host cell followed by tyrosine phosphorylation through host protein kinases. H. pylori induces cellular processes which are either PAI- or CagA-dependent (e.g., cell motility), PAI-dependent, but CagA-independent (e.g., interleukin-8 release), or PAI- and CagA-independent (e.g., cyclooxygenase-2 release). Here, we investigated H. pylori strains mutated in single PAI genes of the wild type strain Hp26695 and their effects on cell motility. We found 17 gene products out of 27 PAI genes playing a superordinated role and five PAI-encoded proteins exhibiting a clearly critical role in motogenic host cell responses, whereas the remaining five PAI gene products had no significant influence on the motogenic response in reaction to H. pylori infection. This study clearly demonstrated that H. pylori-induced cell motility and invasive growth involve type IV secretion system-dependent signalling as well as translocated and phosphorylated CagA. These findings reveal a deeper insight in to the meaning of the T4SS of H. pylori for host cell motility.

Antigens, Bacterial↗

PubMedDate not supplied

Molecular mechanisms of bacterial virulence: type III secretion and pathogenicity islands.

Recently, two novel but widespread themes have emerged in the field of bacterial virulence: type III secretion systems and pathogenicity islands. Type III secretion systems, which are found in various gram-negative organisms, are specialized for the export of virulence factors delivered directly to host cells. These factors subvert normal host cell functions in ways that seem beneficial to invading bacteria. The genes encoding several type III secretion systems reside on pathogenicity islands, which are inserted DNA segments within the chromosome that confer upon the host bacterium a variety of virulence traits, such as the ability to acquire iron and to adhere to or enter host cells. Many of these segments of DNA appear to have been acquired in a single step from a foreign source. The ability to obtain complex virulence traits in one genetic event, rather than by undergoing natural selection for many generations, provides a mechanism for sudden radical changes in bacterial-host interactions. Type III secretion systems and pathogenicity islands must have played critical roles in the evolution of known pathogens and are likely to lead to the emergence of novel infectious diseases in the future.

Bacterial Proteins↗

PubMed2005

Comparative analyses of Xanthomonas and Xylella complete genomes.

Computational analyses of four bacterial genomes of the Xanthomonadaceae family reveal new unique genes that may be involved in adaptation, pathogenicity, and host specificity. The Xanthomonas genus presents 3636 unique genes distributed in 1470 families, while Xylella genus presents 1026 unique genes distributed in 375 families. Among Xanthomonas-specific genes, we highlight a large number of cell wall degrading enzymes, proteases, and iron receptors, a set of energy metabolism genes, second copy of the type II secretion system, type III secretion system, flagella and chemotactic machinery, and the xanthomonadin synthesis gene cluster. Important genes unique to the Xylella genus are an additional copy of a type IV pili gene cluster and the complete machinery of colicin V synthesis and secretion. Intersections of gene sets from both genera reveal a cluster of genes homologous to Salmonella's SPI-7 island in Xanthomonas axonopodis pv citri and Xylella fastidiosa 9a5c, which might be involved in host specificity. Each genome also presents important unique genes, such as an HMS cluster, the kdgT gene, and O-antigen in Xanthomonas axonopodis pv citri; a number of avrBS genes and a distinct O-antigen in Xanthomonas campestris pv campestris, a type I restriction-modification system and a nickase gene in Xylella fastidiosa 9a5c, and a type II restriction-modification system and two genes related to peptidoglycan biosynthesis in Xylella fastidiosa temecula 1. All these differences imply a considerable number of gene gains and losses during the divergence of the four lineages, and are associated with structural genome modifications that may have a direct relation with the mode of transmission, adaptation to specific environments and pathogenicity of each organism.

Bacterial Physiological Phenomena↗

PubMed2006

Crystallization and preliminary X-ray diffraction analysis of BipD, a virulence factor from Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of melioidosis, possesses a protein-secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to secrete virulence-associated proteins into target cells of the host organism. The BipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and most likely functionally analogous to IpaD from Shigella and SipD from Salmonella. Thus, the BipD protein is likely to be a component of a type III protein-secretion system (TTSS) in B. pseudomallei. Proteins in the same class as BipD, such as IpaD and SipD, are thought to act as extracellular chaperones to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and might even link the translocon pore with the secretion needle. There is evidence that the translocator proteins also bind an integrin which stimulates actin-mediated insertion of the bacterium into the host-cell membrane. Native BipD has been crystallized in a monoclinic crystal form that diffracts X-rays to 2.5 angstroms resolution. BipD protein which incorporates selenomethionine (SeMet-BipD) has also been expressed and forms crystals which diffract to a higher resolution of 2.1 angstroms.

Bacterial Proteins↗