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Sample records for protein secretion system

  1. A light-triggered protein secretion system

    PubMed Central

    Chen, Daniel; Gibson, Emily S.

    2013-01-01

    Optical control of protein interactions has emerged as a powerful experimental paradigm for manipulating and studying various cellular processes. Tools are now available for controlling a number of cellular functions, but some fundamental processes, such as protein secretion, have been difficult to engineer using current optical tools. Here we use UVR8, a plant photoreceptor protein that forms photolabile homodimers, to engineer the first light-triggered protein secretion system. UVR8 fusion proteins were conditionally sequestered in the endoplasmic reticulum, and a brief pulse of light triggered robust forward trafficking through the secretory pathway to the plasma membrane. UVR8 was not responsive to excitation light used to image cyan, green, or red fluorescent protein variants, allowing multicolor visualization of cellular markers and secreted protein cargo as it traverses the cellular secretory pathway. We implemented this novel tool in neurons to demonstrate restricted, local trafficking of secretory cargo near dendritic branch points. PMID:23671313

  2. Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae

    PubMed Central

    Krehenbrink, Martin; Downie, J Allan

    2008-01-01

    Background Proteins secreted by bacteria play an important role in infection of eukaryotic hosts. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. Proteins secreted during the infection process by some rhizobial strains can influence infection and modify the plant defence signalling pathways. The aim of this study was to systematically analyse protein secretion in the recently sequenced strain Rhizobium leguminosarum bv. viciae 3841. Results Similarity searches using defined protein secretion systems from other Gram-negative bacteria as query sequences revealed that R. l. bv. viciae 3841 has ten putative protein secretion systems. These are the general export pathway (GEP), a twin-arginine translocase (TAT) secretion system, four separate Type I systems, one putative Type IV system and three Type V autotransporters. Mutations in genes encoding each of these (except the GEP) were generated, but only mutations affecting the PrsDE (Type I) and TAT systems were observed to affect the growth phenotype and the profile of proteins in the culture supernatant. Bioinformatic analysis and mass fingerprinting of tryptic fragments of culture supernatant proteins identified 14 putative Type I substrates, 12 of which are secreted via the PrsDE, secretion system. The TAT mutant was defective for the symbiosis, forming nodules incapable of nitrogen fixation. Conclusion None of the R. l. bv. viciae 3841 protein secretion systems putatively involved in the secretion of proteins to the extracellular space (Type I, Type IV, Type V) is required for establishing the symbiosis with legumes. The PrsDE (Type I) system was shown to be the major route of protein secretion in non-symbiotic cells and to secrete proteins of widely varied size and predicted function. This is in contrast to many Type I systems from other bacteria, which typically secrete specific substrates encoded by genes often localised in close proximity to the genes encoding the

  3. The cargo and the transport system: secreted proteins and protein secretion in Trichoderma reesei (Hypocrea jecorina).

    PubMed

    Saloheimo, Markku; Pakula, Tiina M

    2012-01-01

    Trichoderma reesei (Hypocrea jecorina) is an efficient cell factory for protein production that is exploited by the enzyme industry. Yields of over 100 g secreted protein l(-1) from industrial fermentations have been reported. In this review we discuss the spectrum of proteins secreted by T. reesei and the studies carried out on its protein secretion system. The major enzymes secreted by T. reesei under production conditions are those degrading plant polysaccharides, the most dominant ones being the major cellulases, as demonstrated by the 2D gel analysis of the secretome. According to genome analysis, T. reesei has fewer genes encoding enzymes involved in plant biomass degradation compared with other fungi with sequenced genomes. We also discuss other T. reesei secreted enzymes and proteins that have been studied, such as proteases, laccase, tyrosinase and hydrophobins. Investigation of the T. reesei secretion pathway has included molecular characterization of the pathway components functioning at different stages of the secretion process as well as analysis of the stress responses caused by impaired folding or trafficking in the pathway or by expression of heterologous proteins. Studies on the transcriptional regulation of the secretory pathway have revealed similarities, but also interesting differences, with other organisms, such as a different induction mechanism of the unfolded protein response and the repression of genes encoding secreted proteins under secretion stress conditions. PMID:22053009

  4. Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

    PubMed Central

    Metcalf, Kevin J.; Finnerty, Casey; Azam, Anum; Valdivia, Elias

    2014-01-01

    The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28 ± 9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant. PMID:25038096

  5. The ESX System in Bacillus subtilis Mediates Protein Secretion

    PubMed Central

    Chase, Michael R.; Sarracino, David A.; Fortune, Sarah M.; Burton, Briana M.

    2014-01-01

    Esat-6 protein secretion systems (ESX or Ess) are required for the virulence of several human pathogens, most notably Mycobacterium tuberculosis and Staphylococcus aureus. These secretion systems are defined by a conserved FtsK/SpoIIIE family ATPase and one or more WXG100 family secreted substrates. Gene clusters coding for ESX systems have been identified amongst many organisms including the highly tractable model system, Bacillus subtilis. In this study, we demonstrate that the B. subtilis yuk/yue locus codes for a nonessential ESX secretion system. We develop a functional secretion assay to demonstrate that each of the locus gene products is specifically required for secretion of the WXG100 virulence factor homolog, YukE. We then employ an unbiased approach to search for additional secreted substrates. By quantitative profiling of culture supernatants, we find that YukE may be the sole substrate that depends on the FtsK/SpoIIIE family ATPase for secretion. We discuss potential functional implications for secretion of a unique substrate. PMID:24798022

  6. Identification of protein secretion systems in bacterial genomes

    PubMed Central

    Abby, Sophie S.; Cury, Jean; Guglielmini, Julien; Néron, Bertrand; Touchon, Marie; Rocha, Eduardo P. C.

    2016-01-01

    Bacteria with two cell membranes (diderms) have evolved complex systems for protein secretion. These systems were extensively studied in some model bacteria, but the characterisation of their diversity has lagged behind due to lack of standard annotation tools. We built online and standalone computational tools to accurately predict protein secretion systems and related appendages in bacteria with LPS-containing outer membranes. They consist of models describing the systems’ components and genetic organization to be used with MacSyFinder to search for T1SS-T6SS, T9SS, flagella, Type IV pili and Tad pili. We identified ~10,000 candidate systems in bacterial genomes, where T1SS and T5SS were by far the most abundant and widespread. All these data are made available in a public database. The recently described T6SSiii and T9SS were restricted to Bacteroidetes, and T6SSii to Francisella. The T2SS, T3SS, and T4SS were frequently encoded in single-copy in one locus, whereas most T1SS were encoded in two loci. The secretion systems of diderm Firmicutes were similar to those found in other diderms. Novel systems may remain to be discovered, since some clades of environmental bacteria lacked all known protein secretion systems. Our models can be fully customized, which should facilitate the identification of novel systems. PMID:26979785

  7. Identification of Anaplasma marginale Type IV Secretion System Effector Proteins

    PubMed Central

    Brayton, Kelly A.; Beare, Paul A.; Brown, Wendy C.; Heinzen, Robert A.; Broschat, Shira L.

    2011-01-01

    Background Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now. Results By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system. Conclusions The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work. PMID:22140462

  8. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

    PubMed

    Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

    2013-01-01

    The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion. PMID:23075153

  9. SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli

    PubMed Central

    Deng, Wanyin; Yu, Hong B.; Li, Yuling

    2015-01-01

    ABSTRACT The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. IMPORTANCE Many bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into

  10. Por Secretion System-Dependent Secretion and Glycosylation of Porphyromonas gingivalis Hemin-Binding Protein 35

    PubMed Central

    Shoji, Mikio; Sato, Keiko; Yukitake, Hideharu; Kondo, Yoshio; Narita, Yuka; Kadowaki, Tomoko; Naito, Mariko; Nakayama, Koji

    2011-01-01

    The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study. PMID:21731719

  11. Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants

    PubMed Central

    Hueck, Christoph J.

    1998-01-01

    Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli

  12. Protein secretion systems and adhesins: the molecular armory of Gram-negative pathogens.

    PubMed

    Gerlach, Roman G; Hensel, Michael

    2007-10-01

    Protein secretion is a basic cellular function found in organisms of all kingdoms of life. Gram-negative bacteria have evolved a remarkable number of pathways for the transport of proteins across the cell envelope. The secretion systems fulfill general cellular functions but are also essential for pathogenic bacteria during the interaction with eukaryotic host cells. Secretion systems range from relatively simple structures such as type I secretion systems composed of three subunits that only secrete one substrate protein to complex machines such as type III and IV secretion systems composed of more than 20 subunits that can translocate large sets of effector proteins into eukaryotic target cells. In this review, the main structural and functional features of secretion systems are described. One subgroup of substrate proteins of secretion systems are protein adhesins. Despite the conserved function in binding to host cell ligands or to abiotic surfaces, the assembly of the various bacterial adhesins is highly divergent. Here we give an overview on the recent understanding of the assembly of fimbrial and non-fimbrial adhesins and the role of type I, III and V secretion systems and specialized branches of the general secretion pathway in their biogenesis. PMID:17482513

  13. ROLE OF TYPE III PROTEIN SECRETION SYSTEM IN SINORHIZOBIUM FREDII USDA257 AND SOYBEAN INTERACTIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant and animal pathogenic bacteria have evolved a specialized protein secretion system called type III to directly inject proteins into their host cells. The Type III secretion system (TTSS) plays an important role in plant-microbe interactions since mutation in TTSS causes a loss of bacterial pa...

  14. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems

    PubMed Central

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B.; Patel, Trushar R.; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems. PMID:27029048

  15. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems. PMID:27029048

  16. An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast

    PubMed Central

    Bae, Jung-Hoon; Hyun Sung, Bong; Kim, Hyun-Jin; Park, Soon-Ho; Lim, Kwang-Mook; Kim, Mi-Jin; Lee, Cho-Ryong; Sohn, Jung-Hoon

    2015-01-01

    To produce rarely secreted recombinant proteins in the yeast Saccharomyces cerevisiae, we developed a novel genome-wide optimal translational fusion partner (TFP) screening system that involves recruitment of an optimal secretion signal and fusion partner. A TFP library was constructed from a genomic and truncated cDNA library by using the invertase-based signal sequence trap technique. The efficiency of the system was demonstrated using two rarely secreted proteins, human interleukin (hIL)-2 and hIL-32. Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L. Moreover, numerous uncovered yeast secretion signals and fusion partners were identified, leading to efficient secretion of various recombinant proteins. Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L. This screening technique could provide new methods for the production of various types of difficult-to-express proteins. PMID:26195161

  17. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    DOEpatents

    Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

    2016-08-09

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  18. Protein Secretion Systems in Pseudomonas aeruginosa: An Essay on Diversity, Evolution, and Function.

    PubMed

    Filloux, Alain

    2011-01-01

    Protein secretion systems are molecular nanomachines used by Gram-negative bacteria to thrive within their environment. They are used to release enzymes that hydrolyze complex carbon sources into usable compounds, or to release proteins that capture essential ions such as iron. They are also used to colonize and survive within eukaryotic hosts, causing acute or chronic infections, subverting the host cell response and escaping the immune system. In this article, the opportunistic human pathogen Pseudomonas aeruginosa is used as a model to review the diversity of secretion systems that bacteria have evolved to achieve these goals. This diversity may result from a progressive transformation of cell envelope complexes that initially may not have been dedicated to secretion. The striking similarities between secretion systems and type IV pili, flagella, bacteriophage tail, or efflux pumps is a nice illustration of this evolution. Differences are also needed since various secretion configurations call for diversity. For example, some proteins are released in the extracellular medium while others are directly injected into the cytosol of eukaryotic cells. Some proteins are folded before being released and transit into the periplasm. Other proteins cross the whole cell envelope at once in an unfolded state. However, the secretion system requires conserved basic elements or features. For example, there is a need for an energy source or for an outer membrane channel. The structure of this review is thus quite unconventional. Instead of listing secretion types one after each other, it presents a melting pot of concepts indicating that secretion types are in constant evolution and use basic principles. In other words, emergence of new secretion systems could be predicted the way Mendeleïev had anticipated characteristics of yet unknown elements. PMID:21811488

  19. Protein Secretion Systems in Pseudomonas aeruginosa: An Essay on Diversity, Evolution, and Function

    PubMed Central

    Filloux, Alain

    2011-01-01

    Protein secretion systems are molecular nanomachines used by Gram-negative bacteria to thrive within their environment. They are used to release enzymes that hydrolyze complex carbon sources into usable compounds, or to release proteins that capture essential ions such as iron. They are also used to colonize and survive within eukaryotic hosts, causing acute or chronic infections, subverting the host cell response and escaping the immune system. In this article, the opportunistic human pathogen Pseudomonas aeruginosa is used as a model to review the diversity of secretion systems that bacteria have evolved to achieve these goals. This diversity may result from a progressive transformation of cell envelope complexes that initially may not have been dedicated to secretion. The striking similarities between secretion systems and type IV pili, flagella, bacteriophage tail, or efflux pumps is a nice illustration of this evolution. Differences are also needed since various secretion configurations call for diversity. For example, some proteins are released in the extracellular medium while others are directly injected into the cytosol of eukaryotic cells. Some proteins are folded before being released and transit into the periplasm. Other proteins cross the whole cell envelope at once in an unfolded state. However, the secretion system requires conserved basic elements or features. For example, there is a need for an energy source or for an outer membrane channel. The structure of this review is thus quite unconventional. Instead of listing secretion types one after each other, it presents a melting pot of concepts indicating that secretion types are in constant evolution and use basic principles. In other words, emergence of new secretion systems could be predicted the way Mendeleïev had anticipated characteristics of yet unknown elements. PMID:21811488

  20. Unusual genetic organization of a functional type I protein secretion system in Neisseria meningitidis.

    PubMed

    Wooldridge, Karl G; Kizil, Murat; Wells, Damien B; Ala'aldeen, Dlawer A A

    2005-09-01

    Proteins secreted by Neisseria meningitidis are thought to play important roles in the pathogenesis of meningococcal disease. These proteins include the iron-repressible repeat-in-toxin (RTX) exoprotein FrpC. Related proteins in other pathogens are secreted via a type I secretion system (TOSS), but such a system has not been demonstrated in N. meningitidis. An in silico search of the group B meningococcal genome suggested the presence of a uniquely organized TOSS. Genes encoding homologs of the Escherichia coli HlyB (ATP-binding), HlyD (membrane fusion), and TolC (outer membrane channel) proteins were identified. In contrast to the cistronic organization of the secretion genes in most other rtx operons, the hlyD and tolC genes were adjacent but unlinked to hlyB; neither locus was part of an operon containing genes encoding putative TOSS substrates. Both loci were flanked by genes normally associated with mobile genetic elements. The three genes were shown to be expressed independently. Mutation at either locus resulted in an inability to secrete FrpC and a related protein, here called FrpC2. Successful complementation of these mutations at an ectopic site confirmed the observed phenotypes were caused by loss of function of the putative TOSS genes. We show that genes scattered in the meningococcal genome encode a functional TOSS required for secretion of the meningococcal RTX proteins. PMID:16113272

  1. The Type II secretion system delivers matrix proteins for biofilm formation by Vibrio cholerae.

    PubMed

    Johnson, Tanya L; Fong, Jiunn C; Rule, Chelsea; Rogers, Andrew; Yildiz, Fitnat H; Sandkvist, Maria

    2014-12-01

    Gram-negative bacteria have evolved several highly dedicated pathways for extracellular protein secretion, including the type II secretion (T2S) system. Since substrates secreted via the T2S system include both virulence factors and degradative enzymes, this secretion system is considered a major survival mechanism for pathogenic and environmental species. Previous analyses revealed that the T2S system mediates the export of ≥ 20 proteins in Vibrio cholerae, a human pathogen that is indigenous to the marine environment. Here we demonstrate a new role in biofilm formation for the V. cholerae T2S system, since wild-type V. cholerae was found to secrete the biofilm matrix proteins RbmC, RbmA, and Bap1 into the culture supernatant, while an isogenic T2S mutant could not. In agreement with this finding, the level of biofilm formation in a static microtiter assay was diminished in T2S mutants. Moreover, inactivation of the T2S system in a rugose V. cholerae strain prevented the development of colony corrugation and pellicle formation at the air-liquid interface. In contrast, extracellular secretion of the exopolysaccharide VPS, an essential component of the biofilm matrix, remained unaffected in the T2S mutants. Our results indicate that the T2S system provides a mechanism for the delivery of extracellular matrix proteins known to be important for biofilm formation by V. cholerae. Because the T2S system contributes to the pathogenicity of V. cholerae by secreting proteins such as cholera toxin and biofilm matrix proteins, elucidation of the molecular mechanism of T2S has the potential to lead to the development of novel preventions and therapies. PMID:25266381

  2. The Type II Secretion System Delivers Matrix Proteins for Biofilm Formation by Vibrio cholerae

    PubMed Central

    Johnson, Tanya L.; Fong, Jiunn C.; Rule, Chelsea; Rogers, Andrew; Yildiz, Fitnat H.

    2014-01-01

    Gram-negative bacteria have evolved several highly dedicated pathways for extracellular protein secretion, including the type II secretion (T2S) system. Since substrates secreted via the T2S system include both virulence factors and degradative enzymes, this secretion system is considered a major survival mechanism for pathogenic and environmental species. Previous analyses revealed that the T2S system mediates the export of ≥20 proteins in Vibrio cholerae, a human pathogen that is indigenous to the marine environment. Here we demonstrate a new role in biofilm formation for the V. cholerae T2S system, since wild-type V. cholerae was found to secrete the biofilm matrix proteins RbmC, RbmA, and Bap1 into the culture supernatant, while an isogenic T2S mutant could not. In agreement with this finding, the level of biofilm formation in a static microtiter assay was diminished in T2S mutants. Moreover, inactivation of the T2S system in a rugose V. cholerae strain prevented the development of colony corrugation and pellicle formation at the air-liquid interface. In contrast, extracellular secretion of the exopolysaccharide VPS, an essential component of the biofilm matrix, remained unaffected in the T2S mutants. Our results indicate that the T2S system provides a mechanism for the delivery of extracellular matrix proteins known to be important for biofilm formation by V. cholerae. Because the T2S system contributes to the pathogenicity of V. cholerae by secreting proteins such as cholera toxin and biofilm matrix proteins, elucidation of the molecular mechanism of T2S has the potential to lead to the development of novel preventions and therapies. PMID:25266381

  3. A protein secretion system linked to bacteroidete gliding motility and pathogenesis.

    PubMed

    Sato, Keiko; Naito, Mariko; Yukitake, Hideharu; Hirakawa, Hideki; Shoji, Mikio; McBride, Mark J; Rhodes, Ryan G; Nakayama, Koji

    2010-01-01

    Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum. PMID:19966289

  4. A protein secretion system linked to bacteroidete gliding motility and pathogenesis

    PubMed Central

    Sato, Keiko; Naito, Mariko; Yukitake, Hideharu; Hirakawa, Hideki; Shoji, Mikio; McBride, Mark J.; Rhodes, Ryan G.; Nakayama, Koji

    2009-01-01

    Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum. PMID:19966289

  5. Development of a highly efficient protein-secreting system in recombinant Lactobacillus casei.

    PubMed

    Kajikawa, Akinobu; Ichikawa, Eiko; Igimi, Shizunobu

    2010-02-01

    The available techniques for heterologous protein secretion in Lactobacillus strains are limited. The aim of the present study was to develop an efficient protein-secretion system using recombinant lactobacilli for various applications such as live delivery of biotherapeutics. For the construction of expression vectors, the Lactobacillus brevis slpA promoter, Lactobacillus casei prtP signal sequence, and mouse IL-10 sequences were used as a model system. Interestingly, the slpA promoter exhibited strong activity in L. casei contrary to previous observations. In order to stabilize replication of the plasmid in E. coli, a removable terminator sequence was built into the promoter region. For the improvement of secretion efficiency, a DTNSD oligopeptide was added to the cleavage site of signal peptidase. The resulting plasmids provided remarkably efficient IL-10 secretion. Accumulation of the protein in the culture supernatant varied widely according to the pH conditions. By analysis of the secreted protein, formation of homodimers and biological activity, IL-10 was confirmed to be functional. The presently constructed plasmids could be useful tools for heterologous protein-secretion in L. casei. PMID:20208444

  6. A Novel Periplasmic Protein, VrpA, Contributes to Efficient Protein Secretion by the Type III Secretion System in Xanthomonas spp.

    PubMed

    Zhou, Xiaofeng; Hu, Xiufang; Li, Jinyun; Wang, Nian

    2015-02-01

    Efficient secretion of type III effector proteins from the bacterial cytoplasm to host cell cytosol via a type III secretion system (T3SS) is crucial for virulence of plant-pathogenic bacterium. Our previous study revealed a conserved hypothetical protein, virulence-related periplasm protein A (VrpA), which was identified as a critical virulence factor for Xanthomonas citri subsp. citri. In this study, we demonstrate that mutation of vrpA compromises X. citri subsp. citri virulence and hypersensitive response induction. This deficiency is also observed in the X. campestris pv. campestris strain, suggesting a functional conservation of VrpA in Xanthomonas spp. Our study indicates that VrpA is required for efficient protein secretion via T3SS, which is supported by multiple lines of evidence. A CyaA reporter assay shows that VrpA is involved in type III effector secretion; quantitative reverse-transcription polymerase chain reaction analysis suggests that the vrpA mutant fails to activate citrus-canker-susceptible gene CsLOB1, which is transcriptionally activated by transcription activator-like effector PthA4; in vitro secretion study reveals that VrpA plays an important role in secretion of T3SS pilus, translocon, and effector proteins. Our data also indicate that VrpA in X. citri subsp. citri localizes to bacterial periplasmic space and the periplasmic localization is required for full function of VrpA and X. citri subsp. citri virulence. Protein-protein interaction studies show that VrpA physically interacts with periplasmic T3SS components HrcJ and HrcC. However, the mutation of VrpA does not affect T3SS gene expression. Additionally, VrpA is involved in X. citri subsp. citri tolerance of oxidative stress. Our data contribute to the mechanical understanding of an important periplasmic protein VrpA in Xanthomonas spp. PMID:25338144

  7. An optimized system for expression and purification of secreted bacterial proteins.

    PubMed

    Geisbrecht, Brian V; Bouyain, Samuel; Pop, Mihai

    2006-03-01

    In this report, we describe an optimized system for the efficient overexpression, purification, and refolding of secreted bacterial proteins. Candidate secreted proteins were produced recombinantly in Escherichia coli as Tobacco Etch Virus protease-cleavable hexahistidine-c-myc eptiope fusion proteins. Without regard to their initial solubility, recombinant fusion proteins were extracted from whole cells with guanidium chloride, purified under denaturing conditions by immobilized metal affinity chromatography, and refolded by rapid dilution into a solution containing only Tris buffer and sodium chloride. Following concentration on the same resin under native conditions, each protein was eluted for further purification and/or characterization. Preliminary studies on a test set of 12 secreted proteins ranging in size from 13 to 130 kDa yielded between 10 and 50 mg of fusion protein per liter of induced culture at greater than 90% purity, as judged by Coomassie-stained SDS-PAGE. Of the nine proteins further purified, analytical gel filtration chromatography indicated that each was a monomer in solution and circular dichroism spectroscopy revealed that each had adopted a well-defined secondary structure. While there are many potential applications for this system, the results presented here suggest that it will be particularly useful for investigators employing structural approaches to understand protein function, as attested to by the crystal structures of three proteins purified using this methodology (B.V. Geisbrecht, B.Y. Hamaoka, B. Perman, A. Zemla, D.J. Leahy, J. Biol. Chem. 280 (2005) 17243-17250). PMID:16260150

  8. Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae

    PubMed Central

    2014-01-01

    Background The koji mold, Aspergillus oryzae is widely used for the production of industrial enzymes due to its particularly high protein secretion capacity and ability to perform post-translational modifications. However, systemic analysis of its secretion system is lacking, generally due to the poorly annotated proteome. Results Here we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely related fungal species such as Aspergillus nidulans and Aspergillus niger. To evaluate the defined component list, we performed transcriptome analysis on three α-amylase over-producing strains with varying levels of secretion capacities. Specifically, secretory components involved in the ER-associated processes (including components involved in the regulation of transport between ER and Golgi) were significantly up-regulated, with many of them never been identified for A. oryzae before. Furthermore, we defined a complete list of the putative A. oryzae secretome and monitored how it was affected by overproducing amylase. Conclusion In combination with the transcriptome data, the most complete secretory component list and the putative secretome, we improved the systemic understanding of the secretory machinery of A. oryzae in response to high levels of protein secretion. The roles of many newly predicted secretory components were experimentally validated and the enriched component list provides a better platform for driving more mechanistic studies of the protein secretory pathway in this industrially important fungus. PMID:24961398

  9. Consuming viscous prey: a novel protein-secreting delivery system in neotropical snail-eating snakes

    PubMed Central

    2014-01-01

    Background Efficient venom delivery systems are known to occur only in varanoid lizards and advanced colubroidean snakes among squamate reptiles. Although components of these venomous systems might have been present in a common ancestor, the two lineages independently evolved strikingly different venom gland systems. In snakes, venom is produced exclusively by serous glands in the upper jaw. Within the colubroidean radiation, lower jaw seromucous infralabial glands are known only in two distinct lineages–the basal pareatids and the more advanced Neotropical dipsadines known as “goo-eating snakes”. Goo-eaters are a highly diversified, ecologically specialized clade that feeds exclusively on invertebrates (e.g., gastropod molluscs and annelids). Their evolutionary success has been attributed to their peculiar feeding strategies, which remain surprisingly poorly understood. More specifically, it has long been thought that the more derived Dipsadini genera Dipsas and Sibynomorphus use glandular toxins secreted by their infralabial glands to extract snails from their shells. Results Here, we report the presence in the tribe Dipsadini of a novel lower jaw protein-secreting delivery system effected by a gland that is not functionally related to adjacent teeth, but rather opens loosely on the oral epithelium near the tip of the mandible, suggesting that its secretion is not injected into the prey as a form of envenomation but rather helps control the mucus and assists in the ingestion of their highly viscous preys. A similar protein-secreting system is also present in the goo-eating genus Geophis and may share the same adaptive purpose as that hypothesized for Dipsadini. Our phylogenetic hypothesis suggests that the acquisition of a seromucous infralabial gland represents a uniquely derived trait of the goo-eating clade that evolved independently twice within the group as a functionally complex protein-secreting delivery system. Conclusions The acquisition by snail

  10. Secretion of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase by the LEE-encoded type III secretion system in enteropathogenic Escherichia coli.

    PubMed

    Aguilera, Laura; Ferreira, Elaine; Giménez, Rosa; Fernández, Francisco José; Taulés, Marta; Aguilar, Juan; Vega, M Cristina; Badia, Josefa; Baldomà, Laura

    2012-06-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein secreted by pathogens and involved in adhesion and/or virulence. Previously we reported that enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli secrete GAPDH into the culture medium. This bacterial protein binds human plasminogen and fibrinogen and remains associated with Caco-2 cells upon infection. In these pathogens, GAPDH secretion is not linked to outer membrane vesicles and depends on growth conditions, although the secretion mechanism is still unknown. EPEC is an attaching and effacing pathogen able to secrete and translocate multiple effector proteins into infected cells through a type III secretion system (T3SS). The secretion process is often dependent on a bacterial chaperone. The chaperone CesT displays broad substrate specificity and plays a central role in the recruitment of multiple type III effectors to the T3SS apparatus. Here we provide genetic evidences on GAPDH secretion through T3SS by EPEC grown in DMEM. Secretion of GAPDH is increased in ΔsepD mutants and abolished in mutants defective in the type III ATPase EscN. Complementation with escN gene restores GAPDH secretion. In addition, we prove by means of pull down experiments, overlay immunoblotting and biolayer interferometry a novel interaction between GAPDH and the chaperone CesT. This interaction, which is strong and slow dissociating, may stabilize a population of GAPDH molecules in a secretion competent-state and target them to the type III secretion apparatus. This is the first description of CesT interaction with a housekeeping protein and its export through T3SS. PMID:22433988

  11. The S-layer proteins of Tannerella forsythia are secreted via a type IX secretion system that is decoupled from protein O-glycosylation

    PubMed Central

    Tomek, Markus B.; Neumann, Laura; Nimeth, Irene; Koerdt, Andrea; Andesner, Philipp; Messner, Paul; Mach, Lukas; Potempa, Jan S.; Schäffer, Christina

    2014-01-01

    SUMMARY Conserved C-terminal domains (CTD) have been shown to act as a signal for the translocation of certain proteins across the outer membrane of Bacteroidetes via a type IX secretion system (T9SS). The genome sequence of the periodontal pathogen Tannerella forsythia predicts the presence of the components for a T9SS in conjunction with a suite of CTD proteins. T. forsythia is covered with a 2-dimensional crystalline surface (S-) layer composed of the glycosylated CTD proteins TfsA and TfsB. To investigate if T9SS is functional in T. forsythia, T9SS-deficient mutants were generated by targeting either TF0955 (putative C-terminal signal peptidase) or TF2327 (PorK ortholog), and the mutants were analyzed with respect to secretion, assembly and glycosylation of the S-layer proteins as well as to proteolytic processing of the CTD and biofilm formation. In either mutant, TfsA and TfsB were incapable of translocation, as evidenced by the absence of the S-layer in transmission electron microscopy of ultrathin-sectioned bacterial cells. Despite entrapped within the periplasm, mass spectrometry analysis revealed that the S-layer proteins were modified with the complete, mature glycan found on the secreted proteins, indicating that protein translocation and glycosylation are two independent processes. Further, the T9SS mutants showed a denser biofilm with less voids compared to the wild-type. This study demonstrates the functionality of T9SS and the requirement of CTD for the outer membrane passage of extracellular proteins in T. forsythia, exemplified with the two S-layer proteins. In addition, T9SS protein translocation is decoupled from O-glycan attachment in T. forsythia. PMID:24943676

  12. SseBCD Proteins Are Secreted by the Type III Secretion System of Salmonella Pathogenicity Island 2 and Function as a Translocon

    PubMed Central

    Nikolaus, Thomas; Deiwick, Jörg; Rappl, Catherine; Freeman, Jeremy A.; Schröder, Werner; Miller, Samuel I.; Hensel, Michael

    2001-01-01

    The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is required for systemic infections and intracellular accumulation of Salmonella enterica. This system is induced by intracellular Salmonella and subsequently transfers effector proteins into the host cell. Growth conditions either inducing expression of the type III secretion system or the secretion of substrate proteins were defined. Here we report the identification of a set of substrate proteins consisting of SseB, SseC, and SseD that are secreted by the SPI2 system in vitro. Secretion was observed if bacterial cells were exposed to acidic pH after growth in minimal medium with limitation of Mg2+ or phosphate. SseB, -C, and -D were isolated in a fraction detached from the bacterial cell surface by mechanical shearing, indicating that these proteins are predominantly assembled into complexes on the bacterial cell surface. The three proteins were required for the translocation of SPI2 effector proteins SspH1 and SspH2 into infected host cells. Thus, SseB, SseC, and SseD function as the translocon for effector proteins by intracellular Salmonella. PMID:11567004

  13. Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish

    PubMed Central

    Lu, Jin Fang; Wang, Wei Na; Wang, Gai Ling; Zhang, He; Zhou, Ying; Gao, Zhi Peng

    2015-01-01

    The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens. PMID:26459509

  14. Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish.

    PubMed

    Lu, Jin Fang; Wang, Wei Na; Wang, Gai Ling; Zhang, He; Zhou, Ying; Gao, Zhi Peng; Nie, Pin; Xie, Hai Xia

    2016-01-01

    The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens. PMID:26459509

  15. EffectiveDB—updates and novel features for a better annotation of bacterial secreted proteins and Type III, IV, VI secretion systems

    PubMed Central

    Eichinger, Valerie; Nussbaumer, Thomas; Platzer, Alexander; Jehl, Marc-André; Arnold, Roland; Rattei, Thomas

    2016-01-01

    Protein secretion systems play a key role in the interaction of bacteria and hosts. EffectiveDB (http://effectivedb.org) contains pre-calculated predictions of bacterial secreted proteins and of intact secretion systems. Here we describe a major update of the database, which was previously featured in the NAR Database Issue. EffectiveDB bundles various tools to recognize Type III secretion signals, conserved binding sites of Type III chaperones, Type IV secretion peptides, eukaryotic-like domains and subcellular targeting signals in the host. Beyond the analysis of arbitrary protein sequence collections, the new release of EffectiveDB also provides a ‘genome-mode’, in which protein sequences from nearly complete genomes or metagenomic bins can be screened for the presence of three important secretion systems (Type III, IV, VI). EffectiveDB contains pre-calculated predictions for currently 1677 bacterial genomes from the EggNOG 4.0 database and for additional bacterial genomes from NCBI RefSeq. The new, user-friendly and informative web portal offers a submission tool for running the EffectiveDB prediction tools on user-provided data. PMID:26590402

  16. Cutting edge: Mouse NAIP1 detects the type III secretion system needle protein.

    PubMed

    Rayamajhi, Manira; Zak, Daniel E; Chavarria-Smith, Joseph; Vance, Russell E; Miao, Edward A

    2013-10-15

    The NAIP/NLRC4 inflammasomes activate caspase-1 in response to bacterial type III secretion systems (T3SSs). Inadvertent injection of the T3SS rod protein and flagellin into the cytosol is detected through murine NAIP2 and NAIP5/6, respectively. In this study, we identify the agonist for the orphan murine NAIP1 receptor as the T3SS needle protein. NAIP1 is poorly expressed in resting mouse bone marrow-derived macrophages; however, priming with polyinosinic-polycytidylic acid induces it and confers needle protein sensitivity. Further, overexpression of NAIP1 in immortalized bone marrow-derived macrophages by retroviral transduction enabled needle detection. In contrast, peritoneal cavity macrophages basally express NAIP1 and respond to needle protein robustly, independent of priming. Human macrophages are known to express only one NAIP gene, which detects the needle protein, but not rod or flagellin. Thus, murine NAIP1 is functionally analogous to human NAIP. PMID:24043898

  17. Aeromonas salmonicida Ati2 is an effector protein of the type three secretion system.

    PubMed

    Dallaire-Dufresne, Stéphanie; Barbeau, Xavier; Sarty, Darren; Tanaka, Katherine H; Denoncourt, Alix M; Lagüe, Patrick; Reith, Michael E; Charette, Steve J

    2013-09-01

    The bacterium Aeromonas salmonicida, a fish pathogen, uses the type three secretion system (TTSS) to inject effector proteins into host cells to promote the infection. The study of the genome of A. salmonicida has revealed the existence of Ati2, a potential TTSS effector protein. In the present study, a structure-function analysis of Ati2 has been done to determine its role in the virulence of A. salmonicida. Biochemical assays revealed that Ati2 is secreted into the medium in a TTSS-dependent manner. Protein sequence analyses, molecular modelling and biochemical assays demonstrated that Ati2 is an inositol polyphosphate 5-phosphatase, which hydrolyses PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in a way similar to VPA0450, a protein from Vibrio parahaemolyticus having high sequence similarity with Ati2. Mutants of Ati2 with altered amino acids at two different locations in the catalytic site displayed no phosphatase activity. Wild-type and mutant forms of Ati2 were cloned into expression systems for Dictyostelium discoideum, a soil amoeba used as an alternative host to study A. salmonicida virulence. Expression tests allowed us to demonstrate that Ati2 is toxic for the host cell in a catalytic-dependent manner. Finally, this study demonstrated the existence of a new TTSS effector protein in A. salmonicida. PMID:23832001

  18. Protein secretion in Bacillus species.

    PubMed Central

    Simonen, M; Palva, I

    1993-01-01

    Bacilli secrete numerous proteins into the environment. Many of the secretory proteins, their export signals, and their processing steps during secretion have been characterized in detail. In contrast, the molecular mechanisms of protein secretion have been relatively poorly characterized. However, several components of the protein secretion machinery have been identified and cloned recently, which is likely to lead to rapid expansion of the knowledge of the protein secretion mechanism in Bacillus species. Comparison of the presently known export components of Bacillus species with those of Escherichia coli suggests that the mechanism of protein translocation across the cytoplasmic membrane is conserved among gram-negative and gram-positive bacteria differences are found in steps preceding and following the translocation process. Many of the secretory proteins of bacilli are produced industrially, but several problems have been encountered in the production of Bacillus heterologous secretory proteins. In the final section we discuss these problems and point out some possibilities to overcome them. PMID:8464403

  19. Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells

    PubMed Central

    Galán, Jorge E.; Lara-Tejero, Maria; Marlovits, Thomas C.; Wagner, Samuel

    2015-01-01

    One of the most exciting developments in the field of bacterial pathogenesis in recent years is the discovery that many pathogens utilized complex nanomachines to deliver bacterially encoded effector proteins into target eukaryotic cells. These effector proteins modulate a variety of cellular functions for the pathogen’s benefit. One of these protein-delivery machines is the type III secretion system (T3SS). T3SSs are widespread in nature and are encoded not only by bacteria pathogenic to vertebrates or plants, but also by bacteria that are symbiotic to plants or insects. A central component of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins across the bacterial envelope. Working in conjunction with several cytoplasmic components, the needle complex engages specific substrates in sequential order, moves them across the bacterial envelope, and ultimately delivers them into eukaryotic cells. The central role of T3SSs in pathogenesis makes them great targets for novel antimicrobial strategies. PMID:25002086

  20. Extracellular secretion of recombinant proteins

    SciTech Connect

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  1. Evaluation of Salmonella enterica Type III Secretion System Effector Proteins as Carriers for Heterologous Vaccine Antigens

    PubMed Central

    Hegazy, Wael Abdel Halim; Xu, Xin; Metelitsa, Leonid

    2012-01-01

    Live attenuated strains of Salmonella enterica have a high potential as carriers of recombinant vaccines. The type III secretion system (T3SS)-dependent translocation of S. enterica can be deployed for delivery of heterologous antigens to antigen-presenting cells. Here we investigated the efficacy of various effector proteins of the Salmonella pathogenicity island (SPI2)-encoded T3SS for the translocation of model antigens and elicitation of immune responses. The SPI2 T3SS effector proteins SifA, SteC, SseL, SseJ, and SseF share an endosomal membrane-associated subcellular localization after translocation. We observed that all effector proteins could be used to translocate fusion proteins with the model antigens ovalbumin and listeriolysin into the cytosol of host cells. Under in vitro conditions, fusion proteins with SseJ and SteC stimulated T-cell responses that were superior to those triggered by fusion proteins with SseF. However, in mice vaccinated with Salmonella carrier strains, only fusion proteins based on SseJ or SifA elicited potent T-cell responses. These data demonstrate that the selection of an optimal SPI2 effector protein for T3SS-mediated translocation is a critical parameter for the rational design of effective Salmonella-based recombinant vaccines. PMID:22252866

  2. A vector system for ABC transporter-mediated secretion and purification of recombinant proteins in Pseudomonas species.

    PubMed

    Ryu, Jaewook; Lee, Ukjin; Park, Jiye; Yoo, Do-Hyun; Ahn, Jung Hoon

    2015-03-01

    Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species. PMID:25548043

  3. Detergent Isolation Stabilizes and Activates the Shigella Type III Secretion System Translocator Protein IpaC.

    PubMed

    Bernard, Abram R; Duarte, Shari M; Kumar, Prashant; Dickenson, Nicholas E

    2016-07-01

    Shigella rely on a type III secretion system as the primary virulence factor for invasion and colonization of human hosts. Although there are an estimated 90 million Shigella infections, annually responsible for more than 100,000 deaths worldwide, challenges isolating and stabilizing many type III secretion system proteins have prevented a full understanding of the Shigella invasion mechanism and additionally slowed progress toward a much needed Shigella vaccine. Here, we show that the non-denaturing zwitterionic detergent N, N-dimethyldodecylamine N-oxide (LDAO) and non-ionic detergent n-octyl-oligo-oxyethylene efficiently isolated the hydrophobic Shigella translocator protein IpaC from the co-purified IpaC/IpgC chaperone-bound complex. Both detergents resulted in monomeric IpaC that exhibits strong membrane binding and lysis characteristics while the chaperone-bound complex does not, suggesting that the stabilizing detergents provide a means of following IpaC "activation" in vitro. Additionally, biophysical characterization found that LDAO provides significant thermal and temporal stability to IpaC, protecting it for several days at room temperature and brief exposure to temperatures reaching 90°C. In summary, this work identified and characterized conditions that provide stable, membrane active IpaC, providing insight into key interactions with membranes and laying a strong foundation for future vaccine formulation studies taking advantage of the native immunogenicity of IpaC and the stability provided by LDAO. PMID:27297397

  4. A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in Shigella flexneri

    PubMed Central

    Dohlich, Kim; Zumsteg, Anna Brotcke; Goosmann, Christian; Kolbe, Michael

    2014-01-01

    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. PMID:24453973

  5. Biophysical Characterization of the Type III Secretion System Translocator Proteins and the Translocator Proteins Attached to Bacterium-Like Particles.

    PubMed

    Chen, Xiaotong; Choudhari, Shyamal P; Kumar, Prashant; Toth, Ronald T; Kim, Jae Hyun; Van Roosmalen, Maarten L; Leenhouts, Kees; Middaugh, C Russell; Picking, Wendy L; Picking, William D

    2015-12-01

    Diarrhea caused by Shigella, Salmonella, and Yersinia is an important public health problem, but development of safe and effective vaccines against such diseases is challenging. A new antigen delivery platform called bacterium-like particles (BLPs) was explored as a means for delivering protective antigens from the type III secretion systems (T3SS) of these pathogens. BLPs are peptidoglycan skeletons derived from Lactococcus lactis that are safe for newborns and can carry multiple antigens. Hydrophobic T3SS translocator proteins were fused to a peptidoglycan anchor (PA) for BLP attachment. The proteins and protein-BLP complexes associated with BLPs were characterized and the resulting data used to create three-index empirical phase diagrams (EPDs). On the basis of these EPDs, IpaB (Shigella) and SipB (Salmonella) behave distinctly from YopB (Yersinia) under different environmental stresses. Adding the PA domain appears to enhance the stability of both the PA and translocator proteins, which was confirmed using differential scanning calorimetry, and although the particles dominated the spectroscopic signals in the protein-loaded BLPs, structural changes in the proteins were still detected. The protein-BLPs were most stable near neutral pH, but these proteins' hydrophobicity made them sensitive to environmental stresses. PMID:26422758

  6. VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System.

    PubMed

    Cianfanelli, Francesca R; Alcoforado Diniz, Juliana; Guo, Manman; De Cesare, Virginia; Trost, Matthias; Coulthurst, Sarah J

    2016-06-01

    The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently ('specialised') or non-covalently ('cargo' effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a 'core' T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with

  7. VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System

    PubMed Central

    Cianfanelli, Francesca R.; Alcoforado Diniz, Juliana; Guo, Manman; De Cesare, Virginia; Trost, Matthias; Coulthurst, Sarah J.

    2016-01-01

    The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently (‘specialised’) or non-covalently (‘cargo’ effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a ‘core’ T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the

  8. A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens.

    PubMed

    Mehta, Teena; Childers, Susan E; Glaven, Richard; Lovley, Derek R; Mester, Tünde

    2006-08-01

    Extracellular electron transfer onto Fe(III) oxides in Geobacter sulfurreducens is considered to require proteins that must be exported to the outer surface of the cell. In order to investigate this, the putative gene for OxpG, the pseudopilin involved in a type II general secretion pathway of Gram-negative bacteria, was deleted. The mutant was unable to grow with insoluble Fe(III) oxide as the electron acceptor. Growth on soluble Fe(III) was not affected. An analysis of proteins that accumulated in the periplasm of the oxpG mutant, but not in the wild-type, led to the identification of a secreted protein, OmpB. OmpB is predicted to be a multicopper protein, with highest homology to the manganese oxidase, MofA, from Leptothrix discophora. OmpB contains a potential Fe(III)-binding site and a fibronectin type III domain, suggesting a possible role for this protein in accessing Fe(III) oxides. OmpB was localized to the membrane fraction of G. sulfurreducens and in the supernatant of growing cultures, consistent with the type II secretion system exporting OmpB. A mutant in which ompB was deleted had the same phenotype as the oxpG mutant, suggesting that the failure to export OmpB was responsible for the inability of the oxpG-deficient mutant to reduce Fe(III) oxide. This is the first report that proposes a role for a multicopper oxidase-like protein in an anaerobic organism. These results further emphasize the importance of outer-membrane proteins in Fe(III) oxide reduction and suggest that outer-membrane proteins other than c-type cytochromes are required for Fe(III) oxide reduction in Geobacter species. PMID:16849792

  9. Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System.

    PubMed

    Dubytska, Lidiya P; Rogge, Matthew L; Thune, Ronald L

    2016-01-01

    tons produced annually, and ESC is the leading cause of disease loss in the industry. We have demonstrated the survival and replication of E. ictaluri within channel catfish cells and identified a secretion system that is essential for E. ictaluri intracellular replication and virulence. We have also identified nine proteins encoded in the E. ictaluri genome that we believe are actively transferred from the bacterium to the cytoplasm of the host cell and act to manipulate host cell physiology to the advantage of the bacterium. The data presented here confirm that the proteins are actually transferred during an infection, which will lead to further work on approaches to preventing or controlling ESC. PMID:27303737

  10. Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System

    PubMed Central

    Dubytska, Lidiya P.; Rogge, Matthew L.

    2016-01-01

    tons produced annually, and ESC is the leading cause of disease loss in the industry. We have demonstrated the survival and replication of E. ictaluri within channel catfish cells and identified a secretion system that is essential for E. ictaluri intracellular replication and virulence. We have also identified nine proteins encoded in the E. ictaluri genome that we believe are actively transferred from the bacterium to the cytoplasm of the host cell and act to manipulate host cell physiology to the advantage of the bacterium. The data presented here confirm that the proteins are actually transferred during an infection, which will lead to further work on approaches to preventing or controlling ESC. PMID:27303737

  11. Exocytosis and protein secretion in Trypanosoma

    PubMed Central

    2010-01-01

    Background Human African trypanosomiasis is a lethal disease caused by the extracellular parasite Trypanosoma brucei. The proteins secreted by T. brucei inhibit the maturation of dendritic cells and their ability to induce lymphocytic allogenic responses. To better understand the pathogenic process, we combined different approaches to characterize these secreted proteins. Results Overall, 444 proteins were identified using mass spectrometry, the largest parasite secretome described to date. Functional analysis of these proteins revealed a strong bias toward folding and degradation processes and to a lesser extent toward nucleotide metabolism. These features were shared by different strains of T. brucei, but distinguished the secretome from published T. brucei whole proteome or glycosome. In addition, several proteins had not been previously described in Trypanosoma and some constitute novel potential therapeutic targets or diagnostic markers. Interestingly, a high proportion of these secreted proteins are known to have alternative roles once secreted. Furthermore, bioinformatic analysis showed that a significant proportion of proteins in the secretome lack transit peptide and are probably not secreted through the classical sorting pathway. Membrane vesicles from secretion buffer and infested rat serum were purified on sucrose gradient and electron microscopy pictures have shown 50- to 100-nm vesicles budding from the coated plasma membrane. Mass spectrometry confirmed the presence of Trypanosoma proteins in these microvesicles, showing that an active exocytosis might occur beyond the flagellar pocket. Conclusions This study brings out several unexpected features of the secreted proteins and opens novel perspectives concerning the survival strategy of Trypanosoma as well as possible ways to control the disease. In addition, concordant lines of evidence support the original hypothesis of the involvement of microvesicle-like bodies in the survival strategy allowing

  12. Nuclear Magnetic Resonance Characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa.

    PubMed

    Chaudhury, Sukanya; Nordhues, Bryce A; Kaur, Kawaljit; Zhang, Na; De Guzman, Roberto N

    2015-11-01

    Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS), to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologues are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residue 16 to 41 and from residue 55 to 76. The helices of PcrG are partially formed, have similar backbone dynamics, and are flexible. NMR titrations show that the entire length of PcrG residues from position 9 to 76 is involved in binding to PcrV. PcrG adds to the growing list of partially folded or unstructured proteins with important roles in type III secretion. PMID:26451841

  13. Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

    PubMed Central

    Zhou, Xiaohui; Addwebi, Tarek; Davis, Margaret A.; Orfe, Lisa; Call, Douglas R.; Guard, Jean; Besser, Thomas E.

    2011-01-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne gastroenteritis in humans worldwide. Poultry and poultry products are considered the major vehicles of transmission to humans. Using cell invasiveness as a surrogate marker for pathogenicity, we tested the invasiveness of 53 poultry-associated isolates of S. Enteritidis in a well-differentiated intestinal epithelial cell model (Caco-2). The method allowed classification of the isolates into low (n = 7), medium (n = 18) and high (n = 30) invasiveness categories. Cell invasiveness of the isolates did not correlate with the presence of the virulence-associated gene spvB or the ability of the isolates to form biofilms. Testing of representative isolates with high and low invasiveness in a mouse model revealed that the former were more invasive in vivo and caused more and earlier mortalities, whereas the latter were significantly less invasive in vivo, causing few or no mortalities. Further characterization of representative isolates with low and high invasiveness showed that most of the isolates with low invasiveness had impaired motility and impaired secretion of either flagella-associated proteins (FlgK, FljB and FlgL) or type III secretion system (TTSS)-secreted proteins (SipA and SipD) encoded on Salmonella pathogenicity island-1. In addition, isolates with low invasiveness had impaired ability to invade and/or survive within chicken macrophages. These data suggest that not all isolates of S. Enteritidis recovered from poultry may be equally pathogenic, and that the pathogenicity of S. Enteritidis isolates is associated, in part, with both motility and secretion of TTSS effector proteins. PMID:21292746

  14. Secretion of the respiratory syncytial virus fusion protein from insect cells using the baculovirus expression system.

    PubMed

    Tan, Boon-Huan; Brown, Gaie; Sugrue, Richard J

    2007-01-01

    Sequences derived from the respiratory syncytial virus (RSV) fusion (F) protein were expressed in insect cells as recombinant glutathione-S-transferase (GST)-tagged proteins. The sequence covering the F2 subunit (GST-F2), and a truncated form of the F protein in which the transmembrane domain was removed (GST-F2/F1), were cloned into the baculovirus pAcSecG2T secretory vector. These virus sequences also had the endogenous virus signal sequence removed and replaced with a signal sequence derived from the baculovirus gp67 glycoprotein, which was present in pAcSecG2T. The recombinant RSV glycoproteins were successfully detected in expressing cells by immunofluorescence assay and in the tissue culture medium by western blot analysis. The secreted recombinant GST-F2/F1 protein was further analysed using glycosidases. Our results showed that the GST-F2/F1 protein were sensitive to peptide:N-glycosidase F (PNGase F) treatment, but not to Endoglycosidase H (EndoH) treatment. This indicates that the secreted recombinant proteins were modified by the addition of mature N-linked glycan chains. PMID:17502677

  15. The Host Protein Calprotectin Modulates the Helicobacter pylori cag Type IV Secretion System via Zinc Sequestration

    PubMed Central

    Gaddy, Jennifer A.; Radin, Jana N.; Loh, John T.; Piazuelo, M. Blanca; Kehl-Fie, Thomas E.; Delgado, Alberto G.; Ilca, Florin T.; Peek, Richard M.; Cover, Timothy L.; Chazin, Walter J.; Skaar, Eric P.; Scott Algood, Holly M.

    2014-01-01

    Transition metals are necessary for all forms of life including microorganisms, evidenced by the fact that 30% of all proteins are predicted to interact with a metal cofactor. Through a process termed nutritional immunity, the host actively sequesters essential nutrient metals away from invading pathogenic bacteria. Neutrophils participate in this process by producing several metal chelating proteins, including lactoferrin and calprotectin (CP). As neutrophils are an important component of the inflammatory response directed against the bacterium Helicobacter pylori, a major risk factor for gastric cancer, it was hypothesized that CP plays a role in the host response to H. pylori. Utilizing a murine model of H. pylori infection and gastric epithelial cell co-cultures, the role CP plays in modifying H. pylori -host interactions and the function of the cag Type IV Secretion System (cag T4SS) was investigated. This study indicates elevated gastric levels of CP are associated with the infiltration of neutrophils to the H. pylori-infected tissue. When infected with an H. pylori strain harboring a functional cag T4SS, calprotectin-deficient mice exhibited decreased bacterial burdens and a trend toward increased cag T4SS -dependent inflammation compared to wild-type mice. In vitro data demonstrate that culturing H. pylori with sub-inhibitory doses of CP reduces the activity of the cag T4SS and the biogenesis of cag T4SS-associated pili in a zinc-dependent fashion. Taken together, these data indicate that zinc homeostasis plays a role in regulating the proinflammatory activity of the cag T4SS. PMID:25330071

  16. Construction, characterization and use of Edwardsiella ictaluri flagella-type three secretion system protein arrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enteric septicemia of catfish, caused by Edwardsiella ictaluri, is the leading disease in channel catfish (Ictalurus punctatus) that is responsible for $50 - 60 million economic losses to catfish producers annually in the Southeastern U.S. The flagella and type three secretion system (TTSS) in Gram...

  17. Protein secretion in Pichia pastoris and advances in protein production.

    PubMed

    Damasceno, Leonardo M; Huang, Chung-Jr; Batt, Carl A

    2012-01-01

    Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P. pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production. PMID:22057543

  18. Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins

    SciTech Connect

    Meshcheryakov, Vladimir A.; Kitao, Akio; Matsunami, Hideyuki; Samatey, Fadel A.

    2013-05-01

    Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhB{sub C}). Here, the crystal structures of FlhB{sub C} from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhB{sub C} structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhB{sub C} leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhB{sub C} and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhB{sub C} molecule.

  19. Protein Secretion and the Endoplasmic Reticulum

    PubMed Central

    Benham, Adam M.

    2012-01-01

    In a complex multicellular organism, different cell types engage in specialist functions, and as a result, the secretory output of cells and tissues varies widely. Whereas some quiescent cell types secrete minor amounts of proteins, tissues like the pancreas, producing insulin and other hormones, and mature B cells, producing antibodies, place a great demand on their endoplasmic reticulum (ER). Our understanding of how protein secretion in general is controlled in the ER is now quite sophisticated. However, there remain gaps in our knowledge, particularly when applying insight gained from model systems to the more complex situations found in vivo. This article describes recent advances in our understanding of the ER and its role in preparing proteins for secretion, with an emphasis on glycoprotein quality control and pathways of disulfide bond formation. PMID:22700933

  20. Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins.

    PubMed

    Meshcheryakov, Vladimir A; Kitao, Akio; Matsunami, Hideyuki; Samatey, Fadel A

    2013-05-01

    The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhBC). Here, the crystal structures of FlhBC from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhBC structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhBC leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhBC and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhBC molecule. PMID:23633590

  1. Bacterial Secretion Systems – An overview

    PubMed Central

    Green, Erin R.; Mecsas, Joan

    2015-01-01

    CHAPTER SUMMARY Bacterial pathogens utilize a multitude of methods to invade mammalian hosts, damage tissue sites, and thwart the immune system from responding. One essential component of these strategies for many bacterial pathogens is the secretion of proteins across phospholipid membranes. Secreted proteins can play many roles in promoting bacterial virulence, from enhancing attachment to eukaryotic cells, to scavenging resources in an environmental niche, to directly intoxicating target cells and disrupting their functions. Many pathogens use dedicated protein secretion systems to secrete virulence factors from the cytosol of the bacteria into host cells or the host environment. In general, bacterial protein secretion apparatuses can be divided into different classes, based on their structures, functions, and specificity. Some systems are conserved in all classes of bacteria and secrete a broad array of substrates, while others are only found in a small number of bacterial species and/or are specific to only one or a few proteins. In this chapter, we review the canonical features of several common bacterial protein secretion systems, as well as their roles in promoting the virulence of bacterial pathogens. Additionally, we address recent findings that indicate that the innate immune system of the host can detect and respond to the presence of protein secretion systems during mammalian infection. PMID:26999395

  2. Roles of Hcp family proteins in the pathogenesis of the porcine extraintestinal pathogenic Escherichia coli type VI secretion system.

    PubMed

    Peng, Ying; Wang, Xiangru; Shou, Jin; Zong, Bingbing; Zhang, Yanyan; Tan, Jia; Chen, Jing; Hu, Linlin; Zhu, Yongwei; Chen, Huanchun; Tan, Chen

    2016-01-01

    Hcp (hemolysin-coregulated protein) is considered a vital component of the functional T6SS (Type VI Secretion System), which is a newly discovered secretion system. Our laboratory has previously sequenced the whole genome of porcine extraintestinal pathogenic E. coli (ExPEC) strain PCN033, and identified an integrated T6SS encoding three different hcp family genes. In this study, we first identified a functional T6SS in porcine ExPEC strain PCN033, and demonstrated that the Hcp family proteins were involved in bacterial competition and the interactions with other cells. Interestingly, the three Hcp proteins had different functions. Hcp2 functioned predominantly in bacterial competition; all three proteins were involved in the colonization of mice; and Hcp1 and Hcp3 were predominantly contributed to bacterial-eukaryotic cell interactions. We showed an active T6SS in porcine ExPEC strain PCN033, and the Hcp family proteins had different functions in their interaction with other bacteria or host cells. PMID:27229766

  3. Roles of Hcp family proteins in the pathogenesis of the porcine extraintestinal pathogenic Escherichia coli type VI secretion system

    PubMed Central

    Peng, Ying; Wang, Xiangru; Shou, Jin; Zong, Bingbing; Zhang, Yanyan; Tan, Jia; Chen, Jing; Hu, Linlin; Zhu, Yongwei; Chen, Huanchun; Tan, Chen

    2016-01-01

    Hcp (hemolysin-coregulated protein) is considered a vital component of the functional T6SS (Type VI Secretion System), which is a newly discovered secretion system. Our laboratory has previously sequenced the whole genome of porcine extraintestinal pathogenic E. coli (ExPEC) strain PCN033, and identified an integrated T6SS encoding three different hcp family genes. In this study, we first identified a functional T6SS in porcine ExPEC strain PCN033, and demonstrated that the Hcp family proteins were involved in bacterial competition and the interactions with other cells. Interestingly, the three Hcp proteins had different functions. Hcp2 functioned predominantly in bacterial competition; all three proteins were involved in the colonization of mice; and Hcp1 and Hcp3 were predominantly contributed to bacterial-eukaryotic cell interactions. We showed an active T6SS in porcine ExPEC strain PCN033, and the Hcp family proteins had different functions in their interaction with other bacteria or host cells. PMID:27229766

  4. Ketoglutarate Transport Protein KgtP Is Secreted through the Type III Secretion System and Contributes to Virulence in Xanthomonas oryzae pv. oryzae

    PubMed Central

    Guo, Wei; Cai, Lu-Lu; Zou, Hua-Song; Ma, Wen-Xiu; Liu, Xi-Ling; Zou, Li-Fang; Li, Yu-Rong

    2012-01-01

    The phytopathogenic prokaryote Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight (BB) of rice and utilizes a type III secretion system (T3SS) to deliver T3SS effectors into rice cells. In this report, we show that the ketoglutarate transport protein (KgtP) is secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99A and localizes to the host cell membrane for α-ketoglutaric acid export. kgtP contained an imperfect PIP box (plant-inducible promoter) in the promoter region and was positively regulated by HrpX and HrpG. A kgtP deletion mutant was impaired in bacterial virulence and growth in planta; furthermore, the mutant showed reduced growth in minimal media containing α-ketoglutaric acid or sodium succinate as the sole carbon source. The reduced virulence and the deficiency in α-ketoglutaric acid utilization by the kgtP mutant were restored to wild-type levels by the presence of kgtP in trans. The expression of OsIDH, which is responsible for the synthesis of α-ketoglutaric acid in rice, was enhanced when KgtP was present in the pathogen. To our knowledge, this is the first report demonstrating that KgtP, which is regulated by HrpG and HrpX and secreted by the T3SS in Xanthomonas oryzae pv. oryzae, transports α-ketoglutaric acid when the pathogen infects rice. PMID:22685129

  5. Construction of chromosomally located T7 expression system for production of heterologous secreted proteins in Bacillus subtilis.

    PubMed

    Chen, Po Ting; Shaw, Jei-Fu; Chao, Yun-Peng; David Ho, Tuan-Hua; Yu, Su-May

    2010-05-12

    Bacillus subtilis is most commonly employed for secretion of recombinant proteins. To circumvent the problems caused by using plasmids, the T7 expression system known for its high efficiency was rebuilt in B. subtilis. Accordingly, a markerless and replicon-free method was developed for genomic insertion of DNAs. By the act of homologous recombination via the guide DNA, a suicidal vector carrying the gene of interest was integrated into genomic loci of bacteria. Removal of the inserted selection marker and replicon flanked by FRT sites was mediated by the FLP recombinase. By using the mentioned system, B. subtilis strain PT5 was constructed to harbor a genomic copy of the spac promoter-regulated T7 gene 1 located at wprA (encoding the cell wall-associated protease). Similarly, the T7 promoter-driven nattokinase or endoglucanase E1 of Thermomonospora fusca genes were also integrated into mpr (encoding an extracellular protease) of strain PT5. Consequently, the integrant PT5/Mmp-T7N or PT5/MT1-E1 resulted in a "clean" producer strain deprived of six proteases. After 24 h, the strain receiving induction was able to secret nattokinase and endoglucanase E1 with the volumetric activity reaching 10860 CU/mL and 8.4 U/mL, respectively. This result clearly indicates the great promise of the proposed approach for high secretion of recombinant proteins in B. subtilis. PMID:20377228

  6. EXTRACELLULAR PROTEINS INVOLVED IN SOYBEAN CULTIVAR-SPECIFIC NODULATION ARE ASSOCIATED WITH PILUS-LIKE SURFACE APPENDANGES AND EXPORTED BY A TYPE III PROTEIN SECRETION SYSTEM IN SINORHIZOBIUM FREDII USDA257

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several Gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes signal-responsive proteins (SR proteins) ...

  7. Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

    SciTech Connect

    Davis, Jamaine; Wang, Jiawei; Tropea, Joseph E.; Zhang, Di; Dauter, Zbigniew; Waugh, David S.; Wlodawer, Alexander

    2009-01-28

    VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave {alpha}-tubulin, thus facilitating intracellular spreading. We have now determined the crystal structure of VirA at 3.0 {angstrom} resolution. The shape of the molecule resembles the letter 'V,' with the residues in the N-terminal third of the 45-kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its Escherichia coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.

  8. Txc, a new type II secretion system of Pseudomonas aeruginosa strain PA7, is regulated by the TtsS/TtsR two-component system and directs specific secretion of the CbpE chitin-binding protein.

    PubMed

    Cadoret, Frédéric; Ball, Geneviève; Douzi, Badreddine; Voulhoux, Romé

    2014-07-01

    We present here the functional characterization of a third complete type II secretion system (T2SS) found in newly sequenced Pseudomonas aeruginosa strain PA7. We call this system Txc (third Xcp homolog). This system is encoded by the RGP69 region of genome plasticity found uniquely in strain PA7. In addition to the 11 txc genes, RGP69 contains two additional genes encoding a possible T2SS substrate and a predicted unorthodox sensor protein, TtsS (type II secretion sensor). We also identified a gene encoding a two-component response regulator called TtsR (type II secretion regulator), which is located upstream of the ttsS gene and just outside RGP69. We show that TtsS and TtsR constitute a new and functional two-component system that controls the production and secretion of the RGP69-encoded T2SS substrate in a Txc-dependent manner. Finally, we demonstrate that this Txc-secreted substrate binds chitin, and we therefore name it CbpE (chitin-binding protein E). PMID:24748613

  9. Txc, a New Type II Secretion System of Pseudomonas aeruginosa Strain PA7, Is Regulated by the TtsS/TtsR Two-Component System and Directs Specific Secretion of the CbpE Chitin-Binding Protein

    PubMed Central

    Cadoret, Frédéric; Ball, Geneviève; Douzi, Badreddine

    2014-01-01

    We present here the functional characterization of a third complete type II secretion system (T2SS) found in newly sequenced Pseudomonas aeruginosa strain PA7. We call this system Txc (third Xcp homolog). This system is encoded by the RGP69 region of genome plasticity found uniquely in strain PA7. In addition to the 11 txc genes, RGP69 contains two additional genes encoding a possible T2SS substrate and a predicted unorthodox sensor protein, TtsS (type II secretion sensor). We also identified a gene encoding a two-component response regulator called TtsR (type II secretion regulator), which is located upstream of the ttsS gene and just outside RGP69. We show that TtsS and TtsR constitute a new and functional two-component system that controls the production and secretion of the RGP69-encoded T2SS substrate in a Txc-dependent manner. Finally, we demonstrate that this Txc-secreted substrate binds chitin, and we therefore name it CbpE (chitin-binding protein E). PMID:24748613

  10. Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system.

    PubMed

    Dilsen, S; Paul, W; Sandgathe, A; Tippe, D; Freudl, R; Thömmes, J; Kula, M R; Takors, R; Wandrey, C; Weuster-Botz, D

    2000-09-01

    A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg 1(-1) of the fusion protein (420 mg 1(-1) of the recombinant hCT precursor) within 14 h, reaching 45 g 1(-1) cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor 1(-1) h(-1)) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-1 scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g(-1) yeast extract). PMID:11030573

  11. The LcrG Tip Chaperone Protein of the Yersinia pestis Type III Secretion System Is Partially Folded.

    PubMed

    Chaudhury, Sukanya; de Azevedo Souza, Clarice; Plano, Gregory V; De Guzman, Roberto N

    2015-09-25

    The type III secretion system (T3SS) is essential in the pathogenesis of Yersinia pestis, the causative agent of plague. A small protein, LcrG, functions as a chaperone to the tip protein LcrV, and the LcrG-LcrV interaction is important in regulating protein secretion through the T3SS. The atomic structure of the LcrG family is currently unknown. However, because of its predicted helical propensity, many have suggested that the LcrG family forms a coiled-coil structure. Here, we show by NMR and CD spectroscopy that LcrG lacks a tertiary structure and it consists of three partially folded α-helices spanning residues 7-38, 41-46, and 58-73. NMR titrations of LcrG with LcrV show that the entire length of a truncated LcrG (residues 7-73) is involved in binding to LcrV. However, there is regional variation in how LcrG binds to LcrV. The C-terminal region of a truncated LcrG (residues 52-73) shows tight binding interaction with LcrV while the N-terminal region (residues 7-51) shows weaker interaction with LcrV. This suggests that there are at least two binding events when LcrG binds to LcrV. Biological assays and mutagenesis indicate that the C-terminal region of LcrG (residues 52-73) is important in blocking protein secretion through the T3SS. Our results reveal structural and mechanistic insights into the atomic conformation of LcrG and how it binds to LcrV. PMID:26259880

  12. Non-classical protein secretion in bacteria

    PubMed Central

    Bendtsen, Jannick D; Kiemer, Lars; Fausbøll, Anders; Brunak, Søren

    2005-01-01

    Background We present an overview of bacterial non-classical secretion and a prediction method for identification of proteins following signal peptide independent secretion pathways. We have compiled a list of proteins found extracellularly despite the absence of a signal peptide. Some of these proteins also have known roles in the cytoplasm, which means they could be so-called "moon-lightning" proteins having more than one function. Results A thorough literature search was conducted to compile a list of currently known bacterial non-classically secreted proteins. Pattern finding methods were applied to the sequences in order to identify putative signal sequences or motifs responsible for their secretion. We have found no signal or motif characteristic to any majority of the proteins in the compiled list of non-classically secreted proteins, and conclude that these proteins, indeed, seem to be secreted in a novel fashion. However, we also show that the apparently non-classically secreted proteins are still distinguished from cellular proteins by properties such as amino acid composition, secondary structure and disordered regions. Specifically, prediction of disorder reveals that bacterial secretory proteins are more structurally disordered than their cytoplasmic counterparts. Finally, artificial neural networks were used to construct protein feature based methods for identification of non-classically secreted proteins in both Gram-positive and Gram-negative bacteria. Conclusion We present a publicly available prediction method capable of discriminating between this group of proteins and other proteins, thus allowing for the identification of novel non-classically secreted proteins. We suggest candidates for non-classically secreted proteins in Escherichia coli and Bacillus subtilis. The prediction method is available online. PMID:16212653

  13. Characterization of the Shigella and Salmonella Type III Secretion System Tip-Translocon Protein-Protein Interaction by Paramagnetic Relaxation Enhancement.

    PubMed

    Kaur, Kawaljit; Chatterjee, Srirupa; De Guzman, Roberto N

    2016-04-15

    Many Gram-negative pathogens, such as Shigella and Salmonella, assemble the type III secretion system (T3SS) to inject virulence proteins directly into eukaryotic cells to initiate infectious diseases. The needle apparatus of the T3SS consists of a base, an extracellular needle, a tip protein complex, and a translocon. The atomic structure of the assembled tip complex and the translocon is unknown. Here, we show by NMR paramagnetic relaxation enhancement (PRE) that the mixed α-β domain at the distal region of the Shigella and Salmonella tip proteins interacts with the N-terminal ectodomain of their major translocon proteins. Our results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and provide insights about the assembly of the needle apparatus of the T3SS. PMID:26749041

  14. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor

    PubMed Central

    Kadowaki, Tomoko; Yukitake, Hideharu; Naito, Mariko; Sato, Keiko; Kikuchi, Yuichiro; Kondo, Yoshio; Shoji, Mikio; Nakayama, Koji

    2016-01-01

    The periodontopathogen Porphyromonas gingivalis secretes potent pathogenic proteases, gingipains, via the type IX secretion system (T9SS). This system comprises at least 11 components; however, the regulatory mechanism of their expression has not yet been elucidated. Here, we found that the PorY (PGN_2001)-PorX (PGN_1019)-SigP (PGN_0274) cascade is involved in the regulation of T9SS. Surface plasmon resonance (SPR) analysis revealed a direct interaction between a recombinant PorY (rPorY) and a recombinant PorX (rPorX). rPorY autophosphorylated and transferred a phosphoryl group to rPorX in the presence of Mn2+. These results demonstrate that PorX and PorY act as a response regulator and a histidine kinase, respectively, of a two component system (TCS), although they are separately encoded on the chromosome. T9SS component-encoding genes were down-regulated in a mutant deficient in a putative extracytoplasmic function (ECF) sigma factor, PGN_0274 (SigP), similar to the porX mutant. Electrophoretic gel shift assays showed that rSigP bound to the putative promoter regions of T9SS component-encoding genes. The SigP protein was lacking in the porX mutant. Co-immunoprecipitation and SPR analysis revealed the direct interaction between SigP and PorX. Together, these results indicate that the PorXY TCS regulates T9SS-mediated protein secretion via the SigP ECF sigma factor. PMID:26996145

  15. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor.

    PubMed

    Kadowaki, Tomoko; Yukitake, Hideharu; Naito, Mariko; Sato, Keiko; Kikuchi, Yuichiro; Kondo, Yoshio; Shoji, Mikio; Nakayama, Koji

    2016-01-01

    The periodontopathogen Porphyromonas gingivalis secretes potent pathogenic proteases, gingipains, via the type IX secretion system (T9SS). This system comprises at least 11 components; however, the regulatory mechanism of their expression has not yet been elucidated. Here, we found that the PorY (PGN_2001)-PorX (PGN_1019)-SigP (PGN_0274) cascade is involved in the regulation of T9SS. Surface plasmon resonance (SPR) analysis revealed a direct interaction between a recombinant PorY (rPorY) and a recombinant PorX (rPorX). rPorY autophosphorylated and transferred a phosphoryl group to rPorX in the presence of Mn(2+). These results demonstrate that PorX and PorY act as a response regulator and a histidine kinase, respectively, of a two component system (TCS), although they are separately encoded on the chromosome. T9SS component-encoding genes were down-regulated in a mutant deficient in a putative extracytoplasmic function (ECF) sigma factor, PGN_0274 (SigP), similar to the porX mutant. Electrophoretic gel shift assays showed that rSigP bound to the putative promoter regions of T9SS component-encoding genes. The SigP protein was lacking in the porX mutant. Co-immunoprecipitation and SPR analysis revealed the direct interaction between SigP and PorX. Together, these results indicate that the PorXY TCS regulates T9SS-mediated protein secretion via the SigP ECF sigma factor. PMID:26996145

  16. Proteins Exported via the PrsD-PrsE Type I Secretion System and the Acidic Exopolysaccharide Are Involved in Biofilm Formation by Rhizobium leguminosarum

    PubMed Central

    Russo, Daniela M.; Williams, Alan; Edwards, Anne; Posadas, Diana M.; Finnie, Christine; Dankert, Marcelo; Downie, J. Allan; Zorreguieta, Angeles

    2006-01-01

    The type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles in cellular adhesion in R. leguminosarum. We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria. PMID:16740954

  17. Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosa-infected macrophages.

    PubMed

    Dacheux, D; Goure, J; Chabert, J; Usson, Y; Attree, I

    2001-04-01

    The Pseudomonas aeruginosa cystic fibrosis isolate CHA induces type III secretion system-dependent but ExoU-independent oncosis of neutrophils and macrophages. Time-lapse microscopy of the infection process revealed the rapid accumulation of motile bacteria around infected cells undergoing the process of oncosis, a phenomenon we termed pack swarming. Characterization of the non-chemotactic CHAcheZ mutant showed that pack swarming is a bacterial chemotactic response to infected macrophages. A non-cytotoxic mutant, lacking the type III-secreted proteins PcrV, PopB and PopD, was able to pack swarm only in the presence of the parental strain CHA or when macrophages were pretreated with the pore-forming toxin streptolysin O. Interaction of P. aeruginosa with red blood cells (RBCs) showed that the contact-dependent haemolysis provoked by CHA requires secretion via the type III system and the PcrV, PopB/PopD proteins. The pore inserted into RBC membrane was estimated from osmoprotection experiments to be between 2.8 and 3.5 nm. CHA-infected macrophages could be protected from cell lysis with PEG3350, indicating that the pore introduced into RBC and macrophage membranes is of similar size. The time course uptake of the vital fluorescent dye, Yo-Pro-1, into infected macrophages confirmed that the formation of transmembrane pores by CHA precedes cellular oncosis. Therefore, CHA-induced macrophage death results from a pore-forming activity that is dependent on the intact pcrGVHpopBD operon. PMID:11298277

  18. Protein secretion controlled by a synthetic gene in Escherichia coli.

    PubMed

    Blanchin-Roland, S; Masson, J M

    1989-03-01

    The inability of Escherichia coli to secrete proteins in growth medium is one of the major drawbacks in its use in genetic engineering. A synthetic gene, homologous to the one coding for the kil peptide of pColE1, was made and cloned under the control of the lac promoter, in order to obtain the inducible secretion of homologous or heterologous proteins by E. coli. The efficiency of this synthetic gene to promote secretion was assayed by analysing the production and secretion of two proteins, the R-TEM1 beta-lactamase, and the alpha-amylase from Bacillus licheniformis. This latter protein was expressed in E. coli from its gene either on the same plasmid as the kil gene or on a different plasmid. The primary effect of the induction of the kil gene is the overproduction of the secreted proteins. When expressed at a high level, the kil gene promotes the overproduction of all periplasmic proteins and the total secretion in the culture medium of both the beta-lactamase or the alpha-amylase. This secretion is semi-selective for most periplasmic proteins are not secreted. The kil peptide induces the secretion of homologous or heterologous proteins in two steps, first acting on the cytoplasmic membrane, then permeabilizing the outer membrane. This system, which is now being assayed at the fermentor scale, is the first example of using a synthetic gene to engineer a new property into a bacterial strain. PMID:2652141

  19. Identification and Analysis of Bacterial Protein Secretion Inhibitors Utilizing a SecA-LacZ Reporter Fusion System

    PubMed Central

    Alksne, L. E.; Burgio, P.; Hu, W.; Feld, B.; Singh, M. P.; Tuckman, M.; Petersen, P. J.; Labthavikul, P.; McGlynn, M.; Barbieri, L.; McDonald, L.; Bradford, P.; Dushin, R. G.; Rothstein, D.; Projan, S. J.

    2000-01-01

    Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds for Staphylococcus aureus strain MN8 were found to be ≤128 μg/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity. PMID:10817687

  20. Proteomics of protein secretion by Aggregatibacter actinomycetemcomitans.

    PubMed

    Zijnge, Vincent; Kieselbach, Thomas; Oscarsson, Jan

    2012-01-01

    The extracellular proteome (secretome) of periodontitis-associated bacteria may constitute a major link between periodontitis and systemic diseases. To obtain an overview of the virulence potential of Aggregatibacter actinomycetemcomitans, an oral and systemic human pathogen implicated in aggressive periodontitis, we used a combined LC-MS/MS and bioinformatics approach to characterize the secretome and protein secretion pathways of the rough-colony serotype a strain D7S. LC-MS/MS revealed 179 proteins secreted during biofilm growth. Further to confirming the release of established virulence factors (e.g. cytolethal distending toxin [CDT], and leukotoxin [LtxA]), we identified additional putative virulence determinants in the secretome. These included DegQ, fHbp, LppC, Macrophage infectivity protein (MIP), NlpB, Pcp, PotD, TolB, and TolC. This finding indicates that the number of extracellular virulence-related proteins is much larger than previously demonstrated, which was also supported by in silico analysis of the strain D7S genome. Moreover, our LC-MS/MS and in silico data revealed that at least Type I, II, and V secretion are actively used to excrete proteins directly into the extracellular space, or via two-step pathways involving the Sec/Tat systems for transport across the inner membrane, and outer membrane factors, secretins and auto-transporters, respectively for delivery across the outer membrane. Taken together, our results provide a molecular basis for further elucidating the role of A. actinomycetemcomitans in periodontal and systemic diseases. PMID:22848560

  1. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  2. The Deinococcus radiodurans DR1245 Protein, a DdrB Partner Homologous to YbjN Proteins and Reminiscent of Type III Secretion System Chaperones

    SciTech Connect

    Norais, Cédric; Servant, Pascale; Bouthier-de-la-Tour, Claire; Coureux, Pierre-Damien; Ithurbide, Solenne; Vannier, Françoise; Guerin, Philippe P.; Dulberger, Charles L.; Satyshur, Kenneth A.; Keck, James L.; Armengaud, Jean; Cox, Michael M.; Sommer, Suzanne

    2013-02-18

    The bacterium Deinococcus radiodurans exhibits an extreme resistance to ionizing radiation. A small subset of Deinococcus genus-specific genes were shown to be up-regulated upon exposure to ionizing radiation and to play a role in genome reconstitution. These genes include an SSB-like protein called DdrB. Here, we identified a novel protein encoded by the dr1245gene as an interacting partner of DdrB. A strain devoid of the DR1245 protein is impaired in growth, exhibiting a generation time approximately threefold that of the wild type strain while radioresistance is not affected. We determined the three-dimensional structure of DR1245, revealing a relationship with type III secretion system chaperones and YbjN family proteins. Thus, DR1245 may display some chaperone activity towards DdrB and possibly other substrates.

  3. Type V Protein Secretion Pathway: the Autotransporter Story

    PubMed Central

    Henderson, Ian R.; Navarro-Garcia, Fernando; Desvaux, Mickaël; Fernandez, Rachel C.; Ala'Aldeen, Dlawer

    2004-01-01

    Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function. PMID:15590781

  4. Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species.

    PubMed

    Chatzidaki-Livanis, Maria; Geva-Zatorsky, Naama; Comstock, Laurie E

    2016-03-29

    Type VI secretion systems (T6SSs) are multiprotein complexes best studied in Gram-negative pathogens where they have been shown to inhibit or kill prokaryotic or eukaryotic cells and are often important for virulence. We recently showed that T6SS loci are also widespread in symbiotic human gut bacteria of the order Bacteroidales, and that these T6SS loci segregate into three distinct genetic architectures (GA). GA1 and GA2 loci are present on conserved integrative conjugative elements (ICE) and are transferred and shared among diverse human gut Bacteroidales species. GA3 loci are not contained on conserved ICE and are confined toBacteroides fragilis Unlike GA1 and GA2 T6SS loci, most GA3 loci do not encode identifiable effector and immunity proteins. Here, we studied GA3 T6SSs and show that they antagonize most human gut Bacteroidales strains analyzed, except forB. fragilisstrains with the same T6SS locus. A combination of mutation analyses,trans-protection analyses, and in vitro competition assays, allowed us to identify novel effector and immunity proteins of GA3 loci. These proteins are not orthologous to known proteins, do not contain identified motifs, and most have numerous predicted transmembrane domains. Because the genes encoding effector and immunity proteins are contained in two variable regions of GA3 loci, GA3 T6SSs of the speciesB. fragilisare likely the source of numerous novel effector and immunity proteins. Importantly, we show that the GA3 T6SS of strain 638R is functional in the mammalian gut and provides a competitive advantage to this organism. PMID:26951680

  5. The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

    PubMed Central

    Luu, Dee Dee; Schwessinger, Benjamin; Daudi, Arsalan; Liu, Furong; Ruan, Randy; Fontaine-Bodin, Lisa; Koebnik, Ralf

    2014-01-01

    Pattern recognition receptors (PRRs) play an important role in detecting invading pathogens and mounting a robust defense response to restrict infection. In rice, one of the best characterized PRRs is XA21, a leucine rich repeat receptor-like kinase that confers broad-spectrum resistance to multiple strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). In 2009 we reported that an Xoo protein, called Ax21, is secreted by a type I-secretion system and that it serves to activate XA21-mediated immunity. This report has recently been retracted. Here we present data that corrects our previous model. We first show that Ax21 secretion does not depend on the predicted type I secretion system and that it is processed by the general secretion (Sec) system. We further show that Ax21 is an outer membrane protein, secreted in association with outer membrane vesicles. Finally, we provide data showing that ax21 knockout strains do not overcome XA21-mediated immunity. PMID:24482761

  6. Involvement of an Skp-Like Protein, PGN_0300, in the Type IX Secretion System of Porphyromonas gingivalis

    PubMed Central

    Taguchi, Yuko; Sato, Keiko; Yukitake, Hideharu; Inoue, Tetsuyoshi; Nakayama, Masaaki; Naito, Mariko; Kondo, Yoshio; Kano, Konami; Hoshino, Tomonori; Nakayama, Koji; Takashiba, Shogo

    2015-01-01

    The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence. PMID:26502912

  7. Development of secreted proteins as biotherapeutic agents.

    PubMed

    Bonin-Debs, Angelika L; Boche, Irene; Gille, Hendrik; Brinkmann, Ulrich

    2004-04-01

    As one of the most important classes of proteins, secreted factors account for about one-tenth of the human genome, 3000 - 4000 in total, including factors of signalling pathways, blood coagulation and immune defence, as well as digestive enzymes and components of the extracellular matrix. Secreted proteins are a rich source of new therapeutics and drug targets, and are currently the focus of major drug discovery programmes throughout the industry. Many of the most important novel drugs developed in biotechnology have resulted from the application of secreted proteins as therapeutics. Secreted proteins often circulate throughout the body and, therefore, have access to most organs and tissues. Because of that, many of the factors are themselves therapeutic agents. This paper gives an overview on the features and functions of human secreted proteins and peptides, as well as strategies by which to discover additional therapeutic proteins from the human 'secretome'. Furthermore, a variety of examples are provided for the therapeutic use of recombinant secreted proteins as 'biologicals', including features and applications of recombinant antibodies, erythropoietin, insulin, interferon, plasminogen activators, growth hormone and colony-stimulating factors. PMID:15102604

  8. Proteins Related to the Type I Secretion System Are Associated with Secondary SecA_DEAD Domain Proteins in Some Species of Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi

    PubMed Central

    Kamneva, Olga K.; Poudel, Saroj; Ward, Naomi L.

    2015-01-01

    A number of bacteria belonging to the PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) super-phylum contain unusual ribosome-bearing intracellular membranes. The evolutionary origins and functions of these membranes are unknown. Some proteins putatively associated with the presence of intracellular membranes in PVC bacteria contain signal peptides. Signal peptides mark proteins for translocation across the cytoplasmic membrane in prokaryotes, and the membrane of the endoplasmic reticulum in eukaryotes, by highly conserved Sec machinery. This suggests that proteins might be targeted to intracellular membranes in PVC bacteria via the Sec pathway. Here, we show that canonical signal peptides are significantly over-represented in proteins preferentially present in PVC bacteria possessing intracellular membranes, indicating involvement of Sec translocase in their cellular targeting. We also characterized Sec proteins using comparative genomics approaches, focusing on the PVC super-phylum. While we were unable to detect unique changes in Sec proteins conserved among membrane-bearing PVC species, we identified (1) SecA ATPase domain re-arrangements in some Planctomycetes, and (2) secondary SecA_DEAD domain proteins in the genomes of some Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi. This is the first report of potentially duplicated SecA in Gram-negative bacteria. The phylogenetic distribution of secondary SecA_DEAD domain proteins suggests that the presence of these proteins is not related to the occurrence of PVC endomembranes. Further genomic analysis showed that secondary SecA_DEAD domain proteins are located within genomic neighborhoods that also encode three proteins possessing domains specific for the Type I secretion system. PMID:26030905

  9. Proteins Related to the Type I Secretion System Are Associated with Secondary SecA_DEAD Domain Proteins in Some Species of Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi.

    PubMed

    Kamneva, Olga K; Poudel, Saroj; Ward, Naomi L

    2015-01-01

    A number of bacteria belonging to the PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) super-phylum contain unusual ribosome-bearing intracellular membranes. The evolutionary origins and functions of these membranes are unknown. Some proteins putatively associated with the presence of intracellular membranes in PVC bacteria contain signal peptides. Signal peptides mark proteins for translocation across the cytoplasmic membrane in prokaryotes, and the membrane of the endoplasmic reticulum in eukaryotes, by highly conserved Sec machinery. This suggests that proteins might be targeted to intracellular membranes in PVC bacteria via the Sec pathway. Here, we show that canonical signal peptides are significantly over-represented in proteins preferentially present in PVC bacteria possessing intracellular membranes, indicating involvement of Sec translocase in their cellular targeting. We also characterized Sec proteins using comparative genomics approaches, focusing on the PVC super-phylum. While we were unable to detect unique changes in Sec proteins conserved among membrane-bearing PVC species, we identified (1) SecA ATPase domain re-arrangements in some Planctomycetes, and (2) secondary SecA_DEAD domain proteins in the genomes of some Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi. This is the first report of potentially duplicated SecA in Gram-negative bacteria. The phylogenetic distribution of secondary SecA_DEAD domain proteins suggests that the presence of these proteins is not related to the occurrence of PVC endomembranes. Further genomic analysis showed that secondary SecA_DEAD domain proteins are located within genomic neighborhoods that also encode three proteins possessing domains specific for the Type I secretion system. PMID:26030905

  10. Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2▿

    PubMed Central

    Miki, Tsuyoshi; Shibagaki, Yoshio; Danbara, Hirofumi; Okada, Nobuhiko

    2009-01-01

    The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE(I55G) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery. PMID:19767440

  11. Function of FlhB, a membrane protein implicated in the bacterial flagellar type III secretion system.

    PubMed

    Meshcheryakov, Vladimir A; Barker, Clive S; Kostyukova, Alla S; Samatey, Fadel A

    2013-01-01

    The membrane protein FlhB is a highly conserved component of the flagellar secretion system, and it plays an active role in the regulation of protein export. In this study conserved properties of FlhB that are important for its function were investigated. Replacing the flhB gene (or part of the gene) in Salmonella typhimurium with the flhB gene of the distantly related bacterium Aquifex aeolicus greatly reduces motility. However, motility can be restored to some extent by spontaneous mutations in the part of flhB gene coding for the cytoplasmic domain of Aquifex FlhB. Structural analysis suggests that these mutations destabilize the structure. The secondary structure and stability of the mutated cytoplasmic fragments of FlhB have been studied by circular dichroism spectroscopy. The results suggest that conformational flexibility could be important for FlhB function. An extragenic suppressor mutation in the fliS gene, which decreases the affinity of FliS to FliC, partially restores motility of the FlhB substitution mutants. PMID:23874605

  12. Biochemical Methods to Analyze Wnt Protein Secretion.

    PubMed

    Glaeser, Kathrin; Boutros, Michael; Gross, Julia Christina

    2016-01-01

    Wnt proteins act as potent morphogens in various aspects of embryonic development and adult tissue homeostasis. However, in addition to its physiological importance, aberrant Wnt signaling has been linked to the onset and progression of different types of cancer. On the cellular level, the secretion of Wnt proteins involves trafficking of lipid-modified Wnts from the endoplasmic reticulum (ER) to Golgi and further compartments via the Wnt cargo receptor evenness interrupted. Others and we have recently shown that Wnt proteins are secreted on extracellular vesicles (EVs) such as microvesicles and exosomes. Although more details about specific regulation of Wnt secretion steps are emerging, it remains largely unknown how Wnt proteins are channeled into different release pathways such as lipoprotein particles, EVs and cytonemes. Here, we describe protocols to purify and quantify Wnts from the supernatant of cells by either assessing total Wnt proteins in the supernatant or monitoring Wnt proteins on EVs. Purified Wnts from the supernatant as well as total cellular protein content can be investigated by immunoblotting. Additionally, the relative activity of canonical Wnts in the supernatant can be assessed by a dual-luciferase Wnt reporter assay. Quantifying the amount of secreted Wnt proteins and their activity in the supernatant of cells allows the investigation of intracellular trafficking events that regulate Wnt secretion and the role of extracellular modulators of Wnt spreading. PMID:27590148

  13. Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector proteins of Pseudomonas syringae pv. phaseolicola 1448A

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the Hrp type III secretion system (T3SS). To identity genes encoding type III effectors and other potential virulence factors that are r...

  14. Additive effect of calreticulin and translation initiation factor eIF4E on secreted protein production in the baculovirus expression system.

    PubMed

    Teng, Chao-Yi; van Oers, Monique M; Wu, Tzong-Yuan

    2013-10-01

    The baculovirus expression vector system is widely used for the production of recombinant proteins. However, the yield of membrane-bound or secreted proteins is relatively low when compared with intracellular or nuclear proteins. In a previous study, we had demonstrated that the co-expression of the human chaperones calreticulin (CALR) or β-synuclein (β-syn) increased the production of a secreted protein considerably. A similar effect was also seen when co-expressing insect translation initiation factor eIF4E. In this study, different combinations of the three genes were tested (CALR alone, β-syn + CALR, or β-syn + CALR + eIF4E) to further improve secretory protein production by assessing the expression level of a recombinant secreted alkaline phosphatase (SEFP). An additional 1.8-fold increment of SEFP production was obtained when cells co-expressed all the three "helper" genes, compared to cells, in which only CALR was co-produced with SEFP. Moreover, the duration of the SEFP production lasted much longer in cells that co-expressed these three "helper" genes, up to 10 dpi was observed. Utilization of this "triple-supporters" containing vector offers significant advantages when producing secreted proteins and is likely to have benefits for the production of viral vaccines and other pharmaceutical products. PMID:23900798

  15. Delivery of Therapeutic Proteins as Secretable TAT Fusion Products

    PubMed Central

    Flinterman, Marcella; Farzaneh, Farzin; Habib, Nagy; Malik, Farooq; Gäken, Joop; Tavassoli, Mahvash

    2008-01-01

    The trans-acting activator of transcription (TAT) protein transduction domain (PTD) mediates the transduction of peptides and proteins into target cells. The TAT-PTD has an important potential as a tool for the delivery of therapeutic agents. The production of TAT fusion proteins in bacteria, however, is problematic because of protein insolubility and the absence of eukaryotic post-translational modification. An attractive alternative, both for in vitro protein production and for in vivo applications, is the use of higher eukaryotic cells for secretion of TAT fusion proteins. However, the ubiquitous expression of furin endoprotease (PACE or SPC1) in the Golgi/endoplasmic reticulum, and the presence of furin recognition sequences within TAT-PTD, results in the cleavage and loss of the TAT-PTD domain during its secretory transition through the endoplasmic reticulum and Golgi. In this study, we show the development of a synthetic TATκ-PTD in which mutation of the furin recognition sequences, but retention of protein transduction activity, allows secretion of recombinant proteins, followed by successful uptake of the modified protein, by the target cells. This system was used to successfully secrete marker protein, green fluorescent protein (GFP), and apoptin, a protein with tumor-specific cytotoxicity. Detection of GFP, phosphorylation, and induction of cell death by TATκ-GFP-apoptin indicated that the secreted proteins were functional in target cells. This novel strategy therefore has important potential for the efficient delivery of therapeutic proteins. PMID:19050698

  16. Sorting sweet sorting. Protein secretion.

    PubMed

    Ponnambalam, S; Banting, G

    1996-09-01

    Membrane-spanning, lectin-like proteins in the eukaryotic secretory pathway seem to operate quality-control checkpoints by fine tuning protein exit or retention within each subcompartment. PMID:8805362

  17. Dynamics of protein secretion during adipocyte differentiation.

    PubMed

    Ojima, Koichi; Oe, Mika; Nakajima, Ikuyo; Muroya, Susumu; Nishimura, Takanori

    2016-08-01

    The major functions of adipocytes include both lipid storage and the production of secretory factors. However, the type of proteins released from mouse 3T3-L1 cells during adipocyte differentiation remains poorly understood. We examined the dynamics of secreted proteins during adipocyte differentiation using mass spectrometry (MS) combined with an iTRAQ (®) labeling method that enables the simultaneous analysis of relative protein expression levels. A total of 215 proteins were identified and quantified from approximately 10 000 MS/MS spectra. Of these, approximately 38% were categorized as secreted proteins based on gene ontology classification. Adipokine secretion levels were increased with the progression of differentiation. By contrast, levels of fibril collagen components, such as subunits of type I and III collagens, were decreased during differentiation. Basement membrane components attained their peak levels at day 4 when small lipid droplets accumulated in differentiated 3T3-L1 cells. Simultaneously, peak levels of collagen microfibril components that comprise type V and VI collagen subunits were also observed. Our data demonstrated that extracellular matrix components were predominantly released during the early and middle stages of adipocyte differentiation, with a subsequent increase in the secretion of adipokines. This suggests that 3T3-L1 cells secrete adipokines after their ECM is constructed during adipocyte differentiation. PMID:27516960

  18. THE N-TERMINAL AMPHIPATHIC REGION OF THE ESCHERICHIA COLI TYPE III SECRETION SYSTEM PROTEIN EspD IS REQUIRED FOR MEMBRANE INSERTION AND FUNCTION

    PubMed Central

    Dasanayake, Dayal; Richaud, Manon; Cyr, Normand; Caballero-Franco, Celia; Pitroff, Sabrina; Finn, Ron M.; Ausió, Juan; Luo, Wensheng; Donnenberg, Michael S.; Jardim, Armando

    2011-01-01

    Enterohemorrhagic E. coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N-terminal segment of EspD, encompassing residues 1–171, contains two amphipathic domains spanning residues 24–41 and 66–83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His6-EspD1-171 adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His6-EspD1-171 undergoes a pH depended conformational change that increases the α-helix content of this protein ~7-fold. This change coincides with insertion of the region circumscribing Trp47 into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His6-EspD1-171 forms a homodimer that is postulated to promote EspD-EspD oligomerization and pore formation. Complementation of ΔespD null mutant bacteria with an espDΔ66-83 gene showed that this protein was secreted but non-functional. PMID:21651628

  19. Analysis of secreted proteins using SILAC.

    PubMed

    Henningsen, Jeanette; Blagoev, Blagoy; Kratchmarova, Irina

    2014-01-01

    Secreted proteins serve a crucial role in the communication between cells, tissues, and organs. Proteins released to the extracellular environment exert their function either locally or at distant points of the organism. Proteins are secreted in a highly dynamic fashion by cells and tissues in the body responding to the stimuli and requirements presented by the extracellular milieu. Characterization of secretomes derived from various cell types has been performed using different quantitative mass spectrometry-based proteomics strategies, several of them taking advantage of labeling with stable isotopes. Here, we describe the use of Stable Isotope Labeling by Amino acids in Cell culture (SILAC) for the quantitative analysis of the skeletal muscle secretome during myogenesis. PMID:25059621

  20. Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin

    PubMed Central

    Pukatzki, Stefan; Ma, Amy T.; Revel, Andrew T.; Sturtevant, Derek; Mekalanos, John J.

    2007-01-01

    Genes encoding type VI secretion systems (T6SS) are widely distributed in pathogenic Gram-negative bacterial species. In Vibrio cholerae, T6SS have been found to secrete three related proteins extracellularly, VgrG-1, VgrG-2, and VgrG-3. VgrG-1 can covalently cross-link actin in vitro, and this activity was used to demonstrate that V. cholerae can translocate VgrG-1 into macrophages by a T6SS-dependent mechanism. Protein structure search algorithms predict that VgrG-related proteins likely assemble into a trimeric complex that is analogous to that formed by the two trimeric proteins gp27 and gp5 that make up the baseplate “tail spike” of Escherichia coli bacteriophage T4. VgrG-1 was shown to interact with itself, VgrG-2, and VgrG-3, suggesting that such a complex does form. Because the phage tail spike protein complex acts as a membrane-penetrating structure as well as a conduit for the passage of DNA into phage-infected cells, we propose that the VgrG components of the T6SS apparatus may assemble a “cell-puncturing device” analogous to phage tail spikes to deliver effector protein domains through membranes of target host cells. PMID:17873062

  1. Structural Characterization of the Yersinia pestis Type III Secretion System Needle Protein YscF in Complex with Its Heterodimeric Chaperone YscE/YscG

    SciTech Connect

    Sun, Ping; Tropea, Joseph E.; Austin, Brian P.; Cherry, Scott; Waugh, David S.

    2008-05-03

    The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as 'chaperones' to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 {angstrom} resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.

  2. An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production.

    PubMed

    Low, Kheng Oon; Mahadi, Nor Muhammad; Rahim, Raha Abdul; Rabu, Amir; Abu Bakar, Farah Diba; Murad, Abdul Munir Abdul; Illias, Rosli Md

    2011-09-01

    Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage. PMID:21336875

  3. Structure of a Type IV Secretion System

    PubMed Central

    Rivera-Calzada, Angel; Braun, Nathalie; Connery, Sarah; Dujeancourt, Annick; Lu, Fang; Redzej, Adam; Fronzes, Rémi; Orlova, Elena V.; Waksman, Gabriel

    2014-01-01

    Bacterial type IV secretion (T4S) systems translocate virulence factors into eukaryotic cells1,2, distribute genetic material between bacteria, and have shown potential as a tool for the genetic modification of human cells3. Given the complex choreography of the substrate through the secretion apparatus4, the molecular mechanism of the T4S system has proven difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy (EM) to reconstruct the T4S system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form a ~3 megadalton (MDa) nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex1 connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of twelve VirB4 ATPase subunits organised as side by side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems1,4-6. PMID:24670658

  4. Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria.

    PubMed

    Kim, Jin-Sik; Song, Saemee; Lee, Minho; Lee, Seunghwa; Lee, Kangseok; Ha, Nam-Chul

    2016-03-01

    The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria. PMID:26833388

  5. Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum

    PubMed Central

    VieBrock, Lauren; Evans, Sean M.; Beyer, Andrea R.; Larson, Charles L.; Beare, Paul A.; Ge, Hong; Singh, Smita; Rodino, Kyle G.; Heinzen, Robert A.; Richards, Allen L.; Carlyon, Jason A.

    2015-01-01

    Scrub typhus is an understudied, potentially fatal infection that threatens one billion persons in the Asia-Pacific region. How the causative obligate intracellular bacterium, Orientia tsutsugamushi, facilitates its intracellular survival and pathogenesis is poorly understood. Many intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) that traffic to distinct subcellular locations and modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank repertoires plus T1SS and T4SS components. Whether these potential virulence factors are expressed during infection, how the Anks are potentially secreted, and to where they localize in the host cell are not known. We determined that O. tsutsugamushi transcriptionally expresses 20 unique ank genes as well as genes for both T1SS and T4SS during infection of mammalian host cells. Examination of the Anks' C-termini revealed that the majority of them resemble T1SS substrates. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks in an HlyBD-dependent manner. Thus, O. tsutsugamushi Anks C-termini are T1SS-compatible. Conversely, Coxiella burnetii could not secrete heterologously expressed Anks in a T4SS-dependent manner. Analysis of the subcellular distribution patterns of 20 ectopically expressed Anks revealed that, while 6 remained cytosolic or trafficked to the nucleus, 14 localized to, and in some cases, altered the morphology of the endoplasmic reticulum. This study identifies O. tsutsugamushi Anks as T1SS substrates and indicates that many display a tropism for the host cell secretory pathway. PMID:25692099

  6. Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems

    SciTech Connect

    Samudrala, Ram; Heffron, Fred; McDermott, Jason E.

    2009-04-24

    The type III secretion system is an essential component for virulence in many Gram-negative bacteria. Though components of the secretion system apparatus are conserved, its substrates, effector proteins, are not. We have used a machine learning approach to identify new secreted effectors. The method integrates evolutionary measures, such as the pattern of homologs in a range of other organisms, and sequence-based features, such as G+C content, amino acid composition and the N-terminal 30 residues of the protein sequence. The method was trained on known effectors from Salmonella typhimurium and validated on a corresponding set of effectors from Pseudomonas syringae, after eliminating effectors with detectable sequence similarity. The method was able to identify all of the known effectors in P. syringae with a specificity of 84% and sensitivity of 82%. The reciprocal validation, training on P. syringae and validating on S. typhimurium, gave similar results with a specificity of 86% when the sensitivity level was 87%. These results show that type III effectors in disparate organisms share common features. We found that maximal performance is attained by including an N-terminal sequence of only 30 residues, which agrees with previous studies indicating that this region contains the secretion signal. We then used the method to define the most important residues in this putative secretion signal. Finally, we present novel predictions of secreted effectors in S. typhimurium, some of which have been experimentally validated, and apply the method to predict secreted effectors in the genetically intractable human pathogen Chlamydia trachomatis. This approach is a novel and effective way to identify secreted effectors in a broad range of pathogenic bacteria for further experimental characterization and provides insight into the nature of the type III secretion signal.

  7. Production and secretion of recombinant proteins in Dictyostelium discoideum.

    PubMed

    Dittrich, W; Williams, K L; Slade, M B

    1994-06-01

    We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system. The cellular slime mold Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface associated D. discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme beta-glucuronidase (GUS) from Escherichia coli was cell associated. Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium. The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection. PMID:7764951

  8. HDX-MS and deletion analysis of the type 4 secretion system protein TraF from the Escherichia coli F plasmid.

    PubMed

    Lento, Cristina; Ferraro, Michele; Wilson, Derek; Audette, Gerald F

    2016-02-01

    Conjugative DNA transfer by the F-plasmid is achieved through a type IV secretion system (T4SS) encoded within the plasmid's transfer region; TraF is one of several F-T4SS proteins essential for F-pilus assembly. In order to identify regions of the protein important for TraF function, a series of deletion mutants were assessed for their ability to recover conjugative transfer in a traF knockout. Interestingly, modification of any region of TraF abolishes pilus synthesis, resulting in a loss of rescue of conjugative function. Dynamic analysis of TraF by time-resolved hydrogen-deuterium exchange revealed that the C-terminal region containing the predicted thioredoxin-like domain is quite structured, while the N-terminal region, predicted to interact with TraH in the intact F-T4SS, was more dynamic. PMID:26785931

  9. C-terminal domain of CagX is responsible for its interaction with CagT protein of Helicobacter pylori type IV secretion system.

    PubMed

    Gopal, Gopal Jee; Pal, Jagannath; Kumar, Awanish; Mukhopadhyay, Gauranga

    2015-01-01

    Helicobacter pylori are the well known human pathogen associated with gastric cancer and peptic ulcer. Pathogenesis is mainly due to the presence of 40 kb cagPAI (cag Pathogenicity Island) region that encodes the type IV secretion system (TFSS) consisting of a cytoplasmic part, a middle part/core complex (spans from inner membrane to outer membrane), and an outer membrane associated part. CagX and CagT are two important proteins of TFSS that have homology with virB9 and virB7 of Agrobacterium tumefaciens TFSS. In this study, we have shown that the CagX and CagT interact directly by using co-immunoprecipitation of endogenous CagX and CagT and MBP pull down assay. We further authenticate this observation using yeast two-hybrid assay and co-expression of both the protein coding gene in Escherichia coli. We also observed that the C-terminal region of CagX is important for CagT interaction. We reconfirm that CagT depends on CagX for its stabilization. These observations could contribute in overall visualization of assembly and architecture of TFSS because protein-protein interactions among Cag proteins are likely to have an important role in assembly. Thorough understanding about architecture and mechanism of action of cag-TFSS may lead to design controlled drug delivery system. PMID:25446105

  10. Mutations in the Pseudomonas aeruginosa Needle Protein Gene pscF Confer Resistance to Phenoxyacetamide Inhibitors of the Type III Secretion System

    PubMed Central

    Bowlin, Nicholas O.; Williams, John D.; Knoten, Claire A.; Torhan, Matthew C.; Tashjian, Tommy F.; Li, Bing; Aiello, Daniel; Mecsas, Joan; Hauser, Alan R.; Peet, Norton P.; Bowlin, Terry L.

    2014-01-01

    The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates effector toxins into host cells, impeding the host's rapid innate immune response to infection. Inhibitors of T3SS may be useful as prophylactic or adjunctive therapeutic agents to augment the activity of antibiotics in P. aeruginosa infections, such as pneumonia and bacteremia. One such inhibitor, the phenoxyacetamide MBX 1641, exhibits very responsive structure-activity relationships, including striking stereoselectivity, in its inhibition of P. aeruginosa T3SS. These features suggest interaction with a specific, but unknown, protein target. Here, we identify the apparent molecular target by isolating inhibitor-resistant mutants and mapping the mutation sites by deep sequencing. Selection and sequencing of four independent mutants resistant to the phenoxyacetamide inhibitor MBX 2359 identified the T3SS gene pscF, encoding the needle apparatus, as the only locus of mutations common to all four strains. Transfer of the wild-type and mutated alleles of pscF, together with its chaperone and cochaperone genes pscE and pscG, to a ΔpscF P. aeruginosa strain demonstrated that each of the single-codon mutations in pscF is necessary and sufficient to provide secretion and translocation that is resistant to a variety of phenoxyacetamide inhibitor analogs but not to T3SS inhibitors with different chemical scaffolds. These results implicate the PscF needle protein as an apparent new molecular target for T3SS inhibitor discovery and suggest that three other chemically distinct T3SS inhibitors interact with one or more different targets or a different region of PscF. PMID:24468789

  11. The Versatile Type VI Secretion System

    PubMed Central

    Alteri, Christopher J.; Mobley, Harry L.T.

    2016-01-01

    Summary Bacterial Type VI Secretion Systems (T6SS) function as contractile nanomachines to puncture target cells and deliver lethal effectors. In the ten years since the discovery of the T6SS, much has been learned about the structure and function of this versatile protein secretion apparatus. Most of the conserved protein components that comprise the T6SS apparatus itself have been identified and ascribed specific functions. In addition, numerous effector proteins that are translocated by the T6SS have been identified and characterized. These protein effectors usually represent toxic cargoes that are delivered by the attacker cell to a target cell. The field is beginning to better understand the lifestyle or physiology that dictates when bacteria normally express their T6SS. In this Chapter, we consider what is known about the structure and regulation of the T6SS, the numerous classes of antibacterial effector T6SS substrates, and how the action of the T6SS relates to a given lifestyle or behavior in certain bacteria. PMID:27227310

  12. High-Yield Secretion of Multiple Client Proteins in Aspergillus

    SciTech Connect

    Segato, F.; Damasio, A. R. L.; Goncalves, T. A.; de Lucas, R. C.; Squina, F. M.; Decker, S. R.; Prade, R. A.

    2012-07-15

    Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.

  13. The Role of Pathogen-Secreted Proteins in Fungal Vascular Wilt Diseases

    PubMed Central

    de Sain, Mara; Rep, Martijn

    2015-01-01

    A limited number of fungi can cause wilting disease in plants through colonization of the vascular system, the most well-known being Verticillium dahliae and Fusarium oxysporum. Like all pathogenic microorganisms, vascular wilt fungi secrete proteins during host colonization. Whole-genome sequencing and proteomics screens have identified many of these proteins, including small, usually cysteine-rich proteins, necrosis-inducing proteins and enzymes. Gene deletion experiments have provided evidence that some of these proteins are required for pathogenicity, while the role of other secreted proteins remains enigmatic. On the other hand, the plant immune system can recognize some secreted proteins or their actions, resulting in disease resistance. We give an overview of proteins currently known to be secreted by vascular wilt fungi and discuss their role in pathogenicity and plant immunity. PMID:26473835

  14. A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

    PubMed Central

    Souza, Diorge P.; Andrade, Maxuel O.; Alvarez-Martinez, Cristina E.; Arantes, Guilherme M.; Farah, Chuck S.; Salinas, Roberto K.

    2011-01-01

    Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7XAC2622) and its interaction with VirB9. NMR solution studies show that residues 27–41 of the disordered flexible N-terminal region of VirB7XAC2622 interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7XAC2622 has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7XAC2622 oligomerizes through interactions involving conserved residues in the N0 domain and residues 42–49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7XAC2622 oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring. PMID:21589901

  15. A microdomain for protein secretion in Gram-positive bacteria.

    PubMed

    Rosch, Jason; Caparon, Michael

    2004-06-01

    Gram-positive bacteria face unique challenges in generating biologically active conformations for their exported proteins because they lack a dedicated compartment for folding secreted polypeptides. We have discovered that protein secretion by way of the general secretory (Sec) pathway in the important human pathogen Streptococcus pyogenes proceeds through a single microdomain. Unlike other mechanisms for asymmetry involving the Sec pathway, proteins destined for secretion are targeted to a single locus distal to either cell pole that has specialized to contain the Sec translocons. This subcellular organization may represent a paradigm for secretion common to other Gram-positive pathogens with profound implications for pathogenesis. PMID:15178803

  16. TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System*

    PubMed Central

    Zoued, Abdelrahim; Durand, Eric; Bebeacua, Cecilia; Brunet, Yannick R.; Douzi, Badreddine; Cambillau, Christian; Cascales, Eric; Journet, Laure

    2013-01-01

    The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly. PMID:23921384

  17. Investigation of the role of the BAM complex and SurA chaperone in outer-membrane protein biogenesis and type III secretion system expression in Salmonella.

    PubMed

    Fardini, Yann; Trotereau, Jérôme; Bottreau, Elisabeth; Souchard, Charlène; Velge, Philippe; Virlogeux-Payant, Isabelle

    2009-05-01

    In Escherichia coli, the assembly of outer-membrane proteins (OMP) requires the BAM complex and periplasmic chaperones, such as SurA or DegP. Previous work has suggested a potential link between OMP assembly and expression of the genes encoding type-III secretion systems. In order to test this hypothesis, we studied the role of the different lipoproteins of the BAM complex (i.e. BamB, BamC, BamD and BamE), and the periplasmic chaperones SurA and DegP, in these two phenotypes in Salmonella. Analysis of the corresponding deletion mutants showed that, as previously described with the DeltabamB mutant, BamD, SurA and, to a lesser extent, BamE play a role in outer-membrane biogenesis in Salmonella Enteritidis, while the membrane was not notably disturbed in DeltabamC and DeltadegP mutants. Interestingly, we found that BamD is not essential in Salmonella, unlike its homologues in Escherichia coli and Neisseria gonorrhoeae. In contrast, BamD was the only protein required for full expression of T3SS-1 and flagella, as demonstrated by transcriptional analysis of the genes involved in the biosynthesis of these T3SSs. In line with this finding, bamD mutants showed a reduced secretion of effector proteins by these T3SSs, and a reduced ability to invade HT-29 cells. As DeltasurA and DeltabamE mutants had lower levels of OMPs in their outer membrane, but showed no alteration in T3SS-1 and flagella expression, these results demonstrate the absence of a systematic link between an OMP assembly defect and the downregulation of T3SSs in Salmonella; therefore, this link appears to be related to a more specific mechanism that involves at least BamB and BamD. PMID:19372159

  18. The secreted immunoglobulin domain proteins ZIG-5 and ZIG-8 cooperate with L1CAM/SAX-7 to maintain nervous system integrity.

    PubMed

    Bénard, Claire Y; Blanchette, Cassandra; Recio, Janine; Hobert, Oliver

    2012-01-01

    During nervous system development, neuronal cell bodies and their axodendritic projections are precisely positioned through transiently expressed patterning cues. We show here that two neuronally expressed, secreted immunoglobulin (Ig) domain-containing proteins, ZIG-5 and ZIG-8, have no detectable role during embryonic nervous system development of the nematode Caenorhabditis elegans but are jointly required for neuronal soma and ventral cord axons to maintain their correct position throughout postembryonic life of the animal. The maintenance defects observed upon removal of zig-5 and zig-8 are similar to those observed upon complete loss of the SAX-7 protein, the C. elegans ortholog of the L1CAM family of adhesion proteins, which have been implicated in several neurological diseases. SAX-7 exists in two isoforms: a canonical, long isoform (SAX-7L) and a more adhesive shorter isoform lacking the first two Ig domains (SAX-7S). Unexpectedly, the normally essential function of ZIG-5 and ZIG-8 in maintaining neuronal soma and axon position is completely suppressed by genetic removal of the long SAX-7L isoform. Overexpression of the short isoform SAX-7S also abrogates the need for ZIG-5 and ZIG-8. Conversely, overexpression of the long isoform disrupts adhesion, irrespective of the presence of the ZIG proteins. These findings suggest an unexpected interdependency of distinct Ig domain proteins, with one isoform of SAX-7, SAX-7L, inhibiting the function of the most adhesive isoform, SAX-7S, and this inhibition being relieved by ZIG-5 and ZIG-8. Apart from extending our understanding of dedicated neuronal maintenance mechanisms, these findings provide novel insights into adhesive and anti-adhesive functions of IgCAM proteins. PMID:22829780

  19. Secretion of a bacterial protein by mammalian cells.

    PubMed

    Clément, J M; Jehanno, M

    1995-12-15

    The MalE protein is a periplasmic maltooligosaccharide binding protein from Escherichia coli. This protein is widely used as a model for protein export in bacteria and as a vector for the export and one-step affinity purification of foreign polypeptides. Expression of MalE was studied in various animal cell lines. The protein was exported into the culture medium, following the classical pathway of eukaryotic protein secretion. This was shown by a combination of approaches including the use of inhibitors of the Golgi complex and immunocytological methods. The signal sequence of MalE is required for secretion and a specific signal can be added to MalE that targets it to the endoplasmic reticulum. This work opens the way to the study of the secretion of a bacterial protein and to its use as a vector for protein secretion and purification from mammalian cells. PMID:8590643

  20. Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection

    PubMed Central

    Dolezal, Pavel; Aili, Margareta; Tong, Janette; Jiang, Jhih-Hang; Marobbio, Carlo M.; Lee, Sau fung; Schuelein, Ralf; Belluzzo, Simon; Binova, Eva; Mousnier, Aurelie; Frankel, Gad; Giannuzzi, Giulia; Palmieri, Ferdinando; Gabriel, Kipros; Naderer, Thomas; Hartland, Elizabeth L.; Lithgow, Trevor

    2012-01-01

    The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms. PMID:22241989

  1. Type Three Secretion System Island-Encoded Proteins Required for Colonization by Non-O1/Non-O139 Serogroup Vibrio cholerae

    PubMed Central

    Chaand, Mudit; Miller, Kelly A.; Sofia, Madeline K.; Schlesener, Cory; Weaver, Jacob W. A.; Sood, Vibha

    2015-01-01

    Vibrio cholerae is a genetically diverse species, and pathogenic strains can encode different virulence factors that mediate colonization and secretory diarrhea. Although the toxin-coregulated pilus (TCP) is the primary colonization factor in epidemic-causing V. cholerae strains, other strains do not encode the TCP and instead promote colonization via the activity of a type 3 secretion system (T3SS). Using the infant mouse model and T3SS-positive O39 serogroup strain AM-19226, we sought to determine which of 12 previously identified, T3SS-translocated proteins (Vops) are important for host colonization. We constructed in-frame deletions in each of the 12 loci in strain AM-19226 and identified five Vop deletion strains, including ΔVopM, which were severely attenuated for colonization. Interestingly, a subset of deletion strains was also incompetent for effector protein transport. Our collective data therefore suggest that several translocated proteins may also function as components of the structural apparatus or translocation machinery and indicate that while VopM is critical for establishing an infection, the combined activities of other effectors may also contribute to the ability of T3SS-positive strains to colonize host epithelial cell surfaces. PMID:25939511

  2. Examining marginal sequence similarities between bacterial type III secretion system components and Trypanosoma cruzi surface proteins: horizontal gene transfer or convergent evolution?

    PubMed Central

    Silva, Danielle C. F.; Silva, Richard C.; Ferreira, Renata C.; Briones, Marcelo R. S.

    2013-01-01

    The cell invasion mechanism of Trypanosoma cruzi has similarities with some intracellular bacterial taxa especially regarding calcium mobilization. This mechanism is not observed in other trypanosomatids, suggesting that the molecules involved in this type of cell invasion were a product of (1) acquisition by horizontal gene transfer (HGT); (2) secondary loss in the other trypanosomatid lineages of the mechanism inherited since the bifurcation Bacteria-Neomura (1.9 billion to 900 million years ago); or (3) de novo evolution from non-homologous proteins via convergent evolution. Similar to T. cruzi, several bacterial genera require increased host cell cytosolic calcium for intracellular invasion. Among intracellular bacteria, the mechanism of host cell invasion of genus Salmonella is the most similar to T. cruzi. The invasion of Salmonella occurs by contact with the host's cell surface and is mediated by the type III secretion system (T3SS) that promotes the contact-dependent translocation of effector proteins directly into host's cell cytoplasm. Here we provide evidence of distant sequence similarities and structurally conserved domains between T. cruzi and Salmonella spp T3SS proteins. Exhaustive database searches were directed to a wide range of intracellular bacteria and trypanosomatids, exploring sequence patterns for comparison of structural similarities and Bayesian phylogenies. Based on our data we hypothesize that T. cruzi acquired genes for calcium mobilization mediated invasion by ancient HGT from ancestral Salmonella lineages. PMID:23967008

  3. Identification and characterization of secreted proteins in Eimeria tenella

    NASA Astrophysics Data System (ADS)

    Ramlee, Intan Azlinda; Firdaus-Raih, Mohd; Wan, Kiew-Lian

    2015-09-01

    Eimeria tenella is a protozoan parasite that causes coccidiosis, an economically important disease in the poultry industry. The characterization of proteins that are secreted by parasites have been shown to play important roles in parasite invasion and are considered to be potential control agents. In this study, 775 proteins potentially secreted by E. tenella were identified. These proteins were further filtered to remove mitochondrial proteins. Out of 763 putative secreted proteins, 259 proteins possess transmembrane domains while another 150 proteins have GPI (Glycosylphosphatidylinositol) anchors. Homology search revealed that 315 and 448 proteins have matches with known and hypothetical proteins in the database, respectively. Within this data set, previously characterized secretory proteins such as micronemes, rhoptry kinases and dense granules were detected.

  4. An Experimental Approach for the Identification of Conserved Secreted Proteins in Trypanosomatids

    PubMed Central

    Corrales, Rosa M.; Mathieu-Daudé, Françoise; Garcia, Déborah; Brenière, Simone F.; Sereno, Denis

    2010-01-01

    Extracellular factors produced by Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei are important in the host-parasite relationship. Here, we describe a genome-based approach to identify putative extracellular proteins conserved among trypanosomatids that are likely involved in the classical secretory pathway. Potentially secreted proteins were identified by bioinformatic analysis of the T. cruzi genome. A subset of thirteen genes encoding unknown proteins with orthologs containing a signal peptide sequence in L. infantum, L. major, and T. brucei were transfected into L. infantum. Tagged proteins detected in the extracellular medium confirmed computer predictions in about 25% of the hits. Secretion was confirmed for two L. infantum orthologs proteins using the same experimental system. Infectivity studies of transgenic Leishmania parasites suggest that one of the secreted proteins increases parasite replication inside macrophages. This methodology can identify conserved secreted proteins involved in the classical secretory pathway, and they may represent potential virulence factors in trypanosomatids. PMID:20145711

  5. An intrinsic mechanism of secreted protein aging and turnover

    PubMed Central

    Yang, Won Ho; Aziz, Peter V.; Heithoff, Douglas M.; Mahan, Michael J.; Smith, Jeffrey W.; Marth, Jamey D.

    2015-01-01

    The composition and functions of the secreted proteome are controlled by the life spans of different proteins. However, unlike intracellular protein fate, intrinsic factors determining secreted protein aging and turnover have not been identified and characterized. Almost all secreted proteins are posttranslationally modified with the covalent attachment of N-glycans. We have discovered an intrinsic mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosaminidase B (HexB), possess hydrolytic activities that temporally remodel N-glycan structures, progressively exposing different saccharides with increased protein age. Subsequently, endocytic lectins with distinct binding specificities, including the Ashwell–Morell receptor, integrin αM, and macrophage mannose receptor, are engaged in N-glycan ligand recognition and the turnover of secreted proteins. Glycosidase inhibition and lectin deficiencies increased protein life spans and abundance, and the basal rate of N-glycan remodeling varied among distinct proteins, accounting for differences in their life spans. This intrinsic multifactorial mechanism of secreted protein aging and turnover contributes to health and the outcomes of disease. PMID:26489654

  6. A host-specific virulence protein of Erwinia herbicola pv. gypsophilae is translocated into human epithelial cells by the Type III secretion system of enteropathogenic Escherichia coli.

    PubMed

    Valinsky, Lea; Nisan, Israel; Tu, Xuanlin; Nisan, Gal; Rosenshine, Ilan; Hanski, Emanuel; Barash, Isaac; Manulis, Shulamit

    2002-03-01

    summary HsvG is a virulence factor that determines the host specificity of Erwinia herbicola pathovars gypsophilae and betae on gypsophila. We used the calmodulin adenylate cyclase reporter (CyaA) to demonstrate that HsvG is secreted and translocated into HeLa cells by the type III secretion system (TTSS) of the enteropathogenic Escherichia coli (EPEC). A fusion of HsvG-CyaA containing 271 amino acids of the N-terminus of HsvG were introduced into a wild-type EPEC, espB mutant deficient in translocation and an escV mutant deficient in secretion. A significant secretion was detected in EPEC/HsvG-CyaA and its espB mutant, but not with the escV mutant. Translocation was only observed with the wild-type EPEC, and not with the other two mutants. To localize the secretion and translocation signals of HsvG, fusions containing 39, 11 and 3 amino acids of the N-terminus of HsvG were constructed and expressed in EPEC. A fusion containing the first 39 N-terminal amino acids of HsvG was secreted and translocated at significant level (31-35%) as compared to the original fusion. In contrast, fusions containing the 3 and 11 amino acids failed to be secreted and translocated. PMID:20569314

  7. Evaluation of immunogenicity and protective efficacy of orally delivered Shigella type III secretion system proteins IpaB and IpaD.

    PubMed

    Heine, Shannon J; Diaz-McNair, Jovita; Martinez-Becerra, Francisco J; Choudhari, Shyamal P; Clements, John D; Picking, Wendy L; Pasetti, Marcela F

    2013-06-19

    Shigella spp. are food- and water-borne pathogens that cause shigellosis, a severe diarrheal and dysenteric disease that is associated with a high morbidity and mortality in resource-poor countries. No licensed vaccine is available to prevent shigellosis. We have recently demonstrated that Shigella invasion plasmid antigens (Ipas), IpaB and IpaD, which are components of the bacterial type III secretion system (TTSS), can prevent infection in a mouse model of intranasal immunization and lethal pulmonary challenge. Because they are conserved across Shigella spp. and highly immunogenic, these proteins are excellent candidates for a cross-protective vaccine. Ideally, such a vaccine could be administered to humans orally to induce mucosal and systemic immunity. In this study, we investigated the immunogenicity and protective efficacy of Shigella IpaB and IpaD administered orally with a double mutant of the Escherichia coli heat labile toxin (dmLT) as a mucosal adjuvant. We characterized the immune responses induced by oral vs. intranasal immunization and the protective efficacy using a mouse pulmonary infection model. Serum IgG and fecal IgA against IpaB were induced after oral immunization. These responses, however, were lower than those obtained after intranasal immunization despite a 100-fold dosage increase. The level of protection induced by oral immunization with IpaB and IpaD was 40%, while intranasal immunization resulted in 90% protective efficacy. IpaB- and IpaD-specific IgA antibody-secreting cells in the lungs and spleen and T-cell-derived IL-2, IL-5, IL-17 and IL-10 were associated with protection. These results demonstrate the immunogenicity of orally administered IpaB and IpaD and support further studies in humans. PMID:23644075

  8. In vivo quantification of the secretion rates of the hemolysin A Type I secretion system.

    PubMed

    Lenders, Michael H H; Beer, Tobias; Smits, Sander H J; Schmitt, Lutz

    2016-01-01

    Type 1 secretion systems (T1SS) of Gram-negative bacteria secrete a broad range of substrates into the extracellular space. Common to all substrates is a C-terminal secretion sequence and nonapeptide repeats in the C-terminal part that bind Ca(2+) in the extracellular space, to trigger protein folding. Like all T1SS, the hemolysin A (HlyA) T1SS of Escherichia coli consists of an ABC transporter, a membrane fusion protein and an outer membrane protein allowing the one step translocation of the substrate across both membranes. Here, we analyzed the secretion rate of the HlyA T1SS. Our results demonstrate that the rate is independent of substrate-size and operates at a speed of approximately 16 amino acids per transporter per second. We also demonstrate that the rate is independent of the extracellular Ca(2+) concentration raising the question of the driving force of substrate secretion by T1SS in general. PMID:27616645

  9. Type III secretion needle proteins induce cell signaling and cytokine secretion via Toll-like receptors.

    PubMed

    Jessen, Danielle L; Osei-Owusu, Patrick; Toosky, Melody; Roughead, William; Bradley, David S; Nilles, Matthew L

    2014-06-01

    Pathogens are recognized by hosts by use of various receptors, including the Toll-like receptor (TLR) and Nod-like receptor (NLR) families. Ligands for these varied receptors, including bacterial products, are identified by the immune system, resulting in development of innate immune responses. Only a couple of components from type III secretion (T3S) systems are known to be recognized by TLR or NLR family members. Known T3S components that are detected by pattern recognition receptors (PRRs) are (i) flagellin, detected by TLR5 and NLRC4 (Ipaf); and (ii) T3S rod proteins (PrgJ and homologs) and needle proteins (PrgI and homologs), detected by NAIP and the NLRC4 inflammasome. In this report, we characterize the induction of proinflammatory responses through TLRs by the Yersinia pestis T3S needle protein, YscF, the Salmonella enterica needle proteins PrgI and SsaG, and the Shigella needle protein, MxiH. More specifically, we determine that the proinflammatory responses occur through TLR2 and -4. These data support the hypothesis that T3S needles have an unrecognized role in bacterial pathogenesis by modulating immune responses. PMID:24643544

  10. Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

    PubMed Central

    Simone, Domenico; Bay, Denice C.; Leach, Thorin; Turner, Raymond J.

    2013-01-01

    Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial

  11. Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

    SciTech Connect

    Lee, Je Min; Lee, Sang-Jik; Rose, Jocelyn K.C.; Yeam, Inhwa; Kim, Byung-Dong

    2014-04-18

    Highlights: • Yeast secretion trap (YST) is a valuable tool for mining secretome. • A total of 80 secreted proteins are newly identified via YST in pepper fruits. • The secreted proteins are differentially regulated during pepper development and ripening. • Transient GFP-fusion assay and in planta secretion trap can effectively validate the secretion of proteins. - Abstract: Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function in fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.

  12. Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin

    SciTech Connect

    Leiman, Petr G.; Basler, Marek; Ramagopal, Udupi A.; Bonanno, Jeffrey B.; Sauder, J. Michael; Pukatzki, Stefan; Burley, Stephen K.; Almo, Steven C.; Mekalanos, John J.

    2009-04-22

    Protein secretion is a common property of pathogenic microbes. Gram-negative bacterial pathogens use at least 6 distinct extracellular protein secretion systems to export proteins through their multilayered cell envelope and in some cases into host cells. Among the most widespread is the newly recognized Type VI secretion system (T6SS) which is composed of 15--20 proteins whose biochemical functions are not well understood. Using crystallographic, biochemical, and bioinformatic analyses, we identified 3 T6SS components, which are homologous to bacteriophage tail proteins. These include the tail tube protein; the membrane-penetrating needle, situated at the distal end of the tube; and another protein associated with the needle and tube. We propose that T6SS is a multicomponent structure whose extracellular part resembles both structurally and functionally a bacteriophage tail, an efficient machine that translocates proteins and DNA across lipid membranes into cells.

  13. High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

    PubMed Central

    Dobrijevic, Dragana; Di Liberto, Gaetana; Tanaka, Kosei; de Wouters, Tomas; Dervyn, Rozenn; Boudebbouze, Samira; Binesse, Johan; Blottière, Hervé M.; Jamet, Alexandre; Maguin, Emmanuelle; van de Guchte, Maarten

    2013-01-01

    Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems. PMID

  14. Structural modeling of the flagellum MS ring protein FliF reveals similarities to the type III secretion system and sporulation complex

    PubMed Central

    2016-01-01

    The flagellum is a large proteinaceous organelle found at the surface of many bacteria, whose primary role is to allow motility through the rotation of a long extracellular filament. It is an essential virulence factor in many pathogenic species, and is also a priming component in the formation of antibiotic-resistant biofilms. The flagellum consists of the export apparatus on the cytosolic side; the basal body and rotor, spanning the bacterial membrane(s) and periplasm; and the hook-filament, that protrudes away from the bacterial surface. Formation of the basal body MS ring region, constituted of multiple copies of the protein FliF, is one of the initial steps of flagellum assembly. However, the precise architecture of FliF is poorly understood. Here, I report a bioinformatics analysis of the FliF sequence from various bacterial species, suggesting that its periplasmic region is composed of three globular domains. The first two are homologous to that of the type III secretion system injectisome proteins SctJ, and the third possesses a similar fold to that of the sporulation complex component SpoIIIAG. I also describe that Chlamydia possesses an unusual FliF protein, lacking part of the SctJ homology domain and the SpoIIIAG-like domain, and fused to the rotor component FliG at its C-terminus. Finally, I have combined the sequence analysis of FliF with the EM map of the MS ring, to propose the first atomic model for the FliF oligomer, suggesting that FliF is structurally akin to a fusion of the two injectisome components SctJ and SctD. These results further define the relationship between the flagellum, injectisome and sporulation complex, and will facilitate future structural characterization of the flagellum basal body. PMID:26925337

  15. Francisella novicida Pathogenicity Island Encoded Proteins Were Secreted during Infection of Macrophage-Like Cells

    PubMed Central

    Hare, Rebekah F.; Hueffer, Karsten

    2014-01-01

    Intracellular pathogens and other organisms have evolved mechanisms to exploit host cells for their life cycles. Virulence genes of some intracellular bacteria responsible for these mechanisms are located in pathogenicity islands, such as secretion systems that secrete effector proteins. The Francisella pathogenicity island is required for phagosomal escape, intracellular replication, evasion of host immune responses, virulence, and encodes a type 6 secretion system. We hypothesize that some Francisella novicida pathogenicity island proteins are secreted during infection of host cells. To test this hypothesis, expression plasmids for all Francisella novicida FPI-encoded proteins with C-terminal and N-terminal epitope FLAG tags were developed. These plasmids expressed their respective epitope FLAG-tagged proteins at their predicted molecular weights. J774 murine macrophage-like cells were infected with Francisella novicida containing these plasmids. The FPI proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of the respective genes that were deficient for intramacrophage growth. Using these expression plasmids, the localization of the Francisella pathogenicity island proteins were examined via immuno-fluorescence microscopy within infected macrophage-like cells. Several Francisella pathogenicity island encoded proteins (IglABCDEFGHIJ, PdpACE, DotU and VgrG) were detected extracellularly and they were co-localized with the bacteria, while PdpBD and Anmk were not detected and thus remained inside bacteria. Proteins that were co-localized with bacteria had different patterns of localization. The localization of IglC was dependent on the type 6 secretion system. This suggests that some Francisella pathogenicity island proteins were secreted while others remain within the bacterium during infection of host cells as structural components of the secretion system and were necessary for secretion. PMID:25158041

  16. Francisella novicida pathogenicity island encoded proteins were secreted during infection of macrophage-like cells.

    PubMed

    Hare, Rebekah F; Hueffer, Karsten

    2014-01-01

    Intracellular pathogens and other organisms have evolved mechanisms to exploit host cells for their life cycles. Virulence genes of some intracellular bacteria responsible for these mechanisms are located in pathogenicity islands, such as secretion systems that secrete effector proteins. The Francisella pathogenicity island is required for phagosomal escape, intracellular replication, evasion of host immune responses, virulence, and encodes a type 6 secretion system. We hypothesize that some Francisella novicida pathogenicity island proteins are secreted during infection of host cells. To test this hypothesis, expression plasmids for all Francisella novicida FPI-encoded proteins with C-terminal and N-terminal epitope FLAG tags were developed. These plasmids expressed their respective epitope FLAG-tagged proteins at their predicted molecular weights. J774 murine macrophage-like cells were infected with Francisella novicida containing these plasmids. The FPI proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of the respective genes that were deficient for intramacrophage growth. Using these expression plasmids, the localization of the Francisella pathogenicity island proteins were examined via immuno-fluorescence microscopy within infected macrophage-like cells. Several Francisella pathogenicity island encoded proteins (IglABCDEFGHIJ, PdpACE, DotU and VgrG) were detected extracellularly and they were co-localized with the bacteria, while PdpBD and Anmk were not detected and thus remained inside bacteria. Proteins that were co-localized with bacteria had different patterns of localization. The localization of IglC was dependent on the type 6 secretion system. This suggests that some Francisella pathogenicity island proteins were secreted while others remain within the bacterium during infection of host cells as structural components of the secretion system and were necessary for secretion. PMID:25158041

  17. Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins.

    PubMed

    Larsen, Sasha; Weaver, Jun; de Sa Campos, Katherine; Bulahan, Rhobe; Nguyen, Jackson; Grove, Heather; Huang, Amy; Low, Lauren; Tran, Namphuong; Gomez, Seth; Yau, Jennifer; Ilustrisimo, Thomas; Kawilarang, Jessica; Lau, Jonathan; Tranphung, Maivi; Chen, Irene; Tran, Christina; Fox, Marcia; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2013-11-01

    Although Pichia pastoris is a popular protein expression system, it exhibits limitations in its ability to secrete heterologous proteins. Therefore, a REMI (restriction enzyme mediated insertion) strategy was utilized to select mutant beta-g alactosidase s upersecretion (bgs) strains that secreted increased levels of a β-galactosidase reporter. Many of the twelve BGS genes may have functions in intracellular signaling or vesicle transport. Several of these strains also appeared to contain a more permeable cell wall. Preliminary characterization of four bgs mutants showed that they differed in the ability to enhance the export of other reporter proteins. bgs13, which has a disruption in a gene homologous to Saccharomyces cerevisiae protein kinase C (PKC1), gave enhanced secretion of most recombinant proteins that were tested, raising the possibility that it has the universal super-secreter phenotype needed in an industrial production strain of P. pastoris. PMID:23881328

  18. Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective

    PubMed Central

    Chagnot, Caroline; Zorgani, Mohamed A.; Astruc, Thierry; Desvaux, Mickaël

    2013-01-01

    Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates) is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative), monoderm (archetypal Gram-positive) and diderm-mycolate (archetypal acid-fast) bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors) involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field. PMID:24133488

  19. Signal peptide optimization tool for the secretion of recombinant protein from Saccharomyces cerevisiae.

    PubMed

    Mori, Akihiro; Hara, Shoichi; Sugahara, Tomohiro; Kojima, Takaaki; Iwasaki, Yugo; Kawarasaki, Yasuaki; Sahara, Takehiko; Ohgiya, Satoru; Nakano, Hideo

    2015-11-01

    The secretion efficiency of foreign proteins in recombinant microbes is strongly dependent on the combination of the signal peptides (SPs) used and the target proteins; therefore, identifying the optimal SP sequence for each target protein is a crucial step in maximizing the efficiency of protein secretion in both prokaryotes and eukaryotes. In this study, we developed a novel method, named the SP optimization tool (SPOT), for the generation and rapid screening of a library of SP-target gene fusion constructs to identify the optimal SP for maximizing target protein secretion. In contrast to libraries generated in previous studies, SPOT fusion constructs are generated without adding the intervening sequences associated with restriction enzyme digestion sites. Therefore, no extra amino acids are inserted at the N-terminus of the target protein that might affect its function or conformational stability. As a model system, β-galactosidase (LacA) from Aspergillus oryzae was used as a target protein for secretion from Saccharomyces cerevisiae. In total, 60 SPs were selected from S. cerevisiae secretory proteins and utilized to generate the SP library. While many of the SP-LacA fusions were not secreted, several of the SPs, AGA2, CRH1, PLB1, and MF(alpha)1, were found to enhance LacA secretion compared to the WT sequence. Our results indicate that SPOT is a valuable method for optimizing the bioproduction of any target protein, and could be adapted to many host strains. PMID:25912446

  20. Secreted proteins as a fundamental source for biomarker discovery

    PubMed Central

    Stastna, Miroslava; Van Eyk, Jennifer E.

    2012-01-01

    The proteins secreted by various cells (the secretomes) are a potential rich source of biomarkers since they reflect various states of the cells at real time and at given conditions. To have accessible, sufficient and reliable protein markers is desirable since they mark various stages of disease development and their presence/absence can be used for diagnosis, prognosis, risk stratification and therapeutic monitoring. As direct analysis of blood/plasma, a common and noninvasive patient screening method, can be difficult for candidate protein biomarker identification, the alternative/complementary approaches are required, one of them is the analysis of secretomes in cell conditioned media in vitro. Since the proteins secreted by cells as a response to various stimuli are most likely secreted into blood/plasma, the identification and preselection of candidate protein biomarkers from cell secretomes with subsequent validation of their presence at higher levels in serum/plasma is a promising approach. In this review, we discuss the proteins secreted by three progenitor cell types (smooth muscle, endothelial and cardiac progenitor cells) and two adult cell types (neonatal rat ventrical myocytes and smooth muscle cells) which can be relevant to cardiovascular research and which have been recently published in the literature. We found, at least for secretome studies included in this review, that secretomes of progenitor and adult cells overlap by 48% but the secretomes are very distinct among progenitor cell themselves as well as between adult cells. In addition, we compared secreted proteins to protein identifications listed in the Human Plasma PeptideAtlas and in two reports with cardiovascular-related proteins and we performed the extensive literature search to find if any of these secreted proteins were identified in a biomarker study. As expected, many proteins have been identified as biomarkers in cancer but 18 proteins (out of 62) have been tested as biomarkers in

  1. Localization of growth and secretion of proteins in Aspergillus niger.

    PubMed

    Wösten, H A; Moukha, S M; Sietsma, J H; Wessels, J G

    1991-08-01

    Hyphal growth and secretion of proteins in Aspergillus niger were studied using a new method of culturing the fungus between perforated membranes which allows visualization of both parameters. At the colony level the sites of occurrence of growth and general protein secretion were correlated. In 4-d-old colonies both growth and secretion were localized at the periphery of the colony, whereas in a 5-d-old colony growth and secretion also occurred in a more central zone of the colony where conidiophore differentiation was observed. However, in both cases glucoamylase secretion was mainly detected at the periphery of the colonies. At the hyphal level immunogold labelling showed glucoamylase secretion at the tips of leading hyphae only. Microautoradiography after labelling with N-acetylglucosamine showed that these hyphae were probably all growing. Glucoamylase secretion could not be demonstrated immediately after a temperature shock which stopped growth. These results indicate that glucoamylase secretion is located at the tips of growing hyphae only. PMID:1955876

  2. Pseudomonas syringae lytic transglycosylases coregulated with the type III secretion system contribute to the translocation of effector proteins into plant cells.

    PubMed

    Oh, Hye-Sook; Kvitko, Brian H; Morello, Joanne E; Collmer, Alan

    2007-11-01

    Pseudomonas syringae translocates virulence effector proteins into plant cells via a type III secretion system (T3SS) encoded by hrp (for hypersensitive response and pathogenicity) genes. Three genes coregulated with the Hrp T3SS system in P. syringae pv. tomato DC3000 have predicted lytic transglycosylase domains: PSPTO1378 (here designated hrpH), PSPTO2678 (hopP1), and PSPTO852 (hopAJ1). hrpH is located between hrpR and avrE1 in the Hrp pathogenicity island and is carried in the functional cluster of P. syringae pv. syringae 61 hrp genes cloned in cosmid pHIR11. Strong expression of DC3000 hrpH in Escherichia coli inhibits bacterial growth unless the predicted catalytic glutamate at position 148 is mutated. Translocation tests involving C-terminal fusions with a Cya (Bordetella pertussis adenylate cyclase) reporter indicate that HrpH and HopP1, but not HopAJ1, are T3SS substrates. Pseudomonas fluorescens carrying a pHIR11 derivative lacking hrpH is poorly able to translocate effector HopA1, and this deficiency can be restored by HopP1 and HopAJ1, but not by HrpH(E148A) or HrpH(1-241). DC3000 mutants lacking hrpH or hrpH, hopP1, and hopAJ1 combined are variously reduced in effector translocation, elicitation of the hypersensitive response, and virulence. However, the mutants are not reduced in secretion of T3SS substrates in culture. When produced in wild-type DC3000, the HrpH(E148A) and HrpH(1-241) variants have a dominant-negative effect on the ability of DC3000 to elicit the hypersensitive response in nonhost tobacco and to grow and cause disease in host tomato. The three Hrp-associated lytic transglycosylases in DC3000 appear to have overlapping functions in contributing to T3SS functions during infection. PMID:17827286

  3. Further Characterization of a Type III Secretion System (T3SS) and of a New Effector Protein from a Clinical Isolate of Aeromonas Hydrophila - Part I

    EPA Science Inventory

    A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular cha...

  4. Mutations alter secretion of fukutin-related protein.

    PubMed

    Lu, Pei J; Zillmer, Allen; Wu, XiaoHua; Lochmuller, Hanns; Vachris, Judy; Blake, Derek; Chan, Yiumo Michael; Lu, Qi L

    2010-02-01

    Mutations in the fukutin-related protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) as well as other severe muscle disorders, including Walker-Warburg syndrome, muscle-eye-brain disease, and congenital muscular dystrophy type 1C. The FKRP gene encodes a putative glycosyltransferase, but its precise localization and functions have yet to be determined. In the present study, we demonstrated that normal FKRP is secreted into culture medium and mutations alter the pattern of secretion in CHO cells. L276I mutation associated with mild disease phenotype was shown to reduce the level of secretion whereas P448L and C318Y mutations associated with severe disease phenotype almost abolished the secretion. However, a truncated FKRP mutant protein lacking the entire C-terminal 185 amino acids due to the E310X nonsense mutation was able to secrete as efficiently as the normal FKRP. The N-terminal signal peptide sequence is apparently cleaved from the secreted FKRP proteins. Alteration of the secretion pathway by different mutations and spontaneous read-through of nonsense mutation may contribute to wide variations in phenotypes associated with FKRP-related diseases. PMID:19900540

  5. TraK and TraB are conserved outer membrane proteins of the Neisseria gonorrhoeae Type IV secretion system and are expressed at low levels in wild-type cells.

    PubMed

    Ramsey, Meghan E; Hackett, Kathleen T; Bender, Tobias; Kotha, Chaitra; van der Does, Chris; Dillard, Joseph P

    2014-08-15

    Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion. PMID:24914183

  6. TraK and TraB Are Conserved Outer Membrane Proteins of the Neisseria gonorrhoeae Type IV Secretion System and Are Expressed at Low Levels in Wild-Type Cells

    PubMed Central

    Ramsey, Meghan E.; Hackett, Kathleen T.; Bender, Tobias; Kotha, Chaitra; van der Does, Chris

    2014-01-01

    Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion. PMID:24914183

  7. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Parekh, R N; Wittrup, K D

    1997-01-01

    The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture. PMID:9104035

  8. Sertoli cells secrete both testis-specific and serum proteins.

    PubMed Central

    Wright, W W; Musto, N A; Mather, J P; Bardin, C W

    1981-01-01

    The secretions of the Sertoli cell were examined with two polyvalent antisera--one prepared against proteins in rat serum and the other against testis-specific proteins in rete testis fluid. These antisera detected 12 serum and 9 testis-specific proteins in rete testis fluid. To determine the origin of these proteins, primary cultures enriched in Sertoli cells were incubated with [35S]methionine, and the radiolabeled proteins in the medium were immunoprecipitated. Gel electrophoresis of the two immunoprecipitates resolved eight serum and nine testis-specific proteins. These two sets of proteins were specifically bound to their respective antiserum and were immunologically distinct. Medium from Sertoli cell cultures contained 10 times more of the testis-specific proteins than did cultures enriched for testicular myoid or interstitial cells. The concentration of the serum proteins in Sertoli cell medium was 5 and 10 times greater, respectively, than in myoid or interstitial cell preparations. The proteins from Sertoli cells were next characterized on two-dimensional gels. Seven of the proteins recognized by antiserum against serum proteins had identical molecular weights and isoelectric points as serum proteins. Three of these proteins were ceruloplasmin, transferrin, and glycoprotein 2. In addition to the proteins immunoprecipitated by the two antisera, more than 60 other proteins were detected on two-dimensional gels of the total secretory proteins. We conclude that the Sertoli cell secretes many proteins, some of which are specific to the testis and others of which are similar to serum proteins. Images PMID:6950398

  9. Temporal Expression of Pertussis Toxin and Ptl Secretion Proteins by Bordetella pertussis

    PubMed Central

    Rambow-Larsen, Amy A.; Weiss, Alison A.

    2004-01-01

    Pertussis toxin is an AB5 toxin comprised of protein subunits S1 through S5. The individual subunits are secreted by a Sec-dependent mechanism into the periplasm, where the toxin is assembled. The Ptl type IV secretion system mediates secretion of assembled toxin past the outer membrane. In this study, we examined the time course of protein expression, toxin assembly, and secretion as a function of the bacterial growth cycle. Logarithmic growth was observed after a 1-h lag phase. Secreted toxin was first observed at 3 h. Secretion continued throughout the logarithmic growth phase and decreased as the culture entered the stationary phase after about 24 h. On a per cell basis, toxin secretion occurred at a constant rate of 3 molecules/min/cell from 2 to 18 h. More of toxin subunits S1, S2, and S3 were produced than were secreted, resulting in periplasmic accumulation. Periplasmic S1, S2, and S3 were found to be soluble in the periplasm, as well as membrane associated. About one-half of the periplasmic S1, S2 and S3 subunits were incorporated into holotoxin. Secretion component PtlF was present at a low level at time zero, and the level increased between 2 and 24 h from 30 to 1,000 molecules per cell; however, the initial level of PtlF, 30 molecules per cell, supported maximal secretion. The accumulation of both periplasmic toxin and secretion components suggests that translation rates exceed the rate of secretion and that secretion, not toxin and Ptl complex assembly, is rate limiting. PMID:14679223

  10. Protein secretion and surface display in Gram-positive bacteria

    PubMed Central

    Schneewind, Olaf; Missiakas, Dominique M.

    2012-01-01

    The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983

  11. Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The locus of enterocyte effacement (LEE) encodes a type III secretion system (T3SS) for secreting factors that enable Escherichia coli O157:H7 to produce attaching and effacing lesions (A/E) on epithelial cells. The importance of LEE-encoded proteins in intestinal colonization of cattle is well-stud...

  12. Crystal structure of the Yersinia type III secretion protein YscE

    SciTech Connect

    Phan, Jason; Austin, Brian P.; Waugh, David S.

    2010-12-06

    The plague-causing bacterium Yersinia pestis utilizes a contact-dependent (type III) secretion system (T3SS) to transport virulence factors from the bacterial cytosol directly into the interior of mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. The type III secretion apparatus is composed of 20-25 different Yersinia secretion (Ysc) proteins. We report here the structure of YscE, the smallest Ysc protein, which is a dimer in solution. The probable mode of oligomerization is discussed.

  13. Odorant-Binding Protein: Localization to Nasal Glands and Secretions

    NASA Astrophysics Data System (ADS)

    Pevsner, Jonathan; Sklar, Pamela B.; Snyder, Solomon H.

    1986-07-01

    An odorant-binding protein (OBP) was isolated from bovine olfactory and respiratory mucosa. We have produced polyclonal antisera to this protein and report its immunohistochemical localization to mucus-secreting glands of the olfactory and respiratory mucosa. Although OBP was originally isolated as a pyrazine binding protein, both rat and bovine OBP also bind the odorants [3H]methyldihydrojasmonate and 3,7-dimethyl-octan-1-ol as well as 2-isobutyl-3-[3H]methoxypyrazine. We detect substantial odorant-binding activity attributable to OBP in secreted rat nasal mucus and tears but not in saliva, suggesting a role for OBP in transporting or concentrating odorants.

  14. The bud tip is the cellular hot spot of protein secretion in yeasts.

    PubMed

    Puxbaum, Verena; Gasser, Brigitte; Mattanovich, Diethard

    2016-09-01

    Yeasts are valuable hosts for recombinant protein production. Among them, Pichia pastoris is frequently used for production of secreted proteins, and much effort was made to improve the secretion efficiency of this expression platform. However, the knowledge on the secretion machinery is mainly based on studies in Saccharomyces cerevisiae. Therefore, it is of great interest for targeted improvement of the system to learn more about the secretion process in P. pastoris. Using human serum albumin, a protein which is produced in high quantities in P. pastoris, we show here the secretion pathway of this protein. During passage of the secretory route, the recombinant protein is mainly localized in the endoplasmic reticulum (ER) and in COPII vesicles, and is inherited to the daughter cell via the perinuclear ER. The final release to the cell exterior occurs at the bud, initiating at the bud tip and later spreading over the entire bud surface. The same polarized secretion pattern was observed for a recombinant antibody light chain and the native secretory protein Epx1 of P. pastoris. Clarifying the point of release of secretory proteins will have major impact on engineering the secretory pathway of P. pastoris and other budding yeasts. PMID:27338576

  15. Investigating the dynamics of recombinant protein secretion from a microalgal host.

    PubMed

    Lauersen, Kyle J; Huber, Isabel; Wichmann, Julian; Baier, Thomas; Leiter, Andreas; Gaukel, Volker; Kartushin, Viktor; Rattenholl, Anke; Steinweg, Christian; von Riesen, Lena; Posten, Clemens; Gudermann, Frank; Lütkemeyer, Dirk; Mussgnug, Jan H; Kruse, Olaf

    2015-12-10

    Production of recombinant proteins with microalgae represents an alternative platform over plant- or bacterial-based expression systems for certain target proteins. Secretion of recombinant proteins allows accumulation of the target product physically separate from the valuable algal biomass. To date, there has been little investigation into the dynamics of recombinant protein secretion from microalgal hosts-the culture parameters that encourage secreted product accumulation and stability, while encouraging biomass production. In this work, the efficiency of recombinant protein production was optimized by adjusting cultivation parameters for a strain of Chlamydomonas reinhardtii previously engineered to secrete a functional recombinant Lolium perenne ice binding protein (LpIBP), which has applications as a frozen food texturing and cryopreservation additive, into its culture medium. Three media and several cultivation styles were investigated for effects on secreted LpIBP titres and culture growth. A combination of acetate and carbon dioxide feeding with illumination resulted in the highest overall biomass and recombinant protein titres up to 10mgL(-1) in the culture medium. Pure photoautotrophic production was possible using two media types, with recombinant protein accumulation in all cultivations correlating to culture cell density. Two different cultivation systems were used for scale-up to 10L cultivations, one of which produced yields of secreted recombinant protein up to 12mgL(-1) within six cultivation days. Functional ice recrystallization inhibition (IRI) of the LpIBP from total concentrated extracellular protein extracts was demonstrated in a sucrose solution used as a simplified ice cream model. IRI lasted up to 7 days, demonstrating the potential of secreted products from microalgae for use as food additives. PMID:25975624

  16. Targeting bacterial secretion systems: benefits of disarmament in the microcosm.

    PubMed

    Baron, Christian; Coombes, Brian

    2007-03-01

    Secretion systems are used by many bacterial pathogens for the delivery of virulence factors to the extracellular space or directly into host cells. They are attractive targets for the development of novel anti-virulence drugs as their inactivation would lead to pathogen attenuation or avirulence, followed by clearance of the bacteria by the immune system. This review will present the state of knowledge on the assembly and function of type II, type III and type IV secretion systems in Gram-negative bacteria focusing on insights provided by structural analyses of several key components. The suitability of transcription factors regulating the expression of secretion system components and of ATPases, lytic transglycosylases and protein assembly factors as drug targets will be discussed. Recent progress using innovative in vivo as well as in vitro screening strategies led to a first set of secretion system inhibitors with potential for further development as anti-infectives. The discovery of such inhibitors offers exciting and innovative opportunities to further develop these anti-virulence drugs into monotherapy or in combination with classical antibiotics. Bacterial growth per se would not be inhibited by such drugs so that the selection for mutations causing resistance could be reduced. Secretion system inhibitors may therefore avoid many of the problems associated with classical antibiotics and may constitute a valuable addition to our arsenal for the treatment of bacterial infections. PMID:17346208

  17. Production and Secretion of the Polysaccharide Biodispersan of Acinetobacter calcoaceticus A2 in Protein Secretion Mutants.

    PubMed

    Elkeles, A; Rosenberg, E; Ron, E Z

    1994-12-01

    Biodispersan is an extracellular anionic polysaccharide produced by Acinetobacter calcoaceticus A2 that changes the surface properties of limestone and acts both as a dispersant and as a grinding aid (E. Rosenberg, C. Rubinovitz, A. Gottlieb, S. Rosenhak, and E. Z. Ron, Appl. Environ. Microbiol. 54:317-322, 1988; E. Rosenberg, C. Rubinovitz, R. Legmann, and E. Z. Ron, Appl. Environ. Microbiol. 54:323-326, 1988; E. Rosenberg, Z. Schwartz, A. Tenenbaum, C. Rubinovitz, R. Legmann, and E. Z. Ron, J. Dispersion Sci. Technol. 10:241-250, 1989). Extracellular fluid also contains a high concentration of secreted proteins that create problems in the purification and application of biodispersan. In order to obtain preparations of biodispersan that contained smaller amounts of protein, we selected mutants of strain A2 that were defective in protein secretion. These mutants produced equal, or even higher, levels of total biodispersan compared with those of the parental strain. Moreover, although there was a significant drop in the concentration of extracellular proteins in the medium, the secretion of biodispersan was unaffected. These results suggest that secretion mutants are potentially useful for the production of extracellular polysaccharides. PMID:16349473

  18. Membrane and chaperone recognition by the major translocator protein PopB of the type III secretion system of Pseudomonas aeruginosa.

    PubMed

    Discola, Karen F; Förster, Andreas; Boulay, François; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa; Job, Viviana

    2014-02-01

    The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51-59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly. PMID:24297169

  19. Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa*

    PubMed Central

    Discola, Karen F.; Förster, Andreas; Boulay, François; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa; Job, Viviana

    2014-01-01

    The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51–59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly. PMID:24297169

  20. Mechanism of Action of Secreted Newt Anterior Gradient Protein

    PubMed Central

    Grassme, Kathrin S.; Garza-Garcia, Acely; Delgado, Jean-Paul; Godwin, James W.; Kumar, Anoop; Gates, Phillip B.; Brockes, Jeremy P.

    2016-01-01

    Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family. PMID:27100463

  1. Mechanism of Action of Secreted Newt Anterior Gradient Protein.

    PubMed

    Grassme, Kathrin S; Garza-Garcia, Acely; Delgado, Jean-Paul; Godwin, James W; Kumar, Anoop; Gates, Phillip B; Driscoll, Paul C; Brockes, Jeremy P

    2016-01-01

    Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family. PMID:27100463

  2. A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi.

    PubMed

    Login, Frédéric H; Fries, Markus; Wang, Xiaohui; Pickersgill, Richard W; Shevchik, Vladimir E

    2010-05-01

    The type II secretion system (T2SS) is widely exploited by proteobacteria to secrete enzymes and toxins involved in bacterial survival and pathogenesis. The outer membrane pore formed by the secretin OutD and the inner membrane protein OutC are two key components of the secretion complex, involved in secretion specificity. Here, we show that the periplasmic regions of OutC and OutD interact directly and map the interaction site of OutC to a 20-residue peptide named OutCsip (secretin interacting peptide, residues 139-158). This peptide interacts in vitro with two distinct sites of the periplasmic region of OutD, one located on the N0 subdomain and another overlapping the N2-N3' subdomains. The two interaction sites of OutD have different modes of binding to OutCsip. A single substitution, V143S, located within OutCsip prevents its interaction with one of the two binding sites of OutD and fully inactivates the T2SS. We show that the N0 subdomain of OutD interacts also with a second binding site within OutC located in the region proximal to the transmembrane segment. We suggest that successive interactions between these distinct regions of OutC and OutD may have functional importance in switching the secretion machine. PMID:20444086

  3. Type VI secretion system: secretion by a contractile nanomachine

    PubMed Central

    Basler, Marek

    2015-01-01

    The type VI secretion systems (T6SS) are present in about a quarter of all Gram-negative bacteria. Several key components of T6SS are evolutionarily related to components of contractile nanomachines such as phages and R-type pyocins. The T6SS assembly is initiated by formation of a membrane complex that binds a phage-like baseplate with a sharp spike, and this is followed by polymerization of a long rigid inner tube and an outer contractile sheath. Effectors are preloaded onto the spike or into the tube during the assembly by various mechanisms. Contraction of the sheath releases an unprecedented amount of energy, which is used to thrust the spike and tube with the associated effectors out of the effector cell and across membranes of both bacterial and eukaryotic target cells. Subunits of the contracted sheath are recycled by T6SS-specific unfoldase to allow for a new round of assembly. Live-cell imaging has shown that the assembly is highly dynamic and its subcellular localization is in certain bacteria regulated with a remarkable precision. Through the action of effectors, T6SS has mainly been shown to contribute to pathogenicity and competition between bacteria. This review summarizes the knowledge that has contributed to our current understanding of T6SS mode of action. PMID:26370934

  4. Use of surface-enhanced laser desorption ionization protein chip system to analyze streptococcal exotoxin B activity secreted by Streptococcus pyogenes.

    PubMed

    Boyle, M D; Romer, T G; Meeker, A K; Sledjeski, D D

    2001-08-01

    Ciphergen surface-enhanced laser desorption ionization (SELDI) protein chip technology was used to analyze the secretion and autoactivation of the Streptococcus pyogenes cysteine protease SpeB. This method allowed rapid identification of both the zymogen form of the protein Mr approximately 41,000 and the fully active enzyme Mr approximately 28,500. SpeB production in culture supernatants was demonstrated to be growth-phase regulated and SpeB positive and negative variants of a blood passaged S. pyogenes isolate could readily be distinguished. In kinetic studies of the autoactivation of the zymogen form of SpeB, the sequential generation of four intermediates was detected before the accumulation of the fully active enzyme. The methods described enabled enhanced speed, use of lower sample volumes and concentrations, and a more complete molecular characterization of SpeB than allowed by existing methods of analysis using SDS-PAGE and Western immunoblotting. PMID:11412919

  5. TolC-Dependent Secretion of an Ankyrin Repeat-Containing Protein of Rickettsia typhi

    PubMed Central

    Rahman, M. Sayeedur; Ammerman, Nicole C.; Beier-Sexton, Magda; Ceraul, Shane M.; Gillespie, Joseph J.; Azad, Abdu F.

    2012-01-01

    Rickettsia typhi, the causative agent of murine (endemic) typhus, is an obligate intracellular pathogen with a life cycle involving both vertebrate and invertebrate hosts. In this study, we characterized a gene (RT0218) encoding a C-terminal ankyrin repeat domain-containing protein, named Rickettsia ankyrin repeat protein 1 (RARP-1), and identified it as a secreted effector protein of R. typhi. RT0218 showed differential transcript abundance at various phases of R. typhi intracellular growth. RARP-1 was secreted by R. typhi into the host cytoplasm during in vitro infection of mammalian cells. Transcriptional analysis revealed that RT0218 was cotranscribed with adjacent genes RT0217 (hypothetical protein) and RT0216 (TolC) as a single polycistronic mRNA. Given one of its functions as a facilitator of extracellular protein secretion in some Gram-negative bacterial pathogens, we tested the possible role of TolC in the secretion of RARP-1. Using Escherichia coli C600 and an isogenic tolC insertion mutant as surrogate hosts, our data demonstrate that RARP-1 is secreted in a TolC-dependent manner. Deletion of either the N-terminal signal peptide or the C-terminal ankyrin repeats abolished RARP-1 secretion by wild-type E. coli. Importantly, expression of R. typhi tolC in the E. coli tolC mutant restored the secretion of RARP-1, suggesting that TolC has a role in RARP-1 translocation across the outer membrane. This work implies that the TolC component of the putative type 1 secretion system of R. typhi is involved in the secretion process of RARP-1. PMID:22773786

  6. Identification of Secreted Candida Proteins Using Mass Spectrometry.

    PubMed

    Gómez-Molero, Emilia; Dekker, Henk L; de Boer, Albert D; de Groot, Piet W J

    2016-01-01

    Analysis of fungal secretomes using mass spectrometry is a useful technique in cell biology. Knowledge of the secretome of a human fungal pathogen may yield important information of host-pathogen interactions and may be useful for identifying vaccines candidates or diagnostic markers for antifungal strategies. In this chapter, with a main focus on sample preparation aspects, we describe the methodology that we apply for gel-independent batch identification and quantification of proteins that are secreted during growth in liquid cultures. Using these techniques with Candida and other yeast species, the majority of the identified proteins are classical secretory proteins and cell wall proteins containing N-terminal signal peptides for secretion, although dependent on sample preparation quality and the mass spectrometric analysis also usually, a number of nonsecretory proteins are identified. PMID:26519067

  7. Type VI Secretion System Toxins Horizontally Shared between Marine Bacteria

    PubMed Central

    Salomon, Dor; Klimko, John A.; Trudgian, David C.; Kinch, Lisa N.; Grishin, Nick V.; Mirzaei, Hamid; Orth, Kim

    2015-01-01

    The type VI secretion system (T6SS) is a widespread protein secretion apparatus used by Gram-negative bacteria to deliver toxic effector proteins into adjacent bacterial or host cells. Here, we uncovered a role in interbacterial competition for the two T6SSs encoded by the marine pathogen Vibrio alginolyticus. Using comparative proteomics and genetics, we identified their effector repertoires. In addition to the previously described effector V12G01_02265, we identified three new effectors secreted by T6SS1, indicating that the T6SS1 secretes at least four antibacterial effectors, of which three are members of the MIX-effector class. We also showed that the T6SS2 secretes at least three antibacterial effectors. Our findings revealed that many MIX-effectors belonging to clan V are “orphan” effectors that neighbor mobile elements and are shared between marine bacteria via horizontal gene transfer. We demonstrated that a MIX V-effector from V. alginolyticus is a functional T6SS effector when ectopically expressed in another Vibrio species. We propose that mobile MIX V-effectors serve as an environmental reservoir of T6SS effectors that are shared and used to diversify antibacterial toxin repertoires in marine bacteria, resulting in enhanced competitive fitness. PMID:26305100

  8. Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase.

    PubMed

    Inan, Mehmet; Aryasomayajula, Dinesh; Sinha, Jayanta; Meagher, Michael M

    2006-03-01

    A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins. PMID:16255058

  9. Long-term potentiation in the hippocampal slice: evidence for stimulated secretion of newly synthesized proteins

    SciTech Connect

    Duffy, C.; Teyler, T.J.; Shashoua, V.E.

    1981-06-01

    Long-term potentiation of the hippocampal slice preparation results in an increase in the incorporation of labeled valine into the proteins destined for secretion into the extracellular medium. Double-labeling methods established that the increased secretion of the labeled proteins was limited to the potentiated region of a slice; incorporation of labeled valine was increased in the hippocampus if potentiation was through the Schaffer collaterals and in the dentate if potentiation was through the perforant path. Controls for nonspecific stimulation showed no changes. There appears to be a link between long-term potentiation and the metabolic processes that lead to protein synthesis in the hippocampal slice system.

  10. Architecture of the major component of the type III secretion system export apparatus

    PubMed Central

    Abrusci, Patrizia; Vergara–Irigaray, Marta; Johnson, Steven; Beeby, Morgan D; Hendrixson, David; Roversi, Pietro; Friede, Miriam E; Deane, Janet E; Jensen, Grant J; Tang, Christoph M; Lea, Susan M

    2012-01-01

    Type III secretion systems (T3SSs) are bacterial membrane-embedded secretion nanomachines designed to export specifically targeted sets of proteins from the bacterial cytoplasm. Secretion through T3SS is governed by a subset of inner membrane proteins termed the ‘export apparatus’. We show that a key member of the Shigella flexneri export apparatus, MxiA, assembles into a ring essential for secretion in vivo. The ring forming interfaces are well conserved in both non-flagellar and flagellar homologues, implying that the ring is an evolutionary conserved feature in these systems. Electron cryo-tomography reveals a T3SS-associated cytoplasmic torus of size and shape corresponding to the MxiA ring aligned to the secretion channel located between the secretion pore and the ATPase complex. This defines the molecular architecture of the dominant component of the export apparatus and allows us to propose a model for the molecular mechanisms controlling secretion. PMID:23222644

  11. Characterization of novel secreted proteins from Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa is a bacterium that causes disease of agriculturally important crops, including Pierce’s disease of grapevine. Little is known about virulence factors that are necessary for X. fastidiosa to grow and cause disease in the xylem vessels of a plant host. Any protein secreted by the b...

  12. Bacterial Type IV Secretion Systems: Versatile Virulence Machines

    PubMed Central

    Voth, Daniel E.; Broederdorf, Laura J.; Graham, Joseph G.

    2013-01-01

    Many bacterial pathogens employ multicomponent protein complexes to deliver macromolecules directly into their eukaryotic host cell to promote infection. Some Gram-negative pathogens use a versatile type IV secretion system (T4SS) that can translocate DNA or proteins into host cells. T4SSs represent major bacterial virulence determinants and have recently been the focus of intense research efforts designed to better understand and combat infectious diseases. Interestingly, although the two major classes of T4SSs function in a similar manner to secrete proteins, the translocated “effectors” vary substantially from one organism to another. In fact, differing effector repertoires likely contribute to organism-specific host cell interactions and disease outcomes. In this review, we discuss the current state of T4SS research, with an emphasis on intracellular bacterial pathogens of humans and the diverse array of translocated effectors used to manipulate host cells. PMID:22324993

  13. Proteomic identification of secreted proteins of Propionibacterium acnes

    PubMed Central

    2010-01-01

    Background The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. Results Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE) and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS). A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, β-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP) factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. Conclusions Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence properties of P. acnes isolates

  14. Selection for genes encoding secreted proteins and receptors.

    PubMed Central

    Klein, R D; Gu, Q; Goddard, A; Rosenthal, A

    1996-01-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states. Images Fig. 1 PMID:8692953

  15. Structure of EspB, a secreted substrate of the ESX-1 secretion system of Mycobacterium tuberculosis

    PubMed Central

    Korotkova, Natalia; Piton, Jérémie; Wagner, Jonathan M.; Boy-Röttger, Stefanie; Japaridze, Aleksandre; Evans, Timothy J.; Cole, Stewart T.; Pojer, Florence; Korotkov, Konstantin V.

    2015-01-01

    Mycobacterium tuberculosis secretes multiple virulence factors during infection via the general Sec and Tat pathways, and via specialized ESX secretion systems, also referred to as type VII secretion systems. The ESX-1 secretion system is an important virulence determinant because deletion of ESX-1 leads to attenuation of M. tuberculosis. ESX-1 secreted protein B (EspB) contains putative PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, and a C-terminal domain, which is processed by MycP1 protease during secretion. We determined the crystal structure of PE–PPE domains of EspB, which represents an all-helical, elongated molecule closely resembling the structure of the PE25–PPE41 heterodimer despite limited sequence similarity. Also, we determined the structure of full-length EspB, which does not have interpretable electron density for the C-terminal domain confirming that it is largely disordered. Comparative analysis of EspB in cell lysate and culture filtrates of M. tuberculosis revealed that mature secreted EspB forms oligomers. Electron microscopy analysis showed that the N-terminal fragment of EspB forms donut-shaped particles. These data provide a rationale for the future investigation of EspB's role in M. tuberculosis pathogenesis. PMID:26051906

  16. Synthesis and secretion of plasma proteins by embryonic chick hepatocytes: changing patterns during the first three days of culture

    PubMed Central

    1978-01-01

    A simple model system is described for studying synthesis of plasma proteins. The system is based on chick embryo hepatocytes in primary monolayer culture which synthesize a broad spectrum of plasma proteins and secrete them into the culture medium. The secreted proteins are stable and consist almost exclusively of plasma proteins. The cultured cells are nonproliferating hepatic parenchymal cells whose cell mass remains constant in culture. By a modification of Laurell's rocket immunoelectrophoresis, the secreted plasma proteins can be detected in nanogram amounts in 3 microliter of unconcentrated culture medium. Kinetics of secretion are obtained by sequential assay of proteins accumulating in the medium. In this system it is demonstrated that: (a) intracellular plasma protein levels are equivalent to less than 5% of the daily secretion; (b) synthesis and secretion are continuous; and (c) the overall half-time for plasma protein movement along the secretory pathway is less than 10 min. From these results, it follows that the rate at which the plasma proteins are secreted gives a valid estimate of their rate of synthesis. This feature of the culture and the sensitivity of the assay allow routine measurements of plasma protein synthesis without disruption of the cells and without the use of radioisotopes. It is shown, furthermore, that the overall rate of plasma protein synthesis in cultured hepatocytes is constant over a 3- day period and is similar to that of the intact liver. 3,000,000 cells, containing 1 mg cell protein, synthesize 0.2 mg of plasma proteins daily, amounting to one-fifth of hepatocellular protein synthesis. Under the conditions used, albumin synthesis steadily decreases with culture time whereas the synthesis of many other plasma proteins increases. The observed phenotypic changes and reorganization of plasma protein synthesis illustrate how the system may be exploited for studying the regulatory processes governing plasma protein synthesis. PMID

  17. High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin-arginine translocation system in Escherichia coli.

    PubMed

    Albiniak, Anna M; Matos, Cristina F R O; Branston, Steven D; Freedman, Robert B; Keshavarz-Moore, Eli; Robinson, Colin

    2013-08-01

    The twin-arginine translocation (Tat) system transports folded proteins across the plasma membrane in bacteria, and heterologous proteins can be exported by this pathway if a Tat-type signal peptide is present at the N-terminus. The system thus has potential for biopharmaceutical production in Escherichia coli, where export to the periplasm is often a favoured approach. Previous studies have shown that E. coli cells can export high levels of protein by the Tat pathway, and the protein product accummulates almost exclusively in the periplasm. In this study, we analysed E. coli cells that express the Bacillus subtilis TatAdCd system in place of the native TatABC system. We show that a heterologous model protein, comprising the TorA signal peptide linked to green fluorescent protein (TorA-GFP), is efficiently exported by the TatAdCd system. However, whereas the GFP is exported initially to the periplasm during batch fermentation, the mature protein is increasingly found in the extracellular culture medium. By the end of a 16-h fermentation, ~ 90% of exported GFP is present in the medium as active mature protein. The total protein profiles of the medium and periplasm are essentially identical, confirming that the outer membrane becomes leaky during the fermentation process. The cells are otherwise intact, and there is no large-scale release of cytoplasmic contents. Export levels are relatively high, with ~ 0.35 g GFP·L⁻¹ culture present in the medium. This system thus offers a means of producing recombinant protein in E. coli and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing. PMID:23745597

  18. The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65

    PubMed Central

    Kucknoor, Ashwini S.; Mundodi, Vasanthakrishna; Alderete, John F.

    2007-01-01

    Summary We showed recently that contact of human vaginal epithelial cells (VECs) by Trichomonas vaginalis and incubation with trichomonad proteins in conditioned medium induced expression of VEC genes. We performed 2-D SDS-PAGE followed by MALDI-TOF to identify the major secreted proteins. Based on protein abundance and separation of spots in 2-D gels, 32 major secreted proteins were examined, which gave 19 proteins with accession numbers. These proteins included known secreted cysteine proteinases. In addition, other secreted proteins were enzymes of carbohydrate metabolism, adhesin protein AP65, heat shock proteins, thioredoxin reductase and coronins. We confirmed that the secreted trichomonad proteins induced expression of VEC genes, including interleukin 8 (IL-8), COX-2 and fibronectin. Purified AP65 added to VECs had a pronounced effect only on IL-8 gene expression, which was inhibited in the presence of 12G4 monoclonal antibody to AP65. Moreover, AP65 expressed episomally within epithelial cells was found to enhance the expression of IL-8 and COX-2. This may be the first report of analysis of the secreted proteins of T. vaginalis and of the host epithelial cell response to these proteins and to the prominent adhesin AP65. PMID:17590165

  19. Identification and Characterization of Plant Cell Death-Inducing Secreted Proteins From Ustilaginoidea virens.

    PubMed

    Fang, Anfei; Han, Yanqing; Zhang, Nan; Zhang, Min; Liu, Lijuan; Li, Shuai; Lu, Fen; Sun, Wenxian

    2016-05-01

    Ustilaginoidea virens (Cooke) Takah (telemorph Villosiclava virens) is an ascomycetous fungus that causes rice false smut, one of the most important rice diseases. Fungal effectors often play essential roles in host-pathogen coevolutionary interactions. However, little is known about the functions of U. virens effectors. Here, we performed functional studies on putative effectors in U. virens and demonstrated that 13 of 119 putative effectors caused necrosis or necrosis-like phenotypes in Nicotiana benthamiana. Among them, 11 proteins were confirmed to be secreted, using a yeast secretion system, and the corresponding genes are all highly induced during infection, except UV_44 and UV_4753. Eight secreted proteins were proven to trigger cell death or defenses in rice protoplasts and the secretion signal of these proteins is essential for their cell death-inducing activity. The ability of UV_44 and UV_1423 to trigger cell death is dependent on the predicted serine peptidase and ribonuclease catalytic active sites, respectively. We demonstrated that UV_1423 and UV_6205 are N-glycosylated proteins, which glycosylation has different impacts on their abilities to induce cell death. Collectively, the study identified multiple secreted proteins in U. virens with specific structural motifs that induce cell death or defense machinery in nonhost and host plants. PMID:26927000

  20. Characterization of EssB, a protein required for secretion of ESAT-6 like proteins in Staphylococcus aureus

    PubMed Central

    2012-01-01

    Background Staphylococcus aureus secretes EsxA and EsxB, two small polypeptides of the WXG100 family of proteins. Genetic analyses have shown that production and secretion of EsxA and EsxB require an intact ESAT-6 Secretion System (ESS), a cluster of genes that is conserved in many Firmicutes and encompasses esxA and esxB . Here, we characterize EssB, one of the proteins encoded by the ESS cluster. EssB is highly conserved in Gram-positive bacteria and belongs to the Cluster of Orthologous Groups of protein COG4499 with no known function. Results By generating an internal deletion in essB , we demonstrate that EssB is required for secretion of EsxA. We use a polyclonal antibody to identify EssB and show that the protein fractionates with the plasma membrane of S. aureus . Yet, when produced in Escherichia coli, EssB remains mostly soluble and the purified protein assembles into a highly organized oligomer that can be visualized by electron microscopy. Production of truncated EssB variants in wild-type S. aureus confers a dominant negative phenotype on EsxA secretion. Conclusions The data presented here support the notion that EssB may oligomerize and interact with other membrane components to form the WXG100-specific translocon in S. aureus . PMID:23006124

  1. Xanthomonas campestris pv. vesicatoria Secretes Proteases and Xylanases via the Xps Type II Secretion System and Outer Membrane Vesicles

    PubMed Central

    Solé, Magali; Scheibner, Felix; Hoffmeister, Anne-Katrin; Hartmann, Nadine; Hause, Gerd; Rother, Annekatrin; Jordan, Michael; Lautier, Martine; Arlat, Matthieu

    2015-01-01

    ABSTRACT Many plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system from Xanthomonas campestris pv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates from X. campestris pv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins from X. campestris pv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor of X. campestris pv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence and in planta growth of X. campestris pv. vesicatoria. When analyzed in the related pathogen X. campestris pv. campestris, several T2S substrates from X. campestris pv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, in X. campestris pv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed for X. campestris pv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes from X. campestris pv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles. IMPORTANCE The virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S

  2. Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A2.

    PubMed

    Peroutka, Raymond J; Elshourbagy, Nabil; Piech, Tara; Butt, Tauseef R

    2008-09-01

    SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic post-translational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A(2) proteins (sPLA(2)) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA(2) homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43- and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases. PMID:18539905

  3. Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein in Pichia pastoris.

    PubMed

    Li, Zhiguo; Leung, Wilson; Yon, Amy; Nguyen, John; Perez, Vincent C; Vu, Jane; Giang, William; Luong, Linda T; Phan, Tracy; Salazar, Kate A; Gomez, Seth R; Au, Colin; Xiang, Fan; Thomas, David W; Franz, Andreas H; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2010-07-01

    The Escherichia coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N-terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris, a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N-terminal partner to several C-terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C-terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C-terminal of the MBP domain, including the spacer and cargo regions, but the MBP domain could still act to enhance the secretion of certain cargo proteins. PMID:20230898

  4. Monosodium Urate Activates Src/Pyk2/PI3 Kinase and Cathepsin Dependent Unconventional Protein Secretion From Human Primary Macrophages*

    PubMed Central

    Välimäki, Elina; Miettinen, Juho J.; Lietzén, Niina; Matikainen, Sampsa; Nyman, Tuula A.

    2013-01-01

    Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel the details of early phases of defense responses. Here, we have analyzed the secretome of human primary macrophages stimulated with MSU using quantitative two-dimensional gel electrophoresis based proteomics as well as high-throughput qualitative GeLC-MS/MS approach combining protein separation by SDS-PAGE and protein identification by liquid chromatography-MS/MS. Both methods showed that MSU stimulation induced robust protein secretion from lipopolysaccharide-primed human macrophages. Bioinformatic analysis of the secretome data showed that MSU stimulation strongly activates unconventional, vesicle mediated protein secretion. The unconventionally secreted proteins included pro-inflammatory cytokines like IL-1β and IL-18, interferon-induced proteins, and danger signal proteins. Also active forms of lysosomal proteases cathepsins were secreted on MSU stimulation, and cathepsin activity was essential for MSU-induced unconventional protein secretion. Additionally, proteins associated to phosphorylation events including Src family tyrosine kinases were increased in the secretome of MSU-stimulated cells. Our functional studies demonstrated that Src, Pyk2, and PI3 kinases act upstream of cathepsins to activate the overall protein secretion from macrophages. In conclusion, we provide the first comprehensive characterization of protein secretion pathways activated by MSU in human macrophages, and reveal a novel role for cathepsins and Src, Pyk2, PI3 kinases in the activation of

  5. Quantitative Proteomic Analysis of Burkholderia pseudomallei Bsa Type III Secretion System Effectors Using Hypersecreting Mutants

    PubMed Central

    Vander Broek, Charles W.; Chalmers, Kevin J.; Stevens, Mark P.; Stevens, Joanne M.

    2015-01-01

    Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. We constructed a mutant lacking bsaP, a homolog of the T3SS “gatekeeper” family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hypersecrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion. Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei. PMID:25635268

  6. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Hou, Jin; Osterlund, Tobias; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

    2013-04-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased heterologous α-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously. PMID:23208612

  7. Expression, Extracellular Secretion, and Immunogenicity of the Plasmodium falciparum Sporozoite Surface Protein 2 in Salmonella Vaccine Strains

    PubMed Central

    Gómez-Duarte, Oscar G.; Pasetti, Marcela F.; Santiago, Araceli; Sztein, Marcelo B.; Hoffman, Stephen L.; Levine, Myron M.

    2001-01-01

    Deleting transmembrane α-helix motifs from Plasmodium falciparum sporozoite surface protein (SSP-2) allowed its secretion from Salmonella enterica serovar Typhimurium SL3261 and S. enterica serovar Typhi CVD 908-htrA by the Hly type I secretion system. In mice immunized intranasally, serovar Typhimurium constructs secreting SSP-2 stimulated greater gamma interferon splenocyte responses than did nonsecreting constructs (P = 0.04). PMID:11160021

  8. EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles

    DOE PAGESBeta

    Fan, Yao; Tan, Kemin; Chhor, Gekleng; Butler, Emily K.; Jedrzejczak, Robert P.; Missiakas, Dominique; Joachimiak, Andrzej

    2015-07-03

    The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for twomore » alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.« less

  9. A holin and an endopeptidase are essential for chitinolytic protein secretion in Serratia marcescens

    PubMed Central

    Hamilton, Jaeger J.; Marlow, Victoria L.; Owen, Richard A.; Costa, Marília de Assis Alcoforado; Guo, Manman; Buchanan, Grant; Chandra, Govind; Trost, Matthias; Coulthurst, Sarah J.; Palmer, Tracy; Stanley-Wall, Nicola R.

    2014-01-01

    Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria. PMID:25488919

  10. EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles

    PubMed Central

    Fan, Yao; Tan, Kemin; Chhor, Gekleng; Butler, Emily K; Jedrzejczak, Robert P; Missiakas, Dominique; Joachimiak, Andrzej

    2015-01-01

    The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (∼10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown. PMID:26032645

  11. Metrnl: a secreted protein with new emerging functions

    PubMed Central

    Zheng, Si-li; Li, Zhi-yong; Song, Jie; Liu, Jian-min; Miao, Chao-yu

    2016-01-01

    Secreted proteins play critical roles in physiological and pathological processes and can be used as biomarkers and therapies for aging and disease. Metrnl is a novel secreted protein homologous to the neurotrophin Metrn. But this protein, unlike Metrn that is mainly expressed in the brain, shows a relatively wider distribution in the body with high levels of expression in white adipose tissue and barrier tissues. This protein plays important roles in neural development, white adipose browning and insulin sensitization. Based on its expression and distinct functions, this protein is also called Cometin, Subfatin and Interleukin 39, which refer to its neurotrophic effect, adipokine function and the possible action as a cytokine, respectively. The spectrum of Metrnl functions remains to be determined, and the mechanisms of Metrnl action need to be elucidated. In this review, we focus on the discovery, structural characteristics, expression pattern and physiological functions of Metrnl, which will assist in developing this protein as a new therapeutic target or agent. PMID:27063217

  12. Pollen tube growth and guidance: roles of small, secreted proteins

    PubMed Central

    Chae, Keun; Lord, Elizabeth M.

    2011-01-01

    Background Pollination is a crucial step in angiosperm (flowering plant) reproduction. Highly orchestrated pollen–pistil interactions and signalling events enable plant species to avoid inbreeding and outcrossing as a species-specific barrier. In compatible pollination, pollen tubes carrying two sperm cells grow through the pistil transmitting tract and are precisely guided to the ovules, discharging the sperm cells to the embryo sac for fertilization. Scope In Lilium longiflorum pollination, growing pollen tubes utilize two critical mechanisms, adhesion and chemotropism, for directional growth to the ovules. Among several molecular factors discovered in the past decade, two small, secreted cysteine-rich proteins have been shown to play major roles in pollen tube adhesion and reorientation bioassays: stigma/style cysteine-rich adhesin (SCA, approx. 9·3 kDa) and chemocyanin (approx. 9·8 kDa). SCA, a lipid transfer protein (LTP) secreted from the stylar transmitting tract epidermis, functions in lily pollen tube tip growth as well as in forming the adhesive pectin matrix at the growing pollen tube wall back from the tip. Lily chemocyanin is a plantacyanin family member and acts as a directional cue for reorienting pollen tubes. Recent consecutive studies revealed that Arabidopsis thaliana homologues for SCA and chemocyanin play pivotal roles in tip polarity and directionality of pollen tube growth, respectively. This review outlines the biological roles of various secreted proteins in angiosperm pollination, focusing on plant LTPs and chemocyanin. PMID:21307038

  13. Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila

    EPA Science Inventory

    Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrop...

  14. Molecular Characterization of a Functional Type VI Secretion System from a Clinical Isolate of Aeromonas hydrophilia

    EPA Science Inventory

    Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrop...

  15. A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria

    PubMed Central

    Osswald, Annika; Westermann, Christina; Sun, Zhongke; Riedel, Christian U.

    2015-01-01

    Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria. PMID:26086721

  16. A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria.

    PubMed

    Osswald, Annika; Westermann, Christina; Sun, Zhongke; Riedel, Christian U

    2015-01-01

    Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria. PMID:26086721

  17. Imipramine and citalopram facilitate amyloid precursor protein secretion in vitro.

    PubMed

    Pákáski, Magdolna; Bjelik, Annamária; Hugyecz, Marietta; Kása, Péter; Janka, Zoltán; Kálmán, János

    2005-08-01

    Comorbid depression of Alzheimer's disease (AD) is a common mood disorder in the elderly and a broad spectrum of antidepressants have been used for its treatment. Abeta peptides and other derivatives of the amyloid precursor protein (APP) have been implicated as central to the pathogenesis of AD. However, the functional relationship of APP and its proteolytic derivatives to antidepressant therapy is not known. In this study, Western blotting was used to test the ability of the tricyclic antidepressant (TCA) imipramine or the selective serotonin reuptake inhibitor (SSRI) citalopram to change the release of APP and the protein kinase C (PKC) content. Both antidepressants increased APP secretion in primary rat neuronal cultures. Imipramine or citalopram enhanced the level of secreted APP by 3.2- or 3.4-fold, respectively. Increases in PKC level were observed only after imipramine treatment. These in vitro data suggest that both TCA and SSRI are able to interfere with the APP metabolism. Imipramine promotes the non-amyloidogenic route of APP processing via stimulatory effects on PKC. We propose that PKC is not involved in the mechanism underlying the effects of citalopram on the APP metabolism. Since the secreted APP is not further available for the pathological cleavage of beta- and gamma-secretases, antidepressant medication might be beneficial in AD therapy. PMID:15955598

  18. Aphids evolved novel secreted proteins for symbiosis with bacterial endosymbiont.

    PubMed

    Shigenobu, Shuji; Stern, David L

    2013-01-01

    Aphids evolved novel cells, called bacteriocytes, that differentiate specifically to harbour the obligatory mutualistic endosymbiotic bacteria Buchnera aphidicola. The genome of the host aphid Acyrthosiphon pisum contains many orphan genes that display no similarity with genes found in other sequenced organisms, prompting us to hypothesize that some of these orphan genes are related to lineage-specific traits, such as symbiosis. We conducted deep sequencing of bacteriocytes mRNA followed by whole mount in situ hybridizations of over-represented transcripts encoding aphid-specific orphan proteins. We identified a novel class of genes that encode small proteins with signal peptides, which are often cysteine-rich, that are over-represented in bacteriocytes. These genes are first expressed at a developmental time point coincident with the incorporation of symbionts strictly in the cells that contribute to the bacteriocyte and this bacteriocyte-specific expression is maintained throughout the aphid's life. The expression pattern suggests that recently evolved secretion proteins act within bacteriocytes, perhaps to mediate the symbiosis with beneficial bacterial partners, which is reminiscent of the evolution of novel cysteine-rich secreted proteins of leguminous plants that regulate nitrogen-fixing endosymbionts. PMID:23173201

  19. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties

    PubMed Central

    Fessart, Delphine; Domblides, Charlotte; Avril, Tony; Eriksson, Leif A; Begueret, Hugues; Pineau, Raphael; Malrieux, Camille; Dugot-Senant, Nathalie; Lucchesi, Carlo; Chevet, Eric; Delom, Frederic

    2016-01-01

    The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.13887.001 PMID:27240165

  20. Stimulation of IGF-binding protein-1 secretion by AMP-activated protein kinase.

    PubMed

    Lewitt, M S

    2001-04-20

    Insulin-like growth factor-binding protein-1 (IGFBP-1) is stimulated during intensive exercise and in catabolic conditions to very high concentrations, which are not completely explained by known regulators such as insulin and glucocorticoids. The role of AMP-activated protein kinase (AMPK), an important signaling system in lipid and carbohydrate metabolism, in regulating IGFBP-1 was studied in H4-II-E rat hepatoma cells. Arsenic(III) oxide and 5-aminoimidazole-4-carboxamide-riboside (AICAR) were used as activators. AICAR (150 microM) stimulated IGFBP-1 secretion twofold during a 5-h incubation (P = 0.002). Insulin (100 ng/ml) inhibited IGFBP-1 by 80% (P < 0.001), but this was completely abolished in the presence of 150 microM AICAR. The effect of dexamethasone in stimulating IGFBP-1 threefold was additive to the effect of AICAR (P < 0.001) and, in the presence of AICAR, was incompletely inhibited by insulin. In conclusion AMPK is identified as a novel regulatory pathway for IGFBP-1, stimulating secretion and blocking the inhibitory effect of insulin. PMID:11302732

  1. Identification of a Family of Effectors Secreted by the Type III Secretion System That Are Conserved in Pathogenic Chlamydiae▿

    PubMed Central

    Muschiol, Sandra; Boncompain, Gaelle; Vromman, François; Dehoux, Pierre; Normark, Staffan; Henriques-Normark, Birgitta; Subtil, Agathe

    2011-01-01

    Chlamydiae are Gram-negative, obligate intracellular pathogens that replicate within a membrane-bounded compartment termed an inclusion. Throughout their development, they actively modify the eukaryotic environment. The type III secretion (TTS) system is the main process by which the bacteria translocate effector proteins into the inclusion membrane and the host cell cytoplasm. Here we describe a family of type III secreted effectors that are present in all pathogenic chlamydiae and absent in the environment-related species. It is defined by a common domain of unknown function, DUF582, that is present in four or five proteins in each Chlamydiaceae species. We show that the amino-terminal extremity of DUF582 proteins functions as a TTS signal. DUF582 proteins from C. trachomatis CT620, CT621, and CT711 are expressed at the middle and late phases of the infectious cycle. Immunolocalization further revealed that CT620 and CT621 are secreted into the host cell cytoplasm, as well as within the lumen of the inclusion, where they do not associate with bacterial markers. Finally, we show that DUF582 proteins are present in nuclei of infected cells, suggesting that members of the DUF582 family of effector proteins may target nuclear cell functions. The expansion of this family of proteins in pathogenic chlamydiae and their conservation among the different species suggest that they play important roles in the infectious cycle. PMID:21078856

  2. ESX/type VII secretion systems of mycobacteria: Insights into evolution, pathogenicity and protection.

    PubMed

    Simeone, Roxane; Bottai, Daria; Frigui, Wafa; Majlessi, Laleh; Brosch, Roland

    2015-06-01

    Pathogenesis of Mycobacterium tuberculosis depends on the secretion of key virulence factors, such as the 6 kDa early secreted antigenic target ESAT-6 (EsxA) and its protein partner, the 10 kDa culture filtrate protein CFP-10 (EsxB), via the ESX-1 secretion system. ESX-1 represents the prototype system of the recently named type VII secretion systems that exist in a range of actinobacteria. The M. tuberculosis genome harbours a total of five gene clusters potentially coding for type VII secretion systems, designated ESX-1 - ESX-5, with ESX-4 being the most ancient system from which other ESX systems seem to have evolved by gene duplication and gene insertion events. The five ESX systems show similarity in gene content and gene order but differ in function. ESX-1 and ESX-5 are both crucial virulence determinants of M. tuberculosis, but with different mechanisms. While ESX-1 is implicated in the lysis of the host cell phagosomes, ESX-5 is involved in secretion of the mycobacteria specific PE and PPE proteins and cell wall stability. Research on type VII secretion systems has thus become a large and competitive research topic that is tightly linked to studies of host-pathogen interaction of pathogenic mycobacteria. Insights into this matter are of relevance for redrawing the patho-evolution of M. tuberculosis, which might help improving current strategies for prevention, diagnostics and therapy of tuberculosis as well as elucidating the virulence mechanisms employed by this important human pathogen. PMID:25732627

  3. The rise of the Type VI secretion system

    PubMed Central

    2013-01-01

    Bacterial cells have developed multiple strategies to communicate with their surrounding environment. The intracellular compartment is separated from the milieu by a relatively impermeable cell envelope through which small molecules can passively diffuse, while larger macromolecules, such as proteins, can be actively transported. In Gram-negative bacteria, the cell envelope is a double membrane, which houses several supramolecular protein complexes that facilitate the trafficking of molecules. For example, bacterial pathogens use these types of machines to deliver toxins into target eukaryotic host cells, thus subverting host cellular functions. Six different types of nanomachines, called Type I - Type VI secretion systems (T1SS - T6SS), can be readily identified by their composition and mode of action. A remarkable feature of these protein secretion systems is their similarity to systems with other biological functions, such as motility or the exchange of genetic material. The T6SS has provided a refreshing view on this concept since it shares similarity with the puncturing device of bacteriophages, which is used by these viruses to inject their DNA into bacterial target cells. In contrast, the bacterial T6SS transports toxins into other bacteria, engaging a ferocious competition for the colonization of their environment. Moreover, as with few other secretion systems, the T6SS is capable of injecting toxins into eukaryotic cells, which contributes to a successful infection. This highlights the multifunctional aspects of the T6SS, and our understanding of its mechanistic details is an intense field of investigation with significant implications for ecology, agriculture and medicine. PMID:24381728

  4. Infectious Keratitis: Secreted Bacterial Proteins That Mediate Corneal Damage

    PubMed Central

    Marquart, Mary E.; O'Callaghan, Richard J.

    2013-01-01

    Ocular bacterial infections are universally treated with antibiotics, which can eliminate the organism but cannot reverse the damage caused by bacterial products already present. The three very common causes of bacterial keratitis—Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae—all produce proteins that directly or indirectly cause damage to the cornea that can result in reduced vision despite antibiotic treatment. Most, but not all, of these proteins are secreted toxins and enzymes that mediate host cell death, degradation of stromal collagen, cleavage of host cell surface molecules, or induction of a damaging inflammatory response. Studies of these bacterial pathogens have determined the proteins of interest that could be targets for future therapeutic options for decreasing corneal damage. PMID:23365719

  5. NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103

    PubMed Central

    Medina, Carlos; Ollero, Francisco Javier; López-Baena, Francisco Javier

    2015-01-01

    Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103. PMID:26569401

  6. NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103.

    PubMed

    Jiménez-Guerrero, Irene; Pérez-Montaño, Francisco; Medina, Carlos; Ollero, Francisco Javier; López-Baena, Francisco Javier

    2015-01-01

    Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103. PMID:26569401

  7. A Rapid Method for Determining the Concentration of Recombinant Protein Secreted from Pichia pastoris

    NASA Astrophysics Data System (ADS)

    Sun, L. W.; Zhao, Y.; Niu, L. P.; Jiang, R.; Song, Y.; Feng, H.; feng, K.; Qi, C.

    2011-02-01

    Pichia secretive expression system is one of powerful eukaryotic expression systems in genetic engineering, which is especially suitable for industrial utilization. Because of the low concentration of the target protein in initial experiment, the methods and conditions for expression of the target protein should be optimized according to the protein yield repetitively. It is necessary to set up a rapid, simple and convenient analysis method for protein expression levels instead of the generally used method such as ultrafiltration, purification, dialysis, lyophilization and so on. In this paper, acetone precipitation method was chosen to concentrate the recombinant protein firstly after comparing with four different protein precipitation methods systematically, and then the protein was analyzed by SDS-Polyacrylamide Gel Electrophoresis. The recombinant protein was determined with the feature of protein band by the Automated Image Capture and 1-D Analysis Software directly. With this method, the optimized expression conditions of basic fibroblast growth factor secreted from pichia were obtained, which is as the same as using traditional methods. Hence, a convenient tool to determine the optimized conditions for the expression of recombinant proteins in Pichia was established.

  8. Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis

    PubMed Central

    Mueller, Konrad E.; Fields, Kenneth A.

    2015-01-01

    Chlamydia spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform Chlamydia and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 β-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings provide definitive evidence that C. trachomatis CT695 is secreted during infection. Follow-up indirect immunofluorescence studies confirmed CT695 secretion and indicate that this effector can be secreted at multiple points during the chlamydial developmental cycle. Our results indicate that the BlaM-fusion reporter assay will allow efficacious identification of novel secreted proteins. Moreover, this approach can easily be adapted to enable more sophisticated studies of the secretion process in Chlamydia. PMID:26258949

  9. Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2

    PubMed Central

    Konopatskaya, Olga; Matthews, Sharon A.; Harper, Matthew T.; Gilio, Karen; Cosemans, Judith M. E. M.; Williams, Christopher M.; Navarro, Maria N.; Carter, Deborah A.; Heemskerk, Johan W. M.; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W.

    2016-01-01

    Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion. PMID:21527521

  10. PROTEIN EXPRESSION AND SECRETION BY TRICHODERMA REESEI UNDER LOW ENDOGENOUS PROTEIN BACKGROUND

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Trichoderma reesei (Hypocrea jecorina) is one of the most commonly used fungi for the manufacturing of industrial enzyme products. The fungus is capable of secreting proteins in levels up to 100 grams per liter. A number of homologous and heterologous proteins have been successfully over-expressed...

  11. Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide

    PubMed Central

    Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

    2015-01-01

    Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%–92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp. PMID:26473722

  12. Platelet C1- inhibitor. A secreted alpha-granule protein.

    PubMed Central

    Schmaier, A H; Smith, P M; Colman, R W

    1985-01-01

    In order to characterize which proteins of the contact phase of coagulation interact with platelets, human platelets were studied immunochemically and functionally to determine if they contain C1- inhibitor. By means of monospecific antibody to C1- inhibitor, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to measure directly platelet C1- inhibitor. With the CELISA, from 33 to 115 ng of C1- inhibitor antigen per 10(8) platelets from 15 normal donors was quantified in lysates of washed human platelets solubilized in nonionic detergent. The mean concentration in 10(8) platelets was 62 +/- 33 ng (SD). Plasma C1- inhibitor either in the platelet suspension medium or on the surface of the platelets could account for only from 6.5 to 16% of the total antigen measured in the solubilized platelets. Upon functional studies, platelets contained 84 +/- 36 ng (SD) of C1- inhibitor activity in 10(8) platelets. As assessed by the CELISA, platelet C1- inhibitor antigen was immunochemically identical to plasma and purified C1- inhibitor. In contrast, the mean concentration of platelet C1- inhibitor antigen in platelets from four patients with classical hereditary angioedema was 8.3 ng/10(8) platelets (range, 5.3 to 11.3 ng/10(8) platelets). 25 and 31% of the total platelet C1- inhibitor was secreted without cell lysis from normal platelets after exposure to collagen (20 micrograms/ml) and thrombin (1 U/ml), respectively, and this secretion was blocked by metabolic inhibitors. Platelet subcellular fractionation showed that platelet C1- inhibitor resided mostly in alpha-granules, similar to the location of platelet fibrinogen. Thus, human platelets contained C1- inhibitor, which became available by platelet secretion. The identification of platelet C1- inhibitor suggests that platelets may modulate the activation of the proteins of early blood coagulation and the classical complement pathways. Images PMID:3965505

  13. Phosphatidylethanolamine binding protein 4 (PEBP4) is a secreted protein and has multiple functions.

    PubMed

    He, Huan; Liu, Dan; Lin, Hui; Jiang, Shanshan; Ying, Ying; Chun, Shao; Deng, Haiteng; Zaia, Joseph; Wen, Rong; Luo, Zhijun

    2016-07-01

    Phosphatidylethanolamine binding proteins (PEBP) represent a superfamily of proteins that are conserved from bacteria to humans. In mammals, four members have been identified, PEBP1-4. To determine the functional differences among PEBP1-4 and the underlying mechanism for their actions, we performed a sequence alignment and found that PEBP4 contains a signal peptide and potential glycosylation sites, whereas PEBP1-3 are intracellular proteins. To test if PEBP4 is secreted, we made constructs with Myc epitope at the amino (N) terminus or carboxyl (C) terminus to mask the signal sequence or keep it free, respectively. Our data revealed that both mouse and human PEBP4 were secreted when the epitope was tagged at their C-terminus. To our surprise, secretion was dependent upon the C-terminal conserved domain in addition to the N-terminal signal sequence. When the epitope was placed to the N-terminus, the recombinant protein failed to secrete and instead, was retained in the cytoplasm. Mass spectrometry detected asparagine (N)-glycosylation on the secreted PEBP4. Although overexpression of N-terminal tagged PEBP4 resulted in an inhibition of ERK activation by EGF, that with a C-terminal epitope tag did not have such an effect. Likewise, transfection of PEBP4 shRNA did not appear to affect ERK activation, suggesting that PEBP4 does not participate in the regulation of this pathway. In contrast, PEBP4 siRNA suppressed phosphorylation of Act at S473. Therefore, our results suggest that PEBP4 is a multifunctional protein and can be secreted. It will be important to investigate the mechanism by which PEBP4 is secreted and regulates cellular events. PMID:27033522

  14. Expression and secretion of a CB4-1 scFv-GFP fusion protein by fission yeast.

    PubMed

    Naumann, Julia Maria; Küttner, Gabriele; Bureik, Matthias

    2011-01-01

    There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency virus 1) CB4-1 scFv fused to GFP. After expression of the scFv-GFP fused to an N-terminal Cpy1 secretion signal sequence, fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western blot analysis of cell-free culture supernatants confirmed that the scFv-GFP was efficiently secreted with yields up to 5 mg/L. In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv-GFP protein is fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient protein processing and secretion. PMID:20617397

  15. Enteropathogenic Escherichia coli protein secretion is induced in response to conditions similar to those in the gastrointestinal tract.

    PubMed Central

    Kenny, B; Abe, A; Stein, M; Finlay, B B

    1997-01-01

    The pathogenicity of enteropathogenic Escherichia coli (EPEC) is associated with the expression and secretion of specific bacterial factors. EspB is one such secreted protein which is required to trigger host signaling pathways resulting in effacement of microvilli and cytoskeletal rearrangements. These events presumably contribute to the ensuing diarrhea associated with EPEC infections. EPEC encounters several environmental changes and stimuli during its passage from the external environment into the host gastrointestinal tract. In this paper we show that the secretion of EspB is subject to environmental regulation, and maximal secretion occurs under conditions reminiscent of those in the gastrointestinal tract. Thus, secretion is maximal at 37 degrees C, pH 7, and physiological osmolarity. In addition, maximal secretion requires the presence of sodium bicarbonate and calcium and is stimulated by millimolar concentrations of Fe(NO3)3. The secretion of the four other EPEC-secreted proteins appears to be modulated in a manner similar to that of EspB. Our results also show that secretion is not dependent on CO2, as originally reported by Haigh et al. (FEMS Microbiol. Lett. 129: 63-67, 1995), but that CO2 more likely acts as a component of the medium buffering system, since CO2 dependence was abolished by the use of alternative buffers. PMID:9199427

  16. Mutation of crp mediates Serratia marcescens serralysin and global secreted protein production

    PubMed Central

    Shanks, Robert M.Q.; Stella, Nicholas A.; Arena, Kristin E.; Fender, James E.

    2012-01-01

    The bacterial species Serratia marcescens secretes both beneficial and cytotoxic proteins. Here we report that a crp mutant exhibited elevated secreted protease activity. A genetic screen revealed that the gene coding for the metalloprotease serralysin was necessary for the elevated proteolysis, and this was confirmed by western blot analysis. Proteomic analysis of secreted proteins corroborated increased secretion of serralysin protease by crp mutants compared to the wild type. The crp-mutant-secreted fractions also contained less chitinase and chitin binding protein. These data support the hypothesis that cAMP-CRP is an upstream indirect regulator of serralysin production and they provide novel insight into the S. marcescens secretome. PMID:23072819

  17. Rotating wall vessel exposure alters protein secretion and global gene expression in Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Rosado, Helena; O'Neill, Alex J.; Blake, Katy L.; Walther, Meik; Long, Paul F.; Hinds, Jason; Taylor, Peter W.

    2012-04-01

    Staphylococcus aureus is routinely recovered from air and surface samples taken aboard the International Space Station (ISS) and poses a health threat to crew. As bacteria respond to the low shear forces engendered by continuous rotation conditions in a Rotating Wall Vessel (RWV) and the reduced gravitational field of near-Earth flight by altering gene expression, we examined the effect of low-shear RWV growth on protein secretion and gene expression by three S. aureus isolates. When cultured under 1 g, the total amount of protein secreted by these strains varied up to fourfold; under continuous rotation conditions, protein secretion by all three strains was significantly reduced. Concentrations of individual proteins were differentially reduced and no evidence was found for increased lysis. These data suggest that growth under continuous rotation conditions reduces synthesis or secretion of proteins. A limited number of changes in gene expression under continuous rotation conditions were noted: in all isolates vraX, a gene encoding a polypeptide associated with cell wall stress, was down-regulated. A vraX deletion mutant of S. aureus SH1000 was constructed: no differences were found between SH1000 and ΔvraX with respect to colony phenotype, viability, protein export, antibiotic susceptibility, vancomycin kill kinetics, susceptibility to cold or heat and gene modulation. An ab initio protein-ligand docking simulation suggests a major binding site for β-lactam drugs such as imipenem. If such changes to the bacterial phenotype occur during spaceflight, they will compromise the capacity of staphylococci to cause systemic infection and to circumvent antibacterial chemotherapy.

  18. The Protein Architecture of Human Secretory Vesicles Reveals Differential Regulation of Signaling Molecule Secretion by Protein Kinases

    PubMed Central

    Taupenot, Laurent; Ziegler, Michael; O'Connor, Daniel T.; Ma, Qi; Smoot, Michael; Ideker, Trey; Hook, Vivian

    2012-01-01

    Secretory vesicles are required for release of chemical messengers to mediate intercellular signaling among human biological systems. It is necessary to define the organization of the protein architecture of the ‘human’ dense core secretory vesicles (DCSV) to understand mechanisms for secretion of signaling molecules essential for cellular regulatory processes. This study, therefore, conducted extensive quantitative proteomics and systems biology analyses of human DCSV purified from human pheochromocytoma. Over 600 human DCSV proteins were identified with quantitative evaluation of over 300 proteins, revealing that most proteins participate in producing peptide hormones and neurotransmitters, enzymes, and the secretory machinery. Systems biology analyses provided a model of interacting DCSV proteins, generating hypotheses for differential intracellular protein kinases A and C signaling pathways. Activation of cellular PKA and PKC pathways resulted in differential secretion of neuropeptides, catecholamines, and β-amyloid of Alzheimer's disease for mediating cell-cell communication. This is the first study to define a model of the protein architecture of human DCSV for human disease and health. PMID:22916103

  19. The Structure and Function of Type III Secretion Systems

    PubMed Central

    Notti, Ryan Q.; Stebbins, C. Erec

    2015-01-01

    ARTICLE SUMMARY Type III secretion systems (T3SS) afford gram-negative bacteria a most intimate means of altering the biology of their eukaryotic hosts — the direct delivery of effector proteins from the bacterial cytoplasm to that of the eukaryote. This incredible biophysical feat is accomplished by nanosyringe “injectisomes,” which form a conduit across the three plasma membranes, peptidoglycan layer and extracellular space that form a barrier to the direct delivery of proteins from bacterium to host. The focus of this chapter is T3SS function at the structural level; we will summarize the core findings that have shaped our understanding of the structure and function of these systems and highlight recent developments in the field. In turn, we describe the T3SS secretory apparatus, consider its engagement with secretion substrates, and discuss the post-translational regulation of secretory function. Lastly, we close with a discussion of the future prospects for the interrogation of structure-function relationships in the T3SS. PMID:26999392

  20. The Type III Secretion System of Bradyrhizobium japonicum USDA122 Mediates Symbiotic Incompatibility with Rj2 Soybean Plants

    PubMed Central

    Tsukui, Takahiro; Eda, Shima; Kaneko, Takakazu; Sato, Shusei; Okazaki, Shin; Kakizaki-Chiba, Kaori; Itakura, Manabu; Mitsui, Hisayuki; Yamashita, Akifumi; Terasawa, Kimihiro

    2013-01-01

    The rhcJ and ttsI mutants of Bradyrhizobium japonicum USDA122 for the type III protein secretion system (T3SS) failed to secrete typical effector proteins and gained the ability to nodulate Rj2 soybean plants (Hardee), which are symbiotically incompatible with wild-type USDA122. This suggests that effectors secreted via the T3SS trigger incompatibility between these two partners. PMID:23204412

  1. Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis

    PubMed Central

    Chen, Wen-Jen; Kuo, Tzu-Yen; Hsieh, Feng-Chia; Chen, Pi-Yu; Wang, Chang-Sheng; Shih, Yu-Ling; Lai, Ying-Mi; Liu, Je-Ruei; Yang, Yu-Liang; Shih, Ming-Che

    2016-01-01

    Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive rice diseases worldwide. Therefore, in addition to breeding disease-resistant rice cultivars, it is desirable to develop effective biocontrol agents against Xoo. Here, we report that a soil bacterium Pseudomonas taiwanensis displayed strong antagonistic activity against Xoo. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we identified an iron chelator, pyoverdine, secreted by P. taiwanensis that could inhibit the growth of Xoo. Through Tn5 mutagenesis of P. taiwanensis, we showed that mutations in genes that encode components of the type VI secretion system (T6SS) as well as biosynthesis and maturation of pyoverdine resulted in reduced toxicity against Xoo. Our results indicated that T6SS is involved in the secretion of endogenous pyoverdine. Mutations in T6SS component genes affected the secretion of mature pyoverdine from the periplasmic space into the extracellular medium after pyoverdine precursor is transferred to the periplasm by the inner membrane transporter PvdE. In addition, we also showed that other export systems, i.e., the PvdRT-OpmQ and MexAB-OprM efflux systems (for which there have been previous suggestions of involvement) and the type II secretion system (T2SS), are not involved in pyoverdine secretion. PMID:27605490

  2. Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis.

    PubMed

    Chen, Wen-Jen; Kuo, Tzu-Yen; Hsieh, Feng-Chia; Chen, Pi-Yu; Wang, Chang-Sheng; Shih, Yu-Ling; Lai, Ying-Mi; Liu, Je-Ruei; Yang, Yu-Liang; Shih, Ming-Che

    2016-01-01

    Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive rice diseases worldwide. Therefore, in addition to breeding disease-resistant rice cultivars, it is desirable to develop effective biocontrol agents against Xoo. Here, we report that a soil bacterium Pseudomonas taiwanensis displayed strong antagonistic activity against Xoo. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we identified an iron chelator, pyoverdine, secreted by P. taiwanensis that could inhibit the growth of Xoo. Through Tn5 mutagenesis of P. taiwanensis, we showed that mutations in genes that encode components of the type VI secretion system (T6SS) as well as biosynthesis and maturation of pyoverdine resulted in reduced toxicity against Xoo. Our results indicated that T6SS is involved in the secretion of endogenous pyoverdine. Mutations in T6SS component genes affected the secretion of mature pyoverdine from the periplasmic space into the extracellular medium after pyoverdine precursor is transferred to the periplasm by the inner membrane transporter PvdE. In addition, we also showed that other export systems, i.e., the PvdRT-OpmQ and MexAB-OprM efflux systems (for which there have been previous suggestions of involvement) and the type II secretion system (T2SS), are not involved in pyoverdine secretion. PMID:27605490

  3. An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages.

    PubMed

    Silverman, Judith Maxwell; Clos, Joachim; de'Oliveira, Carolina Camargo; Shirvani, Omid; Fang, Yuan; Wang, Christine; Foster, Leonard J; Reiner, Neil E

    2010-03-15

    Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37 degrees C +/- pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-alpha. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation. PMID:20159964

  4. Regulation of hrp genes and type III protein secretion in Erwinia amylovora by HrpX/HrpY, a novel two-component system, and HrpS.

    PubMed

    Wei, Z; Kim, J F; Beer, S V

    2000-11-01

    Two novel regulatory components, hrpX and hrpY, of the hrp system of Erwinia amylovora were identified. The hrpXY operon is expressed in rich media, but its transcription is increased threefold by low pH, nutrient, and temperature levels--conditions that mimic the plant apoplast. hrpXY is autoregulated and directs the expression of hrpL; hrpL, in turn, activates transcription of other loci in the hrp gene cluster (Z.-M. Wei and S. V. Beer, J. Bacteriol. 177:6201-6210, 1995). The deduced amino -acid sequences of hrpX and hrpY are similar to bacterial two-component regulators including VsrA/VsrD of Pseudomonas (Ralstonia) solanacearum, DegS/DegU of Bacillus subtilis, and UhpB/UhpA and NarX/NarP, NarL of Escherichia coli. The N-terminal signal-input domain of HrpX contains PAS domain repeats. hrpS, located downstream of hrpXY, encodes a protein with homology to WtsA (HrpS) of Erwinia (Pantoea) stewartii, HrpR and HrpS of Pseudomonas syringae, and other delta54-dependent, enhancer-binding proteins. Transcription of hrpS also is induced under conditions that mimic the plant apoplast. However, hrpS is not autoregulated, and its expression is not affected by hrpXY. When hrpS or hrpL were provided on multicopy plasmids, both hrpX and hrpY mutants recovered the ability to elicit the hypersensitive reaction in tobacco. This confirms that hrpS and hrpL are not epistatic to hrpXY. A model of the regulatory cascades leading to the induction of the E. amylovora type III system is proposed. PMID:11059492

  5. Protein expression and secretion in the yeast Yarrowia lipolytica.

    PubMed

    Nicaud, Jean-Marc; Madzak, Catherine; van den Broek, Peter; Gysler, Christof; Duboc, Philippe; Niederberger, Peter; Gaillardin, Claude

    2002-08-01

    Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform. To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence. We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers. The wild-type URA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome. These vectors were used to express the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml(-1) in shake flasks, while a strain containing multiple integrations reached 2000 U ml(-1) in shake flasks, 11500 U ml(-1) in batch and 90500 U ml(-1) in fed batch. Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml(-1) in batch with a mono-copy lapA integrant and 28000 mU ml(-1) in fed batch with a multi-copy transformant. PMID:12702287

  6. Modular Organization of the ESX-5 Secretion System in Mycobacterium tuberculosis

    PubMed Central

    Shah, Swati; Briken, Volker

    2016-01-01

    Mycobacteria utilize type VII secretion systems (T7SS) to export many of their important virulence proteins. The T7SS encompasses five homologous secretion systems (ESX-1 to ESX-5). Most pathogenic mycobacterial species, including the human pathogen Mycobacterium tuberculosis, possess all five ESX systems. The ESX-1, -3, and -5 systems are important for virulence of mycobacteria but the molecular mechanisms of their secretion apparatus and the identity and activity of secreted effector proteins are not well characterized. The different ESX systems show similarities in gene composition due to their common phylogenetic origin but recent studies demonstrate mechanistic as well as functional variations between the systems. For example, the ESX-1 system is involved in lysis of the phagosomal membrane and phagosomal escape of the bacteria while the ESX-5 system is required for mycobacterial cell wall stability and host cell lysis. Mechanistically, the ESX-1 substrates show interdependence during secretion while the ESX-5 system may use a duplicated four-gene region (ESX-5a) as an accessory system for transport of a subset of proteins of the ESX-5 secretome. In the present review we will provide an overview of the molecular components of the T7SS and their function with a particular focus on the ESX-5 system. PMID:27200304

  7. Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum.

    PubMed

    Thavayogarajah, Thuvaraka; Gangopadhyay, Preetish; Rahlfs, Stefan; Becker, Katja; Lingelbach, Klaus; Przyborski, Jude M; Holder, Anthony A

    2015-01-01

    Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process. PMID:25909331

  8. Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Thavayogarajah, Thuvaraka; Gangopadhyay, Preetish; Rahlfs, Stefan; Becker, Katja; Lingelbach, Klaus; Przyborski, Jude M.; Holder, Anthony A.

    2015-01-01

    Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process. PMID:25909331

  9. Protein Kinase C Controls Vesicular Transport and Secretion of Apolipoprotein E from Primary Human Macrophages*

    PubMed Central

    Karunakaran, Denuja; Kockx, Maaike; Owen, Dylan M.; Burnett, John R.; Jessup, Wendy; Kritharides, Leonard

    2013-01-01

    Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/β and not PKCδ, -ϵ, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages. PMID:23288845

  10. a Computational Approach to Explore Protein Translocation Through Type III Secretion Apparatus

    NASA Astrophysics Data System (ADS)

    Rathinavelan, Thenmalarchelvi; Im, Wonpil

    2010-01-01

    Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus (TTSA) that is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. Interestingly, the electronegative channel interior creates an energy barrier for MxiH to enter the channel, while the same may facilitate the ejection of the effectors into host cells. Structurally-known basal regions and ATPase underneath the basal region have also such electronegative interior, while effector proteins have considerable electronegative patches on their surfaces. Based on these observations, we propose a repulsive electrostatic mechanism for protein translocation through the TTSA. This mechanism is supported by the suggestion that an ATPase is required for protein translocation through these nanomachines, which may provide the energy to overcome the initial electrostatic energy barrier. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported.

  11. Solution structure of monomeric BsaL, the type III secretion needle protein of Burkholderia pseudomallei.

    PubMed

    Zhang, Lingling; Wang, Yu; Picking, Wendy L; Picking, William D; De Guzman, Roberto N

    2006-06-01

    Many gram-negative bacteria that are important human pathogens possess type III secretion systems as part of their required virulence factor repertoire. During the establishment of infection, these pathogens coordinately assemble greater than 20 different proteins into a macromolecular structure that spans the bacterial inner and outer membranes and, in many respects, resembles and functions like a syringe. This type III secretion apparatus (TTSA) is used to inject proteins into a host cell's membrane and cytoplasm to subvert normal cellular processes. The external portion of the TTSA is a needle that is composed of a single type of protein that is polymerized in a helical fashion to form an elongated tube with a central channel of 2-3 nm in diameter. TTSA needle proteins from a variety of bacterial pathogens share sequence conservation; however, no atomic structure for any TTSA needle protein is yet available. Here, we report the structure of a TTSA needle protein called BsaL from Burkholderia pseudomallei determined by nuclear magnetic resonance (NMR) spectroscopy. The central part of the protein assumes a helix-turn-helix core domain with two well-defined alpha-helices that are joined by an ordered, four-residue linker. This forms a two-helix bundle that is stabilized by interhelix hydrophobic contacts. Residues that flank this presumably exposed core region are not completely disordered, but adopt a partial helical conformation. The atomic structure of BsaL and its sequence homology with other TTSA needle proteins suggest potentially unique structural dynamics that could be linked with a universal mechanism for control of type III secretion in diverse gram-negative bacterial pathogens. PMID:16631790

  12. Named entity recognition for bacterial Type IV secretion systems.

    PubMed

    Ananiadou, Sophia; Sullivan, Dan; Black, William; Levow, Gina-Anne; Gillespie, Joseph J; Mao, Chunhong; Pyysalo, Sampo; Kolluru, Balakrishna; Tsujii, Junichi; Sobral, Bruno

    2011-01-01

    Research on specialized biological systems is often hampered by a lack of consistent terminology, especially across species. In bacterial Type IV secretion systems genes within one set of orthologs may have over a dozen different names. Classifying research publications based on biological processes, cellular components, molecular functions, and microorganism species should improve the precision and recall of literature searches allowing researchers to keep up with the exponentially growing literature, through resources such as the Pathosystems Resource Integration Center (PATRIC, patricbrc.org). We developed named entity recognition (NER) tools for four entities related to Type IV secretion systems: 1) bacteria names, 2) biological processes, 3) molecular functions, and 4) cellular components. These four entities are important to pathogenesis and virulence research but have received less attention than other entities, e.g., genes and proteins. Based on an annotated corpus, large domain terminological resources, and machine learning techniques, we developed recognizers for these entities. High accuracy rates (>80%) are achieved for bacteria, biological processes, and molecular function. Contrastive experiments highlighted the effectiveness of alternate recognition strategies; results of term extraction on contrasting document sets demonstrated the utility of these classes for identifying T4SS-related documents. PMID:21468321

  13. The twin-arginine signal peptide of Bacillus subtilis YwbN can direct either Tat- or Sec-dependent secretion of different cargo proteins: secretion of active subtilisin via the B. subtilis Tat pathway.

    PubMed

    Kolkman, Marc A B; van der Ploeg, René; Bertels, Michael; van Dijk, Maurits; van der Laan, Joop; van Dijl, Jan Maarten; Ferrari, Eugenio

    2008-12-01

    Proteins that are produced for commercial purposes in Bacillus subtilis are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in B. subtilis. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known B. subtilis Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the B. subtilis TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the Bacillus licheniformis alpha-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an Escherichia coli Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in B. subtilis, as is the case with E. coli. We conclude that future applications for the Tat system of B. subtilis will most likely involve commercially interesting proteins that are Sec incompatible. PMID:18931290

  14. Vacuole Membrane Protein 1 Is an Endoplasmic Reticulum Protein Required for Organelle Biogenesis, Protein Secretion, and Development

    PubMed Central

    Calvo-Garrido, Javier; Carilla-Latorre, Sergio; Lázaro-Diéguez, Francisco; Egea, Gustavo

    2008-01-01

    Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1− Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1− cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised. PMID:18550798

  15. Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells.

    PubMed

    Lee, Jin-Gu; Takahama, Shokichi; Zhang, Guofeng; Tomarev, Stanislav I; Ye, Yihong

    2016-07-01

    To safeguard proteomic integrity, cells rely on the proteasome to degrade aberrant polypeptides, but it is unclear how cells remove defective proteins that have escaped degradation owing to proteasome insufficiency or dysfunction. Here we report a pathway termed misfolding-associated protein secretion, which uses the endoplasmic reticulum (ER)-associated deubiquitylase USP19 to preferentially export aberrant cytosolic proteins. Intriguingly, the catalytic domain of USP19 possesses an unprecedented chaperone activity, allowing recruitment of misfolded proteins to the ER surface for deubiquitylation. Deubiquitylated cargos are encapsulated into ER-associated late endosomes and secreted to the cell exterior. USP19-deficient cells cannot efficiently secrete unwanted proteins, and grow more slowly than wild-type cells following exposure to a proteasome inhibitor. Together, our findings delineate a protein quality control (PQC) pathway that, unlike degradation-based PQC mechanisms, promotes protein homeostasis by exporting misfolded proteins through an unconventional protein secretion process. PMID:27295555

  16. Enhancing activity of N-glycosylation for constitutive proteins secretions in non-polarized cells

    SciTech Connect

    Akiyama, Nobutake; Ohno, Yuji; Fukuda, Takahiro; Manome, Yosinobu; Saito, Saburo

    2009-04-17

    Several fusion proteins of mouse Interleukins (mILs) and the enhanced green fluorescent protein (EGFP) were expressed in fibroblast and epithelial cells. Among these proteins, the mIL-31 derivative was the most efficiently secreted into the medium in a N-glycosylation-dependent manner. From the analysis of deletion mutants, the minimal structure for constitutive secretions consisted of a signal peptide and N-glycosylation. Introduction of the signal sequence from mIL-31 to human p53 protein failed to secrete the products, but further addition of the N-glycosylation site resulted in constitutive secretion of biologically active p53 protein into the medium in the N-glycosylated form. In this report, we showed the importance of N-glycosylation for constitutive protein secretions, especially using non-polarized cells.

  17. Characterization of EspC, a 110-kilodalton protein secreted by enteropathogenic Escherichia coli which is homologous to members of the immunoglobulin A protease-like family of secreted proteins.

    PubMed Central

    Stein, M; Kenny, B; Stein, M A; Finlay, B B

    1996-01-01

    Enteropathogenic Escherichia coli (EPEC) secretes at least five proteins. Two of these proteins, EspA and EspB (previously called EaeB), activate signal transduction pathways in host epithelial cells. While the role of the other three proteins (39, 40, and 110 kDa) remains undetermined, secretion of all five proteins is under the control of perA, a known positive regulator of several EPEC virulence factors. On the basis of amino-terminal protein sequence data, we cloned and sequenced the gene which encodes the 110-kDa secreted protein and examined its possible role in EPEC signaling and interaction with epithelial cells. In accordance with the terminology used for espA and espB, we called this gene espC, for EPEC-secreted protein C. We found significant homology between the predicted EspC protein sequence and a family of immunoglobulin A (IgA) protease-like proteins which are widespread among pathogenic bacteria. Members of this protein family are found in avian pathogenic Escherichia coli (Tsh), Haemophilus influenzae (Hap), and Shigella flexneri (SepA). Although these proteins and EspC do not encode IgA protease activity, they have considerable homology with IgA protease from Neisseria gonorrhoeae and H. influenzae and appear to use a export system for secretion. We found that genes homologous to espC also exist in other pathogenic bacteria which cause attaching and effacing lesions, including Hafnia alvei biotype 19982, Citrobacter freundii biotype 4280, and rabbit diarrheagenic E. coli (RDEC-1). Although these strains secrete various proteins similar in molecular size to the proteins secreted by EPEC, we did not detect secretion of a 110-kDa protein by these strains. To examine the possible role of EspC in EPEC interactions with epithelial cells, we constructed a deletion mutant in espC by allelic exchange and characterized the mutant by standard tissue culture assays. We found that EspC is not necessary for mediating EPEC-induced signal transduction in He

  18. Secreted beta-amyloid precursor protein stimulates mitogen-activated protein kinase and enhances tau phosphorylation.

    PubMed Central

    Greenberg, S M; Koo, E H; Selkoe, D J; Qiu, W Q; Kosik, K S

    1994-01-01

    Biological effects related to cell growth, as well as a role in the pathogenesis of Alzheimer disease, have been ascribed to the beta-amyloid precursor protein (beta-APP). Little is known, however, about the intracellular cascades that mediate these effects. We report that the secreted form of beta-APP potently stimulates mitogen-activated protein kinases (MAPKs). Brief exposure of PC-12 pheochromocytoma cells to beta-APP secreted by transfected Chinese hamster ovary cells stimulated the 43-kDa form of MAPK by > 10-fold. Induction of a dominant inhibitory form of ras in a PC12-derived cell line prevented the stimulation of MAPK by secreted beta-APP, demonstrating the dependence of the effect upon p21ras. Because the microtubule-associated protein tau is hyperphosphorylated in Alzheimer disease, we sought and found a 2-fold enhancement in tau phosphorylation associated with the beta-APP-induced MAPK stimulation. In the ras dominant inhibitory cell line, beta-APP failed to enhance phosphorylation of tau. The data presented here provide a link between secreted beta-APP and the phosphorylation state of tau. Images PMID:8041753

  19. Directionality of substrate translocation of the hemolysin A Type I secretion system

    PubMed Central

    Lenders, Michael H. H.; Weidtkamp-Peters, Stefanie; Kleinschrodt, Diana; Jaeger, Karl-Erich; Smits, Sander H. J.; Schmitt, Lutz

    2015-01-01

    Type 1 secretion systems (T1SS) of Gram-negative bacteria are responsible for the secretion of various proteases, lipases, S-layer proteins or toxins into the extracellular space. The paradigm of these systems is the hemolysin A (HlyA) T1SS of Escherichia coli. This multiple membrane protein complex is able to secrete the toxin HlyA in one step across both E. coli membranes. Common to all secreted T1SS substrates is a C-terminal secretion sequence being necessary as well as sufficient for secretion. However, it is not known whether transport occurs directionally, i.e. the N- or the C-terminus of T1SS substrates is secreted first. We have addressed this question by constructing HlyA fusions with the rapidly folding eGFP resulting in a stalled T1SS. Differential labeling and subsequent fluorescence microscopic detection of C- and N-terminal parts of the fusions allowed us to demonstrate vectorial transport of HlyA through the T1SS with the C-terminus appearing first outside the bacterial cells. PMID:26212107

  20. Staphylococcus aureus and Pseudomonas aeruginosa express and secrete human surfactant proteins.

    PubMed

    Bräuer, Lars; Schicht, Martin; Worlitzsch, Dieter; Bensel, Tobias; Sawers, R Gary; Paulsen, Friedrich

    2013-01-01

    Surfactant proteins (SP), originally known from human lung surfactant, are essential to proper respiratory function in that they lower the surface tension of the alveoli. They are also important components of the innate immune system. The functional significance of these proteins is currently reflected by a very large and growing number of publications. The objective goal of this study was to elucidate whether Staphylococcus aureus and Pseudomonas aeruginosa is able to express surfactant proteins. 10 different strains of S. aureus and P. aeruginosa were analyzed by means of RT-PCR, Western blot analysis, ELISA, immunofluorescence microscopy and immunoelectron microscopy. The unexpected and surprising finding revealed in this study is that different strains of S. aureus and P. aeruginosa express and secrete proteins that react with currently commercially available antibodies to known human surfactant proteins. Our results strongly suggest that the bacteria are either able to express 'human-like' surfactant proteins on their own or that commercially available primers and antibodies to human surfactant proteins detect identical bacterial proteins and genes. The results may reflect the existence of a new group of bacterial surfactant proteins and DNA currently lacking in the relevant sequence and structure databases. At any rate, our knowledge of human surfactant proteins obtained from immunological and molecular biological studies may have been falsified by the presence of bacterial proteins and DNA and therefore requires critical reassessment. PMID:23349731

  1. Staphylococcus aureus and Pseudomonas aeruginosa Express and Secrete Human Surfactant Proteins

    PubMed Central

    Worlitzsch, Dieter; Bensel, Tobias; Sawers, R. Gary; Paulsen, Friedrich

    2013-01-01

    Surfactant proteins (SP), originally known from human lung surfactant, are essential to proper respiratory function in that they lower the surface tension of the alveoli. They are also important components of the innate immune system. The functional significance of these proteins is currently reflected by a very large and growing number of publications. The objective goal of this study was to elucidate whether Staphylococcus aureus and Pseudomonas aeruginosa is able to express surfactant proteins. 10 different strains of S. aureus and P. aeruginosa were analyzed by means of RT-PCR, Western blot analysis, ELISA, immunofluorescence microscopy and immunoelectron microscopy. The unexpected and surprising finding revealed in this study is that different strains of S. aureus and P. aeruginosa express and secrete proteins that react with currently commercially available antibodies to known human surfactant proteins. Our results strongly suggest that the bacteria are either able to express ‘human-like’ surfactant proteins on their own or that commercially available primers and antibodies to human surfactant proteins detect identical bacterial proteins and genes. The results may reflect the existence of a new group of bacterial surfactant proteins and DNA currently lacking in the relevant sequence and structure databases. At any rate, our knowledge of human surfactant proteins obtained from immunological and molecular biological studies may have been falsified by the presence of bacterial proteins and DNA and therefore requires critical reassessment. PMID:23349731

  2. The Rab11 Effector Protein FIP1 Regulates Adiponectin Trafficking and Secretion

    PubMed Central

    Moreno-Navarrete, Jose Maria; Fernandez-Real, Jose Manuel; Mora, Silvia

    2013-01-01

    Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release. PMID:24040321

  3. Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata

    SciTech Connect

    Lee, E.Y.H.; Parry, G.; Bissell, M.J.

    1984-01-01

    It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce ..gamma..-casein, a member of the casein gene family, only if they are maintained on floating collagen gels. In this paper we show: (a) Cells on floating collagen gels secrete not only ..gamma..-casein but also ..cap alpha../sub 1/-, ..cap alpha../sub 2/-, and ..beta..-caseins. These are not secreted by cells on plastic and are secreted to only a very limited extent by cells on attached collagen gels. (b) The floating collagen gel regulates at the level of synthesis and/or stabilization of the caseins rather than at the level of secretion alone. Contraction of the floating gel is important in that cells cultured on floating glutaraldehyde cross-linked gels do not secrete any of the caseins. (c) The secretion of an 80,000-mol-wt protein, most probably transferrin, and a 67,000-mol-wt protein, probably butyrophilin, a major protein of the milk fat globule membrane, are partially modulated by substrata. However, in contrast to the caseins, these are always detectable in media from cells cultured on plastic and attached gels. (d) Whey acidic protein, a major whey protein, is actively secreted by freshly isolated cells but is secreted in extremely limited quantities in cultured cells regardless of the nature of the substratum used. Lactalbumin secretion is also decreased significantly in cultured cells. (e) A previously unreported set of proteins, which may be minor milk proteins, are prominently secreted by the mammary cells on all substrata tested. We conclude that while the substratum profoundly influences the secretion of the caseins, it does not regulate the expression of every milk-specific protein in the same way. The mechanistic implications of these findings are discussed.

  4. Characterisation of novel protein families secreted by muscle stage larvae of Trichinella spiralis☆

    PubMed Central

    Guiliano, David B.; Oksov, Yelena; Lustigman, Sara; Gounaris, Kleoniki; Selkirk, Murray E.

    2009-01-01

    Proteins secreted by Trichinella spiralis have a potential role in remodelling host skeletal muscle. However, whilst many parasite-secreted proteins have been identified, it has rarely been demonstrated that these are secreted into the nurse cell. Using an informatics-based analysis, we have searched the T. spiralis expressed sequence tag (EST) datasets for cDNAs encoding potential secreted proteins. Here we describe the characterisation of three of the top candidates isolated from our analysis, termed secreted from muscle stage larvae (SML)-1, -2 and -3. All three proteins were demonstrated to be secreted by muscle stage larvae, and immunohistochemical analysis established that SML-1 and -2 are secreted into developing nurse cells. We also show that SML-2 is processed from a precursor into smaller peptides by a metalloprotease contained within T. spiralis-secreted products. With the identification of these and other secreted proteins, we now have molecules to test in functional assays designed to dissect molecular features of the developing nurse cell. PMID:18992250

  5. Protein-Phospholipid Interactions in Nonclassical Protein Secretion: Problem and Methods of Study

    PubMed Central

    Prudovsky, Igor; Kumar, Thallapuranam Krishnaswamy Suresh; Sterling, Sarah; Neivandt, David

    2013-01-01

    Extracellular proteins devoid of signal peptides use nonclassical secretion mechanisms for their export. These mechanisms are independent of the endoplasmic reticulum and Golgi. Some nonclassically released proteins, particularly fibroblast growth factors (FGF) 1 and 2, are exported as a result of their direct translocation through the cell membrane. This process requires specific interactions of released proteins with membrane phospholipids. In this review written by a cell biologist, a structural biologist and two membrane engineers, we discuss the following subjects: (i) Phenomenon of nonclassical protein release and its biological significance; (ii) Composition of the FGF1 multiprotein release complex (MRC); (iii) The relationship between FGF1 export and acidic phospholipid externalization; (iv) Interactions of FGF1 MRC components with acidic phospholipids; (v) Methods to study the transmembrane translocation of proteins; (vi) Membrane models to study nonclassical protein release. PMID:23396106

  6. The ESAT-6/CFP-10 secretion system of Mycobacterium marinum modulates phagosome maturation.

    PubMed

    Tan, Tracy; Lee, Warren L; Alexander, David C; Grinstein, Sergio; Liu, Jun

    2006-09-01

    Virulence of Mycobacterium tuberculosis and related pathogenic mycobacteria requires the secretion of early secretory antigenic 6 kDa (ESAT-6) and culture filtrate protein 10 (CFP-10), two small proteins that lack traditional signal sequences and are exported through an alternative secretion pathway encoded primarily by the RD1 genetic locus. Mutations affecting the synthesis or secretion of ESAT-6 or CFP-10 attenuate the virulence of M. tuberculosis in murine models of infection. However, the specific functions of these proteins and of their secretion system are currently unclear. In this study, we isolated a mutant of Mycobacterium marinum defective in the secretion of ESAT-6 and CFP-10. The mutation was localized within MM5446, which is orthologous to Rv3871 of M. tuberculosis H37Rv and encodes an ATPase that is a component of the ESAT-6/CFP-10 secretion system. The mutant bacteria were unable to replicate within J774 macrophages although their growth in 7H9 medium was equivalent to the parental strain. Phagosome maturation and acidification were analysed in infected macrophages by confocal and electron microscopy using the late endosome/lysosome marker LAMP-1, along with various fluid-phase markers such as rhodamine-dextran and ferritin and the acidotropic dye LysoTracker Red. These studies demonstrated that while the wild-type parental strain of M. marinum primarily resides in a poorly acidified, non-lysosomal compartment, a significantly higher percentage of the MM5446 mutant organisms are in acidified compartments. These results suggest that the ESAT-6/CFP-10 secretion system plays a role in preventing phagolysosomal fusion, a novel function that accounts for the ability of bacteria to survive inside host cells. This finding provides a mechanism by which the ESAT-6/CFP-10 secretion system potentiates the virulence of pathogenic mycobacteria. PMID:16922861

  7. Expression, secretion and bactericidal activity of type VI secretion system in Vibrio anguillarum.

    PubMed

    Tang, Lei; Yue, Shu; Li, Gui-Yang; Li, Jie; Wang, Xiao-Ran; Li, Shu-Fang; Mo, Zhao-Lan

    2016-10-01

    The type VI secretion system (T6SS) was recently shown to modulate quorum sensing and the stress response in Vibrio anguillarum serotype O1 strain NB10. It is not known whether there is a functionally active T6SS in other serotypes of V. anguillarum. Here, homologues to T6SS cluster VtsEFGH and hemolysin-coregulated protein (Hcp)-encoding genes were found to be prevalent and conserved in clinical isolates of V. anguillarum from fish, including four O1 and five non-O1 serotype strains. Unexpectedly, only the non-O1 serotype strains expressed VtsEFGH and Hcp under laboratory and marine-like conditions, in contrast to the serotype O1 strains. This suggested that the V. anguillarum non-O1 serotype strains tested have constitutive expression of T6SS. Examination of a representative non-O1 strain, MHK3, showed that Hcp production was growth phase dependent and that maximum Hcp production was observed in the exponential growth phase. Moreover, Hcp production by MHK3 was most active under warm marine-like conditions. Further examination revealed a correlation of the constitutive expression of T6SS with bactericidal activity against Escherichia coli and Edwardsiella tarda. The work presented here suggests that the constitutive expression of T6SS provides V. anguillarum with advantage in microbial competition in marine environments. PMID:27172981

  8. Association and Evidence for Linked Recognition of Type IV Secretion System Proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale

    PubMed Central

    Morse, Kaitlyn; Norimine, Junzo; Palmer, Guy H.; Sutten, Eric L.; Baszler, Timothy V.

    2012-01-01

    Like several other bacterial pathogens, Anaplasma marginale has an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen. A. marginale T4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4+ T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine against A. marginale. PMID:22038917

  9. A functional equivalent of endoplasmic reticulum and Golgi in axons for secretion of locally synthesized proteins

    PubMed Central

    Merianda, Tanuja T.; Lin, Andrew C.; Lam, Joyce S.Y.; Vuppalanchi, Deepika; Willis, Dianna E.; Karin, Norman; Holt, Christine E.; Twiss, Jeffery L.

    2013-01-01

    Subcellular localization of protein synthesis provides a means to regulate the protein composition in far reaches of a cell. This localized protein synthesis gives neuronal processes autonomy to rapidly respond to extracellular stimuli. Locally synthesized axonal proteins enable neurons to respond to guidance cues and can help to initiate regeneration after injury. Most studies of axonal mRNA translation have concentrated on cytoplasmic proteins. While ultrastructural studies suggest that axons do not have rough endoplasmic reticulum or Golgi apparatus, mRNAs for transmembrane and secreted proteins localize to axons. Here, we show that growing axons with protein synthetic activity contain ER and Golgi components needed for classical protein synthesis and secretion. Isolated axons have the capacity to traffic locally synthesized proteins into secretory pathways and inhibition of Golgi function attenuates translation-dependent axonal growth responses. Finally, the capacity for secreting locally synthesized proteins in axons appears to be increased by injury. PMID:19022387

  10. The Evolution of the Secreted Regulatory Protein Progranulin.

    PubMed

    Palfree, Roger G E; Bennett, Hugh P J; Bateman, Andrew

    2015-01-01

    Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide

  11. The Evolution of the Secreted Regulatory Protein Progranulin

    PubMed Central

    Palfree, Roger G. E.; Bennett, Hugh P. J.; Bateman, Andrew

    2015-01-01

    Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide

  12. Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress1[W

    PubMed Central

    Barba-Espín, Gregorio; Dedvisitsakul, Plaipol; Hägglund, Per; Svensson, Birte; Finnie, Christine

    2014-01-01

    The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the aleurone layer as a model system for studying plant protein secretion. PMID:24344171

  13. Trade Secret Law and Information Systems: Can Your Students Keep a Secret?

    ERIC Educational Resources Information Center

    Willey, Lorrie; Ford, Janet C.; White, Barbara Jo; Clapper, Danial L.

    2011-01-01

    The impact of intellectual property (IP) law on information systems (IS) professionals in business cannot be overstated. The IS 2010 model curriculum guidelines for undergraduate IS programs stress the importance of information security and knowledge about IP. While copyright and patents are the most well-known types of IP, another, trade secrets,…

  14. Licoflavonol is an inhibitor of the type three secretion system of Salmonella enterica serovar Typhimurium.

    PubMed

    Guo, Zhixing; Li, Xiaoli; Li, Jianfang; Yang, Xuefei; Zhou, Yuan; Lu, Chunhua; Shen, Yuemao

    2016-09-01

    As an important food-borne human pathogen, Salmonella enterica serovar Typhimurium depends on its type III secretion system (T3SS) as a major virulence factor to cause disease all over the world. The T3SS secretes effector proteins to facilitate invasion into host cells. In this study, twenty prenylated flavonoids (1-20) were screened for their anti-T3SS activity, revealing that several analogs exhibited strong inhibitory effects on the secretion of Salmonella pathogenicity island 1 (SPI-1)-associated effector proteins without affecting the growth of bacteria and the secretion of the flagellar protein FliC. Among the flavonoids 1-20, licoflavonol (20) exhibited a strong inhibitory effect on the secretion of the SPI-1 effector proteins via regulating the transcription of the SicA/InvF genes, and the transportation of the effector protein SipC. In summary, licoflavonol, a novel natural inhibitor of Salmonella T3SS, could be a promising candidate for novel type of anti-virulence drugs. PMID:27387231

  15. Large-Scale Cultivation of Acidophilic Hyperthermophiles for Recovery of Secreted Proteins

    PubMed Central

    Worthington, Penny; Blum, Paul; Perez-Pomares, Francisco; Elthon, Tom

    2003-01-01

    An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins. Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system. These alterations provided accurate temperature and pH control. The bioreactor was used to cultivate selected strains of the archaeon Sulfolobus solfataricus and other species within this genus. Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium components. Substrate induction of synthesis of the S. solfataricus-secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes. An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant. These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins. PMID:12514002

  16. Peripheral nerve axons contain machinery for co-translational secretion of axonally-generated proteins.

    PubMed

    Merianda, Tanuja; Twiss, Jeffery

    2013-08-01

    The axonal compartment of developing neurons and mature peripheral nervous system (PNS) neurons has the capacity to locally synthesize proteins. Axonally-synthesized proteins have been shown to facilitate axonal pathfinding and maintenance in developing central nervous system (CNS) and PNS neurons, and to facilitate the regeneration of mature PNS neurons. RNA-profiling studies of the axons of cultured neurons have shown a surprisingly complex population of mRNAs that encode proteins for a myriad of functions. Although classic-appearing rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (ER) and Golgi apparatus have not been documented in axons by ultrastructural studies, axonal RNA profiling studies show several membrane and secreted protein-encoding mRNAs whose translation products would need access to a localized secretory mechanism. We previously showed that the axons of cultured neurons contain functional equivalents of RER and Golgi apparatus. Here, we show that markers for the signal-recognition particle, RER, ER, and Golgi apparatus are present in PNS axons in vivo. Co-localization of these proteins mirrors that seen for cultured axons where locally-translated proteins are localized to the axoplasmic membrane. Moreover, nerve injury increases the levels and/or aggregation of these proteins, suggesting that the regenerating axon has an increased capacity for membrane targeting of locally synthesized proteins. PMID:23839054

  17. Type VI secretion system sheaths as nanoparticles for antigen display

    PubMed Central

    Del Tordello, Elena; Danilchanka, Olga; McCluskey, Andrew J.; Mekalanos, John J.

    2016-01-01

    The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between cells by a mechanism analogous to phage tail contraction. T6SS sheaths are cytoplasmic tubular structures composed of stable VipA-VipB (named for ClpV-interacting protein A and B) heterodimers. Here, the structure of the VipA/B sheath was exploited to generate immunogenic multivalent particles for vaccine delivery. Sheaths composed of VipB and VipA fused to an antigen of interest were purified from Vibrio cholerae or Escherichia coli and used for immunization. Sheaths displaying heterologous antigens generated better immune responses against the antigen and different IgG subclasses compared with soluble antigen alone. Moreover, antigen-specific antibodies raised against sheaths presenting Neisseria meningitidis factor H binding protein (fHbp) antigen were functional in a serum bactericidal assay. Our results demonstrate that multivalent nanoparticles based on the T6SS sheath represent a versatile scaffold for vaccine applications. PMID:26929342

  18. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris.

    PubMed

    Scorer, C A; Buckholz, R G; Clare, J J; Romanos, M A

    1993-12-22

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120 (ENV), is required in large quantities for immunological studies and as a potential vaccine component. We have expressed the DNA encoding gp120 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. The native gene was found to contain a sequence which resembled a Saccharomyces cerevisiae polyadenylation consensus and acted as a premature polyadenylation site in P. pastoris, resulting in the production of truncated mRNA. As full-length mRNA was produced in S. cerevisiae, this indicates differences in mRNA 3'-end formation between the two yeasts. Inactivation of this site by site-directed mutagenesis revealed several additional fortuitous polyadenylation sites within the gene. We have designed and constructed a 69%-synthetic gene with increased G + C content which overcomes this transcriptional problem, giving rise to full-length mRNA. High levels of intracellular, insoluble, unglycosylated ENV were produced [1.25 mg/ml in high-density (2 x 10(10) cells per ml) fermentations]. ENV also was secreted from P. pastoris using the S. cerevisiae alpha-factor prepro secretion leader and the S. cerevisiae invertase signal sequence. However, a high proportion of the secreted product was found to be hyperglycosylated, in contrast to other foreign proteins secreted from P. pastoris. There also was substantial proteolytic degradation, but this was minimized by maintaining a low pH on induction. Insoluble, yeast-derived ENV proteins are being considered as vaccine antigens and the P. pastoris system offers an efficient method of production. PMID:8293993

  19. A bacterial type III secretion-based protein delivery tool for broad applications in cell biology

    PubMed Central

    Ittig, Simon J.; Schmutz, Christoph; Kasper, Christoph A.; Amstutz, Marlise; Schmidt, Alexander; Sauteur, Loïc; Vigano, M. Alessandra; Low, Shyan Huey; Affolter, Markus; Cornelis, Guy R.; Nigg, Erich A.

    2015-01-01

    Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network. PMID:26598622

  20. Characterization of secreted proteins in HepG2 and LO2 cells by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Lin, Juqiang; Ruan, Qiuyong; Liao, Fadian; Lin, Jinyong; Huang, Zufang; Liu, Nenrong; Chen, Rong

    2014-11-01

    Secreted proteins, the promising source of biomarkers for early detection and diagnosis of cancer, have received considerable attention. Raman spectroscopy and principal component analysis (PCA) were used to characterize the secreted proteins collected from the cell cultures of human hepatoma cell line HepG2 and normal human liver cell line LO2 in this paper. We found the major difference of secreted proteins Raman spectra between HepG2 and LO2 cells were in the range of 1200cm-1-1800cm-1. Compared with LO2 cells, some significant changes based on secondary structure of secreted proteins in HepG2 cells were observed, including the increase in the relative intensity of the band at 1004cm-1, 1445cm-1, 1674cm-1 and the decrease at 1074cm-1. These variations of Raman bands indicated that the species and conformation of secreted proteins in HepG2 cells changed. The measured Raman spectra of the two groups were separated into two distinct clusters with no overlap and high specificity and sensitivity by PCA. These results show that the combination of Raman spectroscopy and PCA analysis may be a powerful tool for distinguishing the secreted proteins between human hepatoma cells and normal human liver cells, provide a new thought to analyze the secreted proteins from cancer cells and find a novel cancer biomarker.

  1. Identification of the minimal region in lipase ABC transporter recognition domain of Pseudomonas fluorescens for secretion and fluorescence of green fluorescent protein

    PubMed Central

    2012-01-01

    Background TliA is a thermostable lipase secreted by the type 1 secretion system (T1SS) of Pseudomonas fluorescens. The secretion is promoted by its secretion/chaperone domain located near the C-terminus, which is composed mainly of four Repeat-in-Toxin (RTX) repeats. In order to identify the minimal region of TliA responsible for its secretion, five different copies of the secretion/chaperone domain, each involving truncated N-terminal residues and a common C-terminus, were acquired and named as lipase ABC transporter recognition domains (LARDs). Each LARD was fused to epidermal growth factor (EGF) or green fluorescent protein (GFP), and the secretion of EGF-LARD or GFP-LARD fusion proteins was assessed in Escherichia coli with ABC transporter. Results Among the fusion proteins, GFP or EGF with 105-residue LARD3 was most efficiently secreted. In addition, GFP-LARD3 emitted wild type GFP fluorescence. Structurally, LARD3 had the 4 RTX repeats exposed at the N-terminus, while other LARDs had additional residues prior to them or missed some of the RTX repeats. LARD3 was both necessary and sufficient for efficient secretion and maintenance of GFP fluorescence in E. coli, which was also confirmed in P. fluorescens and P. fluorescens ▵tliA, a knock-out mutant of tliA. Conclusion LARD3 was a potent secretion signal in T1SS for its fusion flanking RTX motif, which enhanced secretion and preserved the fluorescence of GFP. LARD3-mediated secretion in E. coli or P. fluorescens will enable the development of enhanced protein manufacturing factory and recombinant microbe secreting protein of interest in situ. PMID:22578275

  2. Hear no secrets, see no secrets, speak no secrets: secrecy in the Canadian drug approval system.

    PubMed

    Lexchin, J

    1999-01-01

    Systemic bias in the form of a lack of transparency in the operation of the Canadian drug regulatory agency, the Health Protection Branch, seriously undermines our knowledge of how well the agency is functioning. In recent years this secrecy has combined with deregulation, downsizing, and privatization to compromise safety and could lead to deleterious consequences in the way that drugs are being used. Finally, these forces are threatening the ability of the Health Protection Branch to set priorities for the overall system of drug regulation. This article provides concrete examples of each of these problems. The author then discusses why secrecy is so firmly entrenched in the regulatory approval system, and offers some suggestions on how to tackle this issue. PMID:10079402

  3. Rapid Temporal Dynamics of Transcription, Protein Synthesis, and Secretion during Macrophage Activation*

    PubMed Central

    Eichelbaum, Katrin; Krijgsveld, Jeroen

    2014-01-01

    Macrophages provide the first line of host defense with their capacity to react to an array of cytokines and bacterial components requiring tight regulation of protein expression and secretion to invoke a properly tuned innate immune response. To capture the dynamics of this system, we introduce a novel method combining pulsed stable isotope labeling with amino acids in cell culture (SILAC) with pulse labeling using the methionine analog azidohomoalanine that allows the enrichment of newly synthesized proteins via click-chemistry followed by their identification and quantification by mass spectrometry. We show that this permits the analysis of proteome changes on a rapid time scale, as evidenced by the detection of 4852 newly synthesized proteins after only a 20-min SILAC pulse. We have applied this methodology to study proteome response during macrophage activation in a time-course manner. We have combined this with full proteome, transcriptome, and secretome analyses, producing an integrative analysis of the first 3 h of lipopolysaccharide-induced macrophage activation. We observed the rapid induction of multiple processes well known to TLR4 signaling, as well as anti-inflammatory proteins and proteins not previously associated with immune response. By correlating transcriptional, translational, and secretory events, we derived novel mechanistic principles of processes specifically induced by lipopolysaccharides, including ectodomain shedding and proteolytic processing of transmembrane and extracellular proteins and protein secretion independent of transcription. In conclusion, we demonstrate that the combination of pulsed azidohomoalanine and pulsed SILAC permits the detailed characterization of proteomic events on a rapid time scale. We anticipate that this approach will be very useful in probing the immediate effects of cellular stimuli and will provide mechanistic insight into cellular perturbation in multiple biological systems. The data have been deposited

  4. Biophysical Characterization of the Type III Secretion Tip Proteins and the Tip Proteins Attached to Bacterium-Like Particles

    PubMed Central

    Choudhari, Shyamal P.; Chen, Xiaotong; Kim, Jae Hyun; van Roosmalen, Maarten L.; Greenwood, Jamie C.; Joshi, Sangeeta B.; Picking, William D.; Leenhouts, Kees; Middaugh, C. Russell; Picking, Wendy L.

    2014-01-01

    Bacterium-like particles (BLPs), derived from Lactococcus lactis, offer a self-adjuvanting delivery vehicle for subunit protein vaccines. Proteins can be specifically loaded onto the BLPs via a peptidoglycan anchoring domain (PA). In this study, the tip proteins IpaD, SipD and LcrV belonging to type three secretion systems of Shigella flexneri, Salmonella enterica and Yersinia enterocolitica, respectively, were fused to the PA and loaded onto the BLPs. Herein, we biophysically characterized these nine samples and condensed the spectroscopic results into three-index empirical phase diagrams (EPDs). The EPDs show distinctions between the IpaD/SipD and LcrV subfamilies of tip proteins, based on their physical stability, even upon addition of the PA. Upon attachment to the BLPs, the BLPs become defining moiety in the spectroscopic measurements, leaving the tip proteins to have a subtle yet modulating effect on the structural integrity of the tip proteins-BLPs binding. In summary, this work provides a comprehensive view of physical stability of the tip proteins and tip protein-BLPs and serves as a baseline for screening of excipients to increase the stability of the tip protein-BLPs for future vaccine formulation. PMID:24916512

  5. RTX proteins: a highly diverse family secreted by a common mechanism

    PubMed Central

    Linhartová, Irena; Bumba, Ladislav; Mašín, Jiří; Basler, Marek; Osička, Radim; Kamanová, Jana; Procházková, Kateřina; Adkins, Irena; Hejnová-Holubová, Jana; Sadílková, Lenka; Morová, Jana; Šebo, Peter

    2010-01-01

    Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca2+ ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest. PMID:20528947

  6. Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris.

    PubMed

    Moua, Pachai S; Gonzalez, Alfonso; Oshiro, Kristin T; Tam, Vivian; Li, Zhiguo Harry; Chang, Jennifer; Leung, Wilson; Yon, Amy; Thor, Der; Venkatram, Sri; Franz, Andreas H; Risser, Douglas D; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2016-08-01

    The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris. PMID:27079175

  7. EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles

    SciTech Connect

    Fan, Yao; Tan, Kemin; Chhor, Gekleng; Butler, Emily K.; Jedrzejczak, Robert P.; Missiakas, Dominique; Joachimiak, Andrzej

    2015-07-03

    The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.

  8. Cutting Edge: Regulation of Exosome Secretion by the Integral MAL Protein in T Cells.

    PubMed

    Ventimiglia, Leandro N; Fernández-Martín, Laura; Martínez-Alonso, Emma; Antón, Olga M; Guerra, Milagros; Martínez-Menárguez, José Angel; Andrés, Germán; Alonso, Miguel A

    2015-08-01

    Exosomes secreted by T cells play an important role in coordinating the immune response. HIV-1 Nef hijacks the route of exosome secretion of T cells to modulate the functioning of uninfected cells. Despite the importance of the process, the protein machinery involved in exosome biogenesis is yet to be identified. In this study, we show that MAL, a tetraspanning membrane protein expressed in human T cells, is present in endosomes that travel toward the plasma membrane for exosome secretion. In the absence of MAL, the release of exosome particles and markers was greatly impaired. This effect was accompanied by protein sorting defects at multivesicular endosomes that divert the exosomal marker CD63 to autophagic vacuoles. Exosome release induced by HIV-1 Nef was also dependent on MAL expression. Therefore, MAL is a critical element of the machinery for exosome secretion and may constitute a target for modulating exosome secretion by human T cells. PMID:26109641

  9. The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection.

    PubMed

    Weber, Mary M; Faris, Robert; van Schaik, Erin J; McLachlan, Juanita Thrasher; Wright, William U; Tellez, Andres; Roman, Victor A; Rowin, Kristina; Case, Elizabeth Di Russo; Luo, Zhao-Qing; Samuel, James E

    2016-09-01

    Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection. PMID:27324482

  10. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation.

    PubMed

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-08-27

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  11. A Dynamic Study of Protein Secretion and Aggregation in the Secretory Pathway

    PubMed Central

    Mossuto, Maria Francesca; Sannino, Sara; Mazza, Davide; Fagioli, Claudio; Vitale, Milena; Yoboue, Edgar Djaha; Anelli, Tiziana

    2014-01-01

    Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC. PMID:25279560

  12. A dynamic study of protein secretion and aggregation in the secretory pathway.

    PubMed

    Mossuto, Maria Francesca; Sannino, Sara; Mazza, Davide; Fagioli, Claudio; Vitale, Milena; Yoboue, Edgar Djaha; Sitia, Roberto; Anelli, Tiziana

    2014-01-01

    Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC. PMID:25279560

  13. Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus

    PubMed Central

    Parnasa, Rami; Nagar, Elad; Sendersky, Eleonora; Reich, Ziv; Simkovsky, Ryan; Golden, Susan; Schwarz, Rakefet

    2016-01-01

    Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms. PMID:27558743

  14. Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus.

    PubMed

    Parnasa, Rami; Nagar, Elad; Sendersky, Eleonora; Reich, Ziv; Simkovsky, Ryan; Golden, Susan; Schwarz, Rakefet

    2016-01-01

    Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms. PMID:27558743

  15. Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii.

    PubMed

    Brown, Kevin M; Lourido, Sebastian; Sibley, L David

    2016-04-29

    Microneme secretion is essential for motility, invasion, and egress in apicomplexan parasites. Although previous studies indicate that Ca(2+) and cGMP control microneme secretion, little is known about how these pathways are naturally activated. Here we have developed genetically encoded indicators for Ca(2+) and microneme secretion to better define the signaling pathways that regulate these processes in Toxoplasma gondii We found that microneme secretion was triggered in vitro by exposure to a single host protein, serum albumin. The natural agonist serum albumin induced microneme secretion in a protein kinase G-dependent manner that correlated with increased cGMP levels. Surprisingly, serum albumin acted independently of elevated Ca(2+) and yet it was augmented by artificial agonists that raise Ca(2+), such as ethanol. Furthermore, although ethanol elevated intracellular Ca(2+), it alone was unable to trigger secretion without the presence of serum or serum albumin. This dichotomy was recapitulated by zaprinast, a phosphodiesterase inhibitor that elevated cGMP and separately increased Ca(2+) in a protein kinase G-independent manner leading to microneme secretion. Taken together, these findings reveal that microneme secretion is centrally controlled by protein kinase G and that this pathway is further augmented by elevation of intracellular Ca(2.) PMID:26933037

  16. Transient fusion and selective secretion of vesicle proteins in Phytophthora nicotianae zoospores.

    PubMed

    Zhang, Weiwei; Blackman, Leila M; Hardham, Adrienne R

    2013-01-01

    Secretion of pathogen proteins is crucial for the establishment of disease in animals and plants. Typically, early interactions between host and pathogen trigger regulated secretion of pathogenicity factors that function in pathogen adhesion and host penetration. During the onset of plant infection by spores of the Oomycete, Phytophthora nicotianae, proteins are secreted from three types of cortical vesicles. Following induction of spore encystment, two vesicle types undergo full fusion, releasing their entire contents onto the cell surface. However, the third vesicle type, so-called large peripheral vesicles, selectively secretes a small Sushi domain-containing protein, PnCcp, while retaining a large glycoprotein, PnLpv, before moving away from the plasma membrane. Selective secretion of PnCcp is associated with its compartmentalization within the vesicle periphery. Pharmacological inhibition of dynamin function, purportedly in vesicle fission, by dynasore treatment provides evidence that selective secretion of PnCcp requires transient fusion of the large peripheral vesicles. This is the first report of selective protein secretion via transient fusion outside mammalian cells. Selective secretion is likely to be an important aspect of plant infection by this destructive pathogen. PMID:24392285

  17. Structure of the Type III Secretion Effector Protein ExoU in Complex with Its Chaperone SpcU

    PubMed Central

    Halavaty, Andrei S.; Borek, Dominika; Tyson, Gregory H.; Veesenmeyer, Jeff L.; Shuvalova, Ludmilla; Minasov, George; Otwinowski, Zbyszek

    2012-01-01

    Disease causing bacteria often manipulate host cells in a way that facilitates the infectious process. Many pathogenic gram-negative bacteria accomplish this by using type III secretion systems. In these complex secretion pathways, bacterial chaperones direct effector proteins to a needle-like secretion apparatus, which then delivers the effector protein into the host cell cytosol. The effector protein ExoU and its chaperone SpcU are components of the Pseudomonas aeruginosa type III secretion system. Secretion of ExoU has been associated with more severe infections in both humans and animal models. Here we describe the 1.92 Å X-ray structure of the ExoU–SpcU complex, a full-length type III effector in complex with its full-length cognate chaperone. Our crystallographic data allow a better understanding of the mechanism by which ExoU kills host cells and provides a foundation for future studies aimed at designing inhibitors of this potent toxin. PMID:23166655

  18. Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases.

    PubMed

    Shindo, Takayuki; Kaschani, Farnusch; Yang, Fan; Kovács, Judit; Tian, Fang; Kourelis, Jiorgos; Hong, Tram Ngoc; Colby, Tom; Shabab, Mohammed; Chawla, Rohini; Kumari, Selva; Ilyas, Muhammad; Hörger, Anja C; Alfano, James R; van der Hoorn, Renier A L

    2016-09-01

    Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes. PMID:27603016

  19. Unfolded protein response-induced ERdj3 secretion links ER stress to extracellular proteostasis

    PubMed Central

    Genereux, Joseph C; Qu, Song; Zhou, Minghai; Ryno, Lisa M; Wang, Shiyu; Shoulders, Matthew D; Kaufman, Randal J; Lasmézas, Corinne I; Kelly, Jeffery W; Wiseman, R Luke

    2015-01-01

    The Unfolded Protein Response (UPR) indirectly regulates extracellular proteostasis through transcriptional remodeling of endoplasmic reticulum (ER) proteostasis pathways. This remodeling attenuates secretion of misfolded, aggregation-prone proteins during ER stress. Through these activities, the UPR has a critical role in preventing the extracellular protein aggregation associated with numerous human diseases. Here, we demonstrate that UPR activation also directly influences extracellular proteostasis through the upregulation and secretion of the ER HSP40 ERdj3/DNAJB11. Secreted ERdj3 binds misfolded proteins in the extracellular space, substoichiometrically inhibits protein aggregation, and attenuates proteotoxicity of disease-associated toxic prion protein. Moreover, ERdj3 can co-secrete with destabilized, aggregation-prone proteins in a stable complex under conditions where ER chaperoning capacity is overwhelmed, preemptively providing extracellular chaperoning of proteotoxic misfolded proteins that evade ER quality control. This regulated co-secretion of ERdj3 with misfolded clients directly links ER and extracellular proteostasis during conditions of ER stress. ERdj3 is, to our knowledge, the first metazoan chaperone whose secretion into the extracellular space is regulated by the UPR, revealing a new mechanism by which UPR activation regulates extracellular proteostasis. PMID:25361606

  20. Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

    PubMed Central

    Singer, Hanna M.; Erhardt, Marc; Steiner, Andrew M.; Zhang, Min-Min; Yoshikami, Doju; Bulaj, Grzegorz; Olivera, Baldomero M.; Hughes, Kelly T.

    2012-01-01

    ABSTRACT The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA. PMID:22647788

  1. A Bacterial Pathogen uses Distinct Type III Secretion Systems to Alternate between Host Kingdom

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gram-negative bacterial pathogens of eukaryotes often secrete proteins directly into host cells via a needle-like protein channel called a ‘type III secretion system’ (T3SS). Bacteria that are adapted to either animal or plant hosts use phylogenetically distinct T3SSs for secreting proteins. Here, ...

  2. The Type VI secretion system - a widespread and versatile cell targeting system.

    PubMed

    Coulthurst, Sarah J

    2013-01-01

    The Type VI secretion system (T6SS) is the most recently described of the Gram-negative bacterial secretion systems and is widely distributed amongst diverse species. T6SSs are currently believed to be complex molecular machines which inject effector proteins into target cells and which incorporate a bacteriophage-like cell-puncturing device. T6SSs have been implicated in eukaryotic cell targeting and virulence in a range of important pathogens. More recently, 'antibacterial' T6SSs have been reported, which are used to efficiently target competitor bacterial cells by the injection of antibacterial toxins. Although it is clear that T6SSs can be deployed as versatile weapons to compete with other bacteria or attack simple or higher eukaryotes, much remains to be determined about this intriguing system. PMID:23542428

  3. The Agrobacterium rhizogenes GALLS Gene Encodes Two Secreted Proteins Required for Genetic Transformation of Plants▿

    PubMed Central

    Hodges, Larry D.; Lee, Lan-Ying; McNett, Henry; Gelvin, Stanton B.; Ream, Walt

    2009-01-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes are related pathogens that cause crown gall and hairy root diseases, which result from integration and expression of bacterial genes in the plant genome. Single-stranded DNA (T strands) and virulence proteins are translocated into plant cells by a type IV secretion system. VirD2 nicks a specific DNA sequence, attaches to the 5′ end, and pilots the DNA into plant cells. A. tumefaciens translocates single-stranded DNA-binding protein VirE2 into plant cells where it likely binds T strands and may aid in targeting them into the nucleus. Although some A. rhizogenes strains lack VirE2, they transfer T strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant for tumor formation. Unlike VirE2, full-length GALLS (GALLS-FL) contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. GALLS-FL and VirE2 contain nuclear localization signals (NLS) and secretion signals. Mutations in any of these domains abolish the ability of the GALLS gene to substitute for virE2. Here, we show that the GALLS gene encodes two proteins from one open reading frame: GALLS-FL and a protein comprised of the C-terminal domain, which initiates at an internal in-frame start codon. On some hosts, both GALLS proteins were required to substitute for VirE2. GALLS-FL tagged with yellow fluorescent protein localized to the nucleus of tobacco cells in an NLS-dependent manner. In plant cells, the GALLS proteins interacted with themselves, VirD2, and each other. VirD2 interacted with GALLS-FL and localized inside the nucleus, where its predicted helicase activity may pull T strands into the nucleus. PMID:18952790

  4. A bacterial pathogen uses distinct type III secretion systems to alternate between host kingdoms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (Pnss), the causative agent of Stewart’s bacterial wilt and...

  5. Contribution of Bordetella bronchiseptica Type III secretion system to respiratory disease in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The type III secretion system (TTSS) of gram negative bacteria allows injection of effector proteins directly into the cytosol of eukaryotic cells. Previous studies have demonstrated that the B. bronchiseptica TTSS plays a role in the persistent bacterial colonization of the trachea of m...

  6. Campylobacter fetus subspecies contain conserved type IV secretion systems on multiple genomic islands and plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The features contributing to the differences in pathogenicity of the C. fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode type IV secretion system (T4SS) and fic-domain (filamentation induced by cyclic AMP) proteins. In the genomes of ...

  7. The Type III secretion system of Gram-negative bacteria: a potential therapeutic target?

    PubMed

    Müller, Simone; Feldman, Mario F; Cornelis, Guy R

    2001-06-01

    Several pathogenic Gram-negative bacteria, including Salmonella, Shigella, Yersinia, Pseudomonas aeruginosa and enteropathogenic Escherichia coli harbour a complex attack system called 'Type III secretion' which is, in every case, an essential virulence determinant. This system, activated by contact with an eukaryotic cell membrane, allows bacteria to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane, to reach the cell's cytosol and destroy or subvert the host cell. The Type III virulence mechanism consists of a secretion apparatus, made up of about 25 proteins, and a set of effector proteins released by this apparatus. The mechanism of protein secretion is highly conserved among the different bacteria, although they cause a variety of diseases with different symptoms and severities, from fatal septicaemia to mild diarrhoea or from fulgurant diarrhoea to chronic infection of the lung. This review focuses on the proteins that make up the secretion machinery and examine if it could be a potential target for novel antimicrobials. PMID:12540268

  8. Protein kinase D negatively regulates hepatitis C virus secretion through phosphorylation of oxysterol-binding protein and ceramide transfer protein.

    PubMed

    Amako, Yutaka; Syed, Gulam H; Siddiqui, Aleem

    2011-04-01

    Hepatitis C virus (HCV) RNA replicates its genome on specialized endoplasmic reticulum modified membranes termed membranous web and utilizes lipid droplets for initiating the viral nucleocapsid assembly. HCV maturation and/or the egress pathway requires host sphingolipid synthesis, which occur in the Golgi. Ceramide transfer protein (CERT) and oxysterol-binding protein (OSBP) play a crucial role in sphingolipid biosynthesis. Protein kinase D (PKD), a serine/threonine kinase, is recruited to the trans-Golgi network where it influences vesicular trafficking to the plasma membrane by regulation of several important mediators via phosphorylation. PKD attenuates the function of both CERT and OSBP by phosphorylation at their respective Ser(132) and Ser(240) residues (phosphorylation inhibition). Here, we investigated the functional role of PKD in HCV secretion. Our studies show that HCV gene expression down-regulated PKD activation. PKD depletion by shRNA or inhibition by pharmacological inhibitor Gö6976 enhanced HCV secretion. Overexpression of a constitutively active form of PKD suppressed HCV secretion. The suppression by PKD was subverted by the ectopic expression of nonphosphorylatable serine mutant CERT S132A or OSBP S240A. These observations imply that PKD negatively regulates HCV secretion/release by attenuating OSBP and CERT functions by phosphorylation inhibition. This study identifies the key role of the Golgi components in the HCV maturation process. PMID:21285358

  9. Avian renal proximal tubule urate secretion is inhibited by cellular stress-induced AMP-activated protein kinase.

    PubMed

    Bataille, Amy M; Maffeo, Carla L; Renfro, J Larry

    2011-06-01

    Urate is a potent antioxidant at high concentrations but it has also been associated with a wide variety of health risks. Plasma urate concentration is determined by ingestion, production, and urinary excretion; however, factors that regulate urate excretion remain uncertain. The objective of this study was to determine whether cellular stress, which has been shown to affect other renal transport properties, modulates urate secretion in the avian renal proximal tubule. Chick kidney proximal tubule epithelial cell primary culture monolayers were used to study the transepithelial transport of radiolabeled urate. This model allowed examination of the processes, such as multidrug resistance protein 4 (Mrp4, Abcc4), which subserve urate secretion in a functional, intact, homologous system. Our results show that the recently implicated urate efflux transporter, breast cancer resistance protein (ABCG2), does not significantly contribute to urate secretion in this system. Exposure to a high concentration of zinc for 6 h induced a cellular stress response and a striking decrease in transepithelial urate secretion. Acute exposure to zinc had no effect on transepithelial urate secretion or isolated membrane vesicle urate transport, suggesting involvement of a cellular stress adaptation. Activation of AMP-activated protein kinase (AMPK), a candidate modulator of ATP-dependent urate efflux, by 5'-aminoimidazole-4-carboxamide 1-β-d-ribo-furanoside caused a decrease in urate secretion similar to that seen with zinc-induced cellular stress. This effect was prevented with the AMPK inhibitor compound C. Notably, the decrease in urate secretion seen with zinc-induced cellular stress was also prevented by compound C, implicating AMPK in regulation of renal uric acid excretion. PMID:21429974

  10. Proteins of the exocytotic core complex mediate platelet alpha-granule secretion. Roles of vesicle-associated membrane protein, SNAP-23, and syntaxin 4.

    PubMed

    Flaumenhaft, R; Croce, K; Chen, E; Furie, B; Furie, B C

    1999-01-22

    To understand the molecular basis of granule release from platelets, we examined the role of vesicle-associated membrane protein, SNAP-23, and syntaxin 4 in alpha-granule secretion. A vesicle-associated membrane protein, SNAP-23, and syntaxin 4 were detected in platelet lysate. These proteins form a SDS-resistant complex that disassembles upon platelet activation. To determine whether these proteins are involved in alpha-granule secretion, we developed a streptolysin O-permeabilized platelet model of alpha-granule secretion. Streptolysin O-permeabilized platelets released alpha-granules, as measured by surface expression of P-selectin, in response to Ca2+ up to 120 min after permeabilization. Incubation of streptolysin O-permeabilized platelets with an antibody directed against vesicle-associated membrane protein completely inhibited Ca2+-induced alpha-granule release. Tetanus toxin cleaved platelet vesicle-associated membrane protein and inhibited Ca2+-induced alpha-granule secretion from streptolysin O-permeabilized platelets. An antibody to syntaxin 4 also inhibited Ca2+-induced alpha-granule release by approximately 75% in this system. These results show that vesicle-associated membrane protein, SNAP-23, and syntaxin 4 form a heterotrimeric complex in platelets that disassembles with activation and demonstrate that alpha-granule release is dependent on vesicle SNAP receptor-target SNAP receptor (vSNARE-tSNARE) interactions. PMID:9891020

  11. Gliding Motility and Por Secretion System Genes Are Widespread among Members of the Phylum Bacteroidetes

    PubMed Central

    Zhu, Yongtao

    2013-01-01

    The phylum Bacteroidetes is large and diverse, with rapid gliding motility and the ability to digest macromolecules associated with many genera and species. Recently, a novel protein secretion system, the Por secretion system (PorSS), was identified in two members of the phylum, the gliding bacterium Flavobacterium johnsoniae and the nonmotile oral pathogen Porphyromonas gingivalis. The components of the PorSS are not similar in sequence to those of other well-studied bacterial secretion systems. The F. johnsoniae PorSS genes are a subset of the gliding motility genes, suggesting a role for the secretion system in motility. The F. johnsoniae PorSS is needed for assembly of the gliding motility apparatus and for secretion of a chitinase, and the P. gingivalis PorSS is involved in secretion of gingipain protease virulence factors. Comparative analysis of 37 genomes of members of the phylum Bacteroidetes revealed the widespread occurrence of gliding motility genes and PorSS genes. Genes associated with other bacterial protein secretion systems were less common. The results suggest that gliding motility is more common than previously reported. Microscopic observations confirmed that organisms previously described as nonmotile, including Croceibacter atlanticus, “Gramella forsetii,” Paludibacter propionicigenes, Riemerella anatipestifer, and Robiginitalea biformata, exhibit gliding motility. Three genes (gldA, gldF, and gldG) that encode an apparent ATP-binding cassette transporter required for F. johnsoniae gliding were absent from two related gliding bacteria, suggesting that the transporter may not be central to gliding motility. PMID:23123910

  12. A 64-kilodalton membrane protein of Bacillus subtilis covered by secreting ribosomes.

    PubMed Central

    Horiuchi, S; Tai, P C; Davis, B D

    1983-01-01

    The complexed (ribosome-bearing) membrane fraction of Bacillus subtilis contains several proteins (CM-proteins) that are virtually absent from the ribosome-free fraction and hence might be components of the apparatus of protein secretion. We have determined, by trypsin digestion and by labeling with a nonpenetrating reagent (diazoiodosulfanilic acid), the accessibility of four of these proteins on the two surfaces of the membrane, as exposed either in protoplasts or in inverted membrane vesicles. The 68-kilodalton protein is a transmembrane protein and the 45-kilodalton protein faces only the external surface, whereas the 31-kilodalton protein is inaccessible from either side. Of particular interest is the 64-kilodalton protein: it can be digested by trypsin, and can bind antibody, on the cytoplasmic surface, but only after the ribosomes have been released. This protein is thus evidently a component of the apparatus of protein secretion, closely covered by secreting ribosomes. Whether the other CM-proteins are also involved in protein secretion is uncertain. Images PMID:6407010

  13. The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

    PubMed Central

    Whitaker, Neal; Chen, Yuqing; Jakubowski, Simon J.; Sarkar, Mayukh K.; Li, Feng

    2015-01-01

    ABSTRACT Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD), and a sequence-variable helical bundle called the all-alpha domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Here, we defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer, whereas production of the AAD in otherwise wild-type donor strains diminished the transfer of cognate but not heterologous substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished the transfer of cognate DNA substrates, although strikingly, the VirD4-AADPcfC chimera (VirD4 with the PcfC AAD) supported the transfer of a mobilizable plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 and PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Our findings support a model in which the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. IMPORTANCE For conjugative transfer of mobile DNA elements, members of the VirD4/TraG/TrwB receptor superfamily bind cognate DNA substrates through mechanisms that are

  14. PscI is a type III secretion needle anchoring protein with in vitro polymerization capacities.

    PubMed

    Monlezun, Laura; Liebl, David; Fenel, Daphna; Grandjean, Teddy; Berry, Alice; Schoehn, Guy; Dessein, Rodrigue; Faudry, Eric; Attree, Ina

    2015-04-01

    The export of bacterial toxins across the bacterial envelope requires the assembly of complex, membrane-embedded protein architectures. Pseudomonas aeruginosa employs type III secretion (T3S) injectisome to translocate exotoxins directly into the cytoplasm of a target eukaryotic cell. This multi-protein channel crosses two bacterial membranes and extends further as a needle through which the proteins travel. We show in this work that PscI, proposed to form the T3S system (T3SS) inner rod, possesses intrinsic properties to polymerize into flexible and regularly twisted fibrils and activates IL-1β production in mouse bone marrow macrophages in vitro. We also found that point mutations within C-terminal amphipathic helix of PscI alter needle assembly in vitro and T3SS function in cell infection assays, suggesting that this region is essential for an efficient needle assembly. The overexpression of PscF partially compensates for the absence of the inner rod in PscI-deficient mutant by forming a secretion-proficient injectisome. All together, we propose that the polymerized PscI in P. aeruginosa optimizes the injectisome function by anchoring the needle within the envelope-embedded complex of the T3S secretome and - contrary to its counterpart in Salmonella - is not involved in substrate switching. PMID:25614137

  15. Campylobacter jejuni survival within human epithelial cells is enhanced by the secreted protein CiaI

    PubMed Central

    Buelow, Daelynn R.; Christensen, Jeffrey E.; Neal-McKinney, Jason M.; Konkel, Michael E.

    2011-01-01

    Summary Although it is known that Campylobacter jejuni invade the cells that line the human intestinal tract, the bacterial proteins that enable this pathogen to survive within Campylobacter-containing vacuoles (CCV) have not been identified. Here, we describe the identification and characterization of a protein that we termed CiaI for Campylobacter invasion antigen involved in Intracellular survival. We show that CiaI harbors an amino-terminal type III secretion (T3S) sequence and is secreted from C. jejuni through the flagellar T3S system. In addition, the ciaI mutant was impaired in intracellular survival when compared to a wild-type strain, as judged by the gentamicin-protection assay. Fluorescence microscopy examination of epithelial cells infected with the C. jejuni ciaI mutant revealed that the CCV were more frequently co-localized with Cathepsin D (a lysosomal marker) than the CCV in cells infected with a C. jejuni wild-type strain. Ectopic expression of CiaI-GFP in epithelial cells yielded a punctate phenotype not observed with the other C. jejuni genes, and this phenotype was abolished by mutation of a dileucine motif located in the carboxy-terminus of the protein. Based on the data, we conclude that CiaI contributes to the ability of C. jejuni to survive within epithelial cells. PMID:21435039

  16. A bacterial type III secretion assay for delivery of fungal effector proteins into wheat.

    PubMed

    Upadhyaya, Narayana M; Mago, Rohit; Staskawicz, Brian J; Ayliffe, Michael A; Ellis, Jeffrey G; Dodds, Peter N

    2014-03-01

    Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector-Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat. PMID:24156769

  17. Secreted and immunogenic proteins produced by the honeybee bacterial pathogen, Paenibacillus larvae.

    PubMed

    Antúnez, Karina; Anido, Matilde; Evans, Jay D; Zunino, Pablo

    2010-03-24

    American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae during larval infection. The aim of the present work was to perform a first approach to the identification and characterization of P. larvae secretome. P. larvae secreted proteins were analyzed by SDS-PAGE and identified by MALDI-TOF. Protein toxicity was evaluated using an experimental model based on feeding of A. mellifera larvae and immunogenicity was evaluated by Western blot, using an antiserum raised against cells and spores of P. larvae. Ten different proteins were identified among P. larvae secreted proteins, including proteins involved in transcription, metabolism, translation, cell envelope, transport, protein folding, degradation of polysaccharides and motility. Although most of these proteins are cytosolic, many of them have been previously detected in the extracellular medium of different Bacillus spp. cultures and have been related to virulence. The secreted proteins resulted highly toxic and immunogenic when larvae were exposed using an experimental model. This is the first description of proteins secreted by the honeybee pathogen P. larvae. This information may be relevant for the elucidation of bacterial pathogenesis mechanisms. PMID:19781868

  18. Studies on the Golgi apparatus. Cumulative inhibition of protein and glycoprotein secretion by d-galactosamine

    PubMed Central

    Bauer, Christian H.; Lukaschek, Rainer; Reutter, Werner G.

    1974-01-01

    1. The administration of d-galactosamine leads to inhibition of protein and glycoprotein secretion by rat liver. To test the secretory function, the secretion times for galactose-and fucose-containing glycoproteins were determined; they were lengthened from 6 to 9min and from 8 to 13min respectively. 2. The Golgi apparatus was enriched 100–120-fold relative to the homogenate. A new linked-assay system for the marker enzyme, UDP-galactose–N-acetyl-d-glucosamine galactosyltransferase, is presented. The activity of the enzyme was measured spectrophotometrically by following the formation of UDP coupled to nicotinamide nucleotide reduction. The Michaelis constants were calculated to be 0.11mm for UDP-galactose with N-acetyl-d-glucosamine as exogenous acceptor and 19mm for N-acetyl-d-glucosamine itself. 3. The physiological substrate of the galactosyltransferase, UDP-galactose, can be replaced by UDP-galactosamine, which accumulates after d-galactosamine administration. Under conditions in vitro the rate of d-galactosamine transfer to an endogenous acceptor protein of the Golgi fraction reaches 9% of that with d-galactose; this finding is noteworthy, because normally a non-acetylated amino sugar does not occur in glycoproteins. 4. The albumin content of the Golgi-rich fraction was diminished to 55% of the reference value 6h after the injection of 375mg of d-galactosamine hydrochloride/kg body wt. The transfer of d-[1-14C]galactose to an endogenous acceptor protein fell to 60% compared with Golgi-rich fractions from untreated animals. Analysis of the Golgi-rich fraction by polyacrylamide-gel electrophoresis showed a decrease or loss of several protein bands. 5. Protein synthesis can be restored by up to 80% if the UTP pool, decreased after d-galactosamine administration, is filled up by several injections of uridine. 6. From the results presented it can be concluded that the disturbed secretion of proteins and glycoproteins was due to a cumulative effect of

  19. Live attenuated Salmonella vaccines against Mycobacterium tuberculosis with antigen delivery via the type III secretion system.

    PubMed

    Juárez-Rodríguez, María Dolores; Arteaga-Cortés, Lourdes T; Kader, Rebin; Curtiss, Roy; Clark-Curtiss, Josephine E

    2012-02-01

    Tuberculosis remains a global health threat, and there is dire need to develop a vaccine that is safe and efficacious and confers long-lasting protection. In this study, we constructed recombinant attenuated Salmonella vaccine (RASV) strains with plasmids expressing fusion proteins consisting of the 80 amino-terminal amino acids of the type 3 secretion system effector SopE of Salmonella and the Mycobacterium tuberculosis antigens early secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10). We demonstrated that the SopE-mycobacterial antigen fusion proteins were translocated into the cytoplasm of INT-407 cells in cell culture assays. Oral immunization of mice with RASV strains synthesizing SopE-ESAT-6-CFP-10 fusion proteins resulted in significant protection of the mice against aerosol challenge with M. tuberculosis H37Rv that was similar to the protection afforded by immunization with Mycobacterium bovis bacillus Calmette-Guérin (BCG) administered subcutaneously. In addition, oral immunization with the RASV strains specifying these mycobacterial antigens elicited production of significant antibody titers to ESAT-6 and production of ESAT-6- or CFP-10-specific gamma interferon (IFN-γ)-secreting and tumor necrosis factor alpha (TNF-α)-secreting splenocytes. PMID:22144486

  20. An Optimized Approach to Recover Secreted Proteins from Fibroblast Conditioned-Media for Secretomic Analysis

    PubMed Central

    Paré, Bastien; Deschênes, Lydia T.; Pouliot, Roxane; Dupré, Nicolas; Gros-Louis, Francois

    2016-01-01

    The proteins secreted by a particular type of cell, the secretome, play important roles in the regulation of many physiological processes via paracrine/autocrine mechanisms, and they are of increasing interest to help understanding rare diseases and to identify potential biomarkers and therapeutic targets. To facilitate ongoing research involving secreted proteins, we revisited cell culture protocols and whole secreted protein enrichment protocols. A reliable method for culturing and precipitating secreted protein from patient-derived fibroblast conditioned-medium was established. The method is based on the optimization of cell confluency and incubation time conditions. The well-established carrier-based TCA-DOC protein precipitation method was consistently found to give higher protein recovery yield. According to our results, we therefore propose that protein enrichment should be performed by TCA-DOC precipitation method after 48 h at 95% of confluence in a serum-deprived culture medium. Given the importance of secreted proteins as a source to elucidate the pathogenesis of rare diseases, especially neurological disorders, this approach may help to discover novel candidate biomarkers with potential clinical significance. PMID:27064649

  1. Secretion of a pneumococcal type II secretion system pilus correlates with DNA uptake during transformation

    PubMed Central

    Balaban, Murat; Bättig, Patrick; Muschiol, Sandra; Tirier, Stephan M.; Wartha, Florian; Normark, Staffan; Henriques-Normark, Birgitta

    2014-01-01

    Streptococcus pneumoniae is a major human pathogen that successfully adapts to the host environment via an efficient uptake system for free DNA liberated from other organisms in the upper respiratory tract, facilitating immune evasion and drug resistance. Although the initial signaling events leading to pneumococcal competence for DNA transformation and the fate of DNA when it has been taken up have been extensively studied, the actual mechanism by which DNA in the environment may traverse the thick capsular and cell wall layers remains unknown. Here we visualize that induction of competence results in the formation of a native morphologically distinct pilus structure on the bacterial surface. This plaited pilus is encoded by the competence (com)G locus, and, after assembly, it is rapidly released into the surrounding medium. Heterologous pneumococcal pilus expression in Escherichia coli was obtained by replacing the pulE-K putative pilin genes of the Klebsiella oxytoca type II secretion system with the complete comG locus. In the pneumococcus, the coordinated secretion of pili from the cells correlates to DNA transformation. A model for DNA transformation is proposed whereby pilus assembly “drills” a channel across the thick cell wall that becomes transiently open by secretion of the pilus, providing the entry port for exogenous DNA to gain access to DNA receptors associated with the cytoplasmic membrane. PMID:24550320

  2. Effect of phosphorus levels on the protein profiles of secreted protein and root surface protein of rice.

    PubMed

    Shinano, Takuro; Yoshimura, Tomoko; Watanabe, Toshihiro; Unno, Yusuke; Osaki, Mitsuru; Nanjo, Yohei; Komatsu, Setsuko

    2013-11-01

    Plant roots are complicated organs that absorb water and nutrients from the soil. Roots also play an essential role in protecting plants from attack by soil pathogens and develop a beneficial role with some soil microorganisms. Plant-derived rhizosphere proteins (e.g., root secretory proteins and root surface binding proteins) are considered to play important roles in developing mutual relationships in the rhizosphere. In the rhizosphere, where plant roots meet the surrounding environment, it has been suggested that root secretory protein and root surface binding protein are important factors. Furthermore, it is not known how the physiological status of the plant affects the profile of these proteins. In this study, rice plants were grown aseptically, with or without phosphorus nutrition, and proteins were obtained from root bathing solution (designated as root secretory proteins) and obtained using 0.2 M CaCl2 solution (designated as root surface binding proteins). The total number of identified proteins in the root bathing solution was 458, and the number of root surface binding proteins was 256. More than half of the proteins were observed in both fractions. Most of the proteins were categorized as either having signal peptides or no membrane transport helix sites. The functional categorization suggested that most of the proteins seemed to have secretory pathways and were involved in defense/disease-related functions. These characteristics seem to be unique to rhizosphere proteins, and the latter might be part of the plants strategy to defeat pathogens in the soil. The low phosphorus treatment significantly increased the number of pathogenesis-related proteins in the root secretory proteins, whereas the change was small in the case of the root surface binding proteins. The results suggested that the roots are actively and selectively secreting protein into the rhizosphere. PMID:24083427

  3. Expression and secretion of Aspergillus fumigatus proteases are regulated in response to different protein substrates

    PubMed Central

    Farnell, Edward; Rousseau, Karine; Thornton, David J.; Bowyer, Paul; Herrick, Sarah E.

    2012-01-01

    The ubiquitous filamentous fungus Aspergillus fumigatus secretes a number of allergens with protease activity and has been linked to a variety of allergic conditions such as Severe Asthma with Fungal Sensitization (SAFS) and Allergic Bronchopulmonary Aspergillosis (ABPA). However, it is unclear which allergen proteases are being secreted during fungal invasion and whether the local biological environment regulates their expression. Understanding the dynamic expression of allergen proteases during growth of A. fumigatus may lead to further characterisation of the pathogenesis of these disorders as well as improved standardisation in the commercial production of these allergens. Secretion of proteases during germination and early growth of A. fumigatus was investigated in response to various complex protein sources (pig lung homogenate, mucin or casein). Protease inhibitor studies demonstrated that A. fumigatus (AF293 strain) secretes predominately serine proteases during growth in pig lung based medium and mainly metalloproteases during growth in casein based medium but suppressed protease secretion in unmodified Vogel's minimal medium and secreted both types in mucin based medium. Analysis of gene transcription and protein identification by mass spectrometry showed that the matrix metalloprotease, Mep/Asp f 5 and the serine protease, Alp1/Asp f 13, were upregulated and secreted during growth in pig lung medium, whereas Alp1 was predominately expressed and secreted in mucin based medium. In casein medium, the matrix metalloprotease, Lap1, was also upregulated and secreted in addition to Mep and Alp1. These findings suggest that A. fumigatus is able to detect different complex proteins available as substrates in its environment and regulate protease secretion accordingly. There is a requirement for the standardisation of A. fumigatus allergen extracts used both in clinical diagnosis of A. fumigatus allergy and in research studies. PMID:22954343

  4. Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis

    PubMed Central

    Le Loir, Y.; Nouaille, S.; Commissaire, J.; Brétigny, L.; Gruss, A.; Langella, P.

    2001-01-01

    Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SPNuc) by that of L. lactis protein Usp45 (SPUsp) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SPUsp than when directed by SPNuc. This SPUsp effect on Nuc secretion is not due to a better antifolding activity, since SPUsp:Nuc precursor proteins display enzymatic activity in vitro, while SPNuc:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895–1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SPUsp and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis. PMID:11526014

  5. Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii

    PubMed Central

    Ribeiro, Orquídea; Magalhães, Frederico; Aguiar, Tatiana Q; Wiebe, Marilyn G; Penttilä, Merja; Domingues, Lucília

    2013-01-01

    To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities.   A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants. PMID:23644277

  6. Secret sharing with a single d -level quantum system

    NASA Astrophysics Data System (ADS)

    Tavakoli, Armin; Herbauts, Isabelle; Żukowski, Marek; Bourennane, Mohamed

    2015-09-01

    We give an example of a wide class of problems for which quantum-information protocols based on multisystem entanglement can be mapped into much simpler ones involving one system. Secret sharing is a cryptographic primitive which plays a central role in various secure multiparty computation tasks and management of keys in cryptography. In secret sharing protocols, a classical message is divided into shares given to recipient parties in such a way that some number of parties need to collaborate in order to reconstruct the message. Quantum protocols for the task commonly rely on multipartite GHZ entanglement. We present a multiparty secret sharing protocol which requires only sequential communication of a single quantum d -level system (for any prime d ). It has huge advantages in scalability and can be realized with state-of-the-art technology.

  7. Delivery of a secreted soluble protein to the vacuole via a membrane anchor

    SciTech Connect

    Barrieu, F.; Chrispeels, M.J.

    1999-08-01

    To further understand how membrane proteins are sorted in the secretory system, the authors devised a strategy that involves the expression of a membrane-anchored yeast invertase in transgenic plants. The construct consisted of a signal peptide followed by the coding region of yeast invertase and the transmembrane domain and cytoplasmic tail of calnexin. The substitution of a lysine near the C terminus of calnexin with a glutamic acid residue ensured progression through the secretory system rather than retention in or return to the endoplasmic reticulum. In the transformed plants, invertase activity and a 70-kD cross-reacting protein were found in the vacuoles. This yeast invertase had plant-specific complex glycans, indicating that transport to the vacuole was mediated by the Golgi apparatus. The microsomal fraction contained a membrane-anchored 90-kD cross-reacting polypeptide, but was devoid of invertase activity. Their results indicate that this membrane-anchored protein proceeds in the secretory system beyond the point where soluble proteins are sorted for secretion, and is detached from its membrane anchor either just before or just after delivery to the vacuole.

  8. Brucella outer membrane protein Omp25 induces microglial cells in vitro to secrete inflammatory cytokines and inhibit apoptosis.

    PubMed

    Ma, Qiao-Li; Liu, Ai-Cui; Ma, Xiao-Juan; Wang, Yan-Bai; Hou, Yu-Ting; Wang, Zhen-Hai

    2015-01-01

    Omp25 protein, an outer membrane protein of Brucella, can cause damage to the central nervous system. As one type of macrophage, microglial cells play a role in immune surveillance and immune protection in the central nervous system; therefore, they are major targets of bacterial attack. The present study examined BV2 mouse microglial cells that were stimulated with different concentrations of Omp25 recombinant protein, and the secretion of inflammatory cytokines by the BV2 cells as well as their level of apoptosis were observed. The objective of the study was to preliminarily illustrate the possible mechanism that Omp25 uses to damage the central nervous system. Mouse BV2 microglial cells were incubated with different concentrations of Omp25 for 24 h, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines interleukin (IL)-6, tumour necrosis factor (TNF)-α and HMGB1 (high mobility group box-1 protein); reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TLR4 (Toll-like receptor 4) mRNA; Annexin V-fluorescein isothiocyanate (FITC) double staining was used to detect apoptosis in the BV2 cells. After the BV2 cells were stimulated with different concentrations of Omp25, the levels of IL-6, TNF-α and HMGB1 was increased, and the difference was statistically significant compared with the control group (P<0.05). The secretion of TNF-α and HMGB1 showed a trend toward an initial increase followed by a decrease. The expression level of TLR4 mRNA was increased. Omp25 protein can inhibit apoptosis in BV2 cells. The outer membrane protein Omp25 of Brucella promotes microglial cells to secrete inflammatory cytokines and inhibit apoptosis. TLR4 may be involved in the immune response of the central nervous system to Brucella infection. PMID:26770344

  9. Brucella outer membrane protein Omp25 induces microglial cells in vitro to secrete inflammatory cytokines and inhibit apoptosis

    PubMed Central

    Ma, Qiao-Li; Liu, Ai-Cui; Ma, Xiao-Juan; Wang, Yan-Bai; Hou, Yu-Ting; Wang, Zhen-Hai

    2015-01-01

    Omp25 protein, an outer membrane protein of Brucella, can cause damage to the central nervous system. As one type of macrophage, microglial cells play a role in immune surveillance and immune protection in the central nervous system; therefore, they are major targets of bacterial attack. The present study examined BV2 mouse microglial cells that were stimulated with different concentrations of Omp25 recombinant protein, and the secretion of inflammatory cytokines by the BV2 cells as well as their level of apoptosis were observed. The objective of the study was to preliminarily illustrate the possible mechanism that Omp25 uses to damage the central nervous system. Mouse BV2 microglial cells were incubated with different concentrations of Omp25 for 24 h, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines interleukin (IL)-6, tumour necrosis factor (TNF)-α and HMGB1 (high mobility group box-1 protein); reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TLR4 (Toll-like receptor 4) mRNA; Annexin V-fluorescein isothiocyanate (FITC) double staining was used to detect apoptosis in the BV2 cells. After the BV2 cells were stimulated with different concentrations of Omp25, the levels of IL-6, TNF-α and HMGB1 was increased, and the difference was statistically significant compared with the control group (P<0.05). The secretion of TNF-α and HMGB1 showed a trend toward an initial increase followed by a decrease. The expression level of TLR4 mRNA was increased. Omp25 protein can inhibit apoptosis in BV2 cells. The outer membrane protein Omp25 of Brucella promotes microglial cells to secrete inflammatory cytokines and inhibit apoptosis. TLR4 may be involved in the immune response of the central nervous system to Brucella infection. PMID:26770344

  10. 76 FR 63316 - Prospective Grant of Exclusive License: Secreted Frizzled Related Protein-1 (sFRP-1) and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-12

    ... Related Protein-1 (sFRP-1) and derivatives thereof and their Use In Therapeutic Applications AGENCY... exclusive license relates to a protein designated secreted Frizzled Related Protein-1 (sFRP-1). sFRP-1, also known as SARP-2 (Secreted Apoptosis Related Protein-2). The IP covers various sFRP-1 compositions...

  11. Cytoskeletal proteins in gastric H/sup +/ secretion: cAMP dependent phosphorylation, immunolocalization, and protein blotting

    SciTech Connect

    Cuppoletti, J.; Sachs, G.; Malinowska, D.H.

    1986-05-01

    The rabbit gastric parietal cell is an excellent model for the study of regulation of secretion and the role of cytoskeleton in secretion. Changes in morphology (appearance of expanded secretory canaliculi lined with microvilli) accompany H/sup +/ secretion stimulated by histamine (cAMP mediated). Parietal cells contain immunoreactive tubulin and are highly enriched in F-actin at secretory canaliculi, detected with fluorescently labelled phallacidin. They have previously shown increased protein phosphorylation in histamine-stimulated purified parietal cells concommitant with increases in H/sup +/ secretion. They report here possible functions of the phosphoproteins. Four of these proteins of apparent size on SDS PAGE of 24, 30, 48 and 130 Kd were membrane associated. /sup 125/I-actin binding to three proteins (24, 30 and 48 Kd) was shown using overlays. A 130 Kd protein reacted with anti-vinculin monoclonal antibody on immunoblots, and was immunolocalized at secretory canaliculi. As a working hypothesis, parietal cells possess membrane-associated proteins which change their state of phosphorylation upon stimulation of H/sup +/. These proteins may be cytoskeletal elements involved in regulation of H/sup +/ secretion. The 130 Kd vinculin-like protein may serve a microfilament-membrane linking role.

  12. The Erwinia chrysanthemi Type III Secretion System Is Required for Multicellular Behavior

    PubMed Central

    Yap, Mee-Ngan; Yang, Ching-Hong; Barak, Jeri D.; Jahn, Courtney E.; Charkowski, Amy O.

    2005-01-01

    Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a σ54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36°C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors. PMID:15629935

  13. Structure of the Type VI secretion system contractile sheath

    PubMed Central

    Kudryashev, Mikhail; Wang, Ray Yu-Ruei; Brackmann, Maximilian; Scherer, Sebastian; Maier, Timm; Baker, David; DiMaio, Frank; Stahlberg, Henning; Egelman, Edward H.; Basler, Marek

    2015-01-01

    Summary Bacteria use rapid contraction of a long sheath of the Type VI secretion system (T6SS) to deliver effectors into a target cell. Here we present an atomic resolution structure of a native contracted Vibrio cholerae sheath determined by cryo-electron microscopy. The sheath subunits, composed of tightly interacting proteins VipA and VipB, assemble into a six-start helix. The helix is stabilized by a core domain assembled from four β-strands donated by one VipA and two VipB molecules. The fold of inner and middle layers is conserved between T6SS and phage sheaths. However, the structure of the outer layer is distinct and suggests a mechanism of interaction of the bacterial sheath with an accessory ATPase, ClpV, that facilitates multiple rounds of effector delivery. Our results provide a mechanistic insight into assembly of contractile nanomachines that bacteria and phages use to translocate macromolecules across membranes. PMID:25723169

  14. A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins*

    PubMed Central

    Crosnier, Cécile; Wanaguru, Madushi; McDade, Brian; Osier, Faith H.; Marsh, Kevin; Rayner, Julian C.; Wright, Gavin J.

    2013-01-01

    Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and

  15. Microfluidic perfusion systems for secretion fingerprint analysis of pancreatic islets: applications, challenges and opportunities.

    PubMed

    Castiello, F Rafael; Heileman, Khalil; Tabrizian, Maryam

    2016-02-01

    A secretome signature is a heterogeneous profile of secretions present in a single cell type. From the secretome signature a smaller panel of proteins, namely a secretion fingerprint, can be chosen to feasibly monitor specific cellular activity. Based on a thorough appraisal of the literature, this review explores the possibility of defining and using a secretion fingerprint to gauge the functionality of pancreatic islets of Langerhans. It covers the state of the art regarding microfluidic perfusion systems used in pancreatic islet research. Candidate analytical tools to be integrated within microfluidic perfusion systems for dynamic secretory fingerprint monitoring were identified. These analytical tools include patch clamp, amperometry/voltametry, impedance spectroscopy, field effect transistors and surface plasmon resonance. Coupled with these tools, microfluidic devices can ultimately find applications in determining islet quality for transplantation, islet regeneration and drug screening of therapeutic agents for the treatment of diabetes. PMID:26732665

  16. Correlation of secretion of retinol and protein by the lacrimal gland

    SciTech Connect

    Ubels, J.L.; Rismondo, V.

    1986-03-01

    Retinol, which is present in tears, is secreted by the lacrimal gland. Retinol secretion is stimulated by cholinergic drugs and vasoactive intestinal peptide with characteristics very similar to the exocytotic secretion of protein by the lacrimal gland, suggesting that retinol and protein are secreted by similar mechanisms. The authors investigated this by cannulating the lacrimal gland ducts of rabbits and collecting lacrimal gland fluid (LGF) under conditions of maximal flow stimulated by IV injection of pilocarpine (400 ..mu..g/kg) every 20 min for 4.5 hr. Over this period LGF protein concentration decreased 36.4% from 22.8 +/- 1.94 mg/ml to 8.29 1.86 mg/ml while retinol decreased 37% from 55.1 +/- 16.2 ng/ml to 20.4 +/- 6.5 ng/ml. The retinol/protein ratio remained constant at 2.88 ng/mg. This demonstrates a strong correlation between retinol and protein secretion, suggesting that retinol may be protein bound. To investigate binding of retinol to LGF protein, LGF was incubated with /sup 3/H-retinol. The bound and unbound retinol were separated on a Lipidex 1000 column. Retinol binding was linear over a range of 1.25-200 nM /sup 3/H-retinol. Binding was not inhibited by PCMBS or addition of a 100-fold excess of unlabeled retinol and was not increased by prior extraction of endogenous retinol from the LGF. This indicates that the binding of retinol to LGF protein is non-specific. Retinol therefore appears to be secreted by the lacrimal gland cells in non-specific association with protein.

  17. Dengue virus envelope domain III protein based on a tetravalent antigen secreted from insect cells: Potential use for serological diagnosis.

    PubMed

    Niu, Guoyu; Pang, Zheng; Guan, Chun; Qi, Jun; Li, Dexin

    2015-04-01

    In the present study, we developed a tetravalent protein by connecting the receptor-binding envelope domain III (EDIII) of the four dengue virus serotypes in the order of D1-D3-D4-D2. Using a baculovirus expression system, the protein was secreted into the supernatant of infected sf9 cells in a stable form with preserved native conformation. Using immobilized affinity chromatography, the recombinant EDIII (rEDIII) protein was purified with a yield of 300μg per 10(6) cells. The purity and reactivity of the protein were determined via SDS-PAGE and Western blot respectively. A MAC-ELISA method based on the secreted rEDIII protein was subsequently established and evaluated using a panel of pre-characterized dengue IgM-positive and -negative human sera. We obtained a specificity of 100% and sensitivity of 93% using this method. Our data collectively suggest that the secreted tetravalent rEDIII protein has potential utility in the diagnosis of dengue virus infections. PMID:25697685

  18. Control of airway tube diameter and integrity by secreted chitin-binding proteins in Drosophila.

    PubMed

    Tiklová, Katarína; Tsarouhas, Vasilios; Samakovlis, Christos

    2013-01-01

    The transporting function of many branched tubular networks like our lungs and circulatory system depend on the sizes and shapes of their branches. Understanding the mechanisms of tube size control during organ development may offer new insights into a variety of human pathologies associated with stenoses or cystic dilations in tubular organs. Here, we present the first secreted luminal proteins involved in tube diametric expansion in the Drosophila airways. obst-A and gasp are conserved among insect species and encode secreted proteins with chitin binding domains. We show that the widely used tracheal marker 2A12, recognizes the Gasp protein. Analysis of obst-A and gasp single mutants and obst-A; gasp double mutant shows that both genes are primarily required for airway tube dilation. Similarly, Obst-A and Gasp control epidermal cuticle integrity and larval growth. The assembly of the apical chitinous matrix of the airway tubes is defective in gasp and obst-A mutants. The defects become exaggerated in double mutants indicating that the genes have partially redundant functions in chitin structure modification. The phenotypes in luminal chitin assembly in the airway tubes are accompanied by a corresponding reduction in tube diameter in the mutants. Conversely, overexpression of Obst-A and Gasp causes irregular tube expansion and interferes with tube maturation. Our results suggest that the luminal levels of matrix binding proteins determine the extent of diametric growth. We propose that Obst-A and Gasp organize luminal matrix assembly, which in turn controls the apical shapes of adjacent cells during tube diameter expansion. PMID:23826295

  19. Human Surfactant Protein A2 Gene Mutations Impair Dimmer/Trimer Assembly Leading to Deficiency in Protein Sialylation and Secretion

    PubMed Central

    Shen, Haitao; Li, Hui; Yang, Wenbing; Pan, Bing; Huang, Guowei; Lin, Guangyu; Ma, Lian; Willard, Belinda; Gu, Jiang; Zheng, Lemin; Wang, Yongyu

    2012-01-01

    Surfactant protein A2 (SP-A2) plays an essential role in surfactant metabolism and lung host defense. SP-A2 mutations in the carbohydrate recognition domain have been related to familial pulmonary fibrosis and can lead to a recombinant protein secretion deficiency in vitro. In this study, we explored the molecular mechanism of protein secretion deficiency and the subsequent biological effects in CHO-K1 cells expressing both wild-type and several different mutant forms of SP-A2. We demonstrate that the SP-A2 G231V and F198S mutants impair the formation of dimmer/trimer SP-A2 which contributes to the protein secretion defect. A deficiency in sialylation, but not N-linked glycosylation, is critical to the observed dimmer/trimer impairment-induced secretion defect. Furthermore, both mutant forms accumulate in the ER and form NP-40-insoluble aggregates. In addition, the soluble mutant SP-A2 could be partially degraded through the proteasome pathway but not the lysosome or autophagy pathway. Intriguingly, 4-phenylbutyrate acid (4-PBA), a chemical chaperone, alleviates aggregate formation and partially rescued the protein secretion of SP-A2 mutants. In conclusion, SP-A2 G231V and F198S mutants impair the dimmer/trimer assembly, which contributes to the protein sialylation and secretion deficiency. The intracellular protein mutants could be partially degraded through the proteasome pathway and also formed aggregates. The treatment of the cells with 4-PBA resulted in reduced aggregation and rescued the secretion of mutant SP-A2. PMID:23056344

  20. Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils

    SciTech Connect

    Wilson, E.; Arnold, R.R.; Merrill, A.H.; Lambeth, J.D.

    1987-05-01

    Sphingoid long-chain bases (sphinganine and sphingosine(So)) have recently been shown to inhibit protein kinase C (PK-C) in vitro and to block activation of the oxidative burst in intact neutrophils (PMN) by inhibiting this enzyme. In the present study, the authors have used So to investigate the role of protein kinase C in stimulus-induced secretion of PMN granule contents. Secretion of the specific granule component lactoferrin (Lf) is completely inhibited by pretreatment with So when either PMA or fLMP is used as the secretogogue. Secretion of lysozyme, a component of both the azurophilic and specific granules, is completely inhibited by So when PMA is used, but only 40% inhibited with fMLP. The secretion of the azurophilic granule markers US -glucuronidase and myeloperoxidase was not affected by So regardless of the agonist used. Data indicate that both PK-C-dependent and -independent pathways participate in the neutrophil secretory response.

  1. Secretion of human interleukin-2 fused with green fluorescent protein in recombinant Pichia pastoris.

    PubMed

    Cha, Hyung Joon; Dalal, Nimish N; Bentley, William E

    2005-07-01

    Methylotrophic yeast Pichia pastoris is convenient for the expression of eukaryotic foreign proteins owing to its potential for posttranslational modifications, protein folding, and facile culturing. In this work, human interleukin (hIL)-2 was successfully produced as a secreted fusion form in recombinant P. pastoris. By employing green fluorescent protein (GFP) as a monitoring fusion partner, clear identification of fusion protein expression and quantification of intracellular hIL-2 were possible even though there was no correlation between culture supernatant fluorescence and secreted hIL-2 owing to high media interference. Importantly, by the addition of casamino acids in basal medium, we were able to enhance threefold amount of secreted hIL-2, which was present both as a fusion and as a clipped fragment. PMID:16014994

  2. Short-chain fatty acids regulate IGF-binding protein secretion by intestinal epithelial cells.

    PubMed

    Nishimura, A; Fujimoto, M; Oguchi, S; Fusunyan, R D; MacDermott, R P; Sanderson, I R

    1998-07-01

    Gastrointestinal epithelial cells secrete insulin-like growth factor (IGF)-binding proteins (IGFBPs), which modulate the actions of IGFs on cell proliferation and differentiation. Short-chain fatty acids are bacterial metabolites from unabsorbed carbohydrate (including fiber). We hypothesized that they may alter the pattern of IGFBPs secreted by epithelial cells as part of a wider phenomenon by which luminal molecules regulate gastrointestinal epithelial cell signaling. The intestinal epithelial cell line, Caco-2, predominantly secretes IGFBP-3; however, butyrate increased the secretion of IGFBP-2 in a dose-dependent and reversible manner. Butyrate decreased the secretion of IGFBP-3. Butyrate altered only the synthesis and not the cell sorting of IGFBPs because 1) the secretion of IGFBPs remained polarized despite changes in their rates of production, and 2) IGFBP secretion corresponded to mRNA accumulation. The ability of short-chain fatty acids or the fungicide trichostatin A to stimulate IGFBP-2 correlated with their actions on histone acetylation. In conclusion, intestinal epithelial cells respond to short-chain fatty acids by altering secretion of IGFBPs. PMID:9688874

  3. Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome

    PubMed Central

    Tjalsma, Harold; Antelmann, Haike; Jongbloed, Jan D.H.; Braun, Peter G.; Darmon, Elise; Dorenbos, Ronald; Dubois, Jean-Yves F.; Westers, Helga; Zanen, Geeske; Quax, Wim J.; Kuipers, Oscar P.; Bron, Sierd; Hecker, Michael; van Dijl, Jan Maarten

    2004-01-01

    Secretory proteins perform a variety of important “remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ∼90 extracellular proteins were identified. Analysis of these proteins disclosed various “secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only ∼50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance. PMID:15187182

  4. A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

    PubMed Central

    Park, Bo R.; Zielke, Ryszard A.; Wierzbicki, Igor H.; Mitchell, Kristie C.; Withey, Jeffrey H.

    2015-01-01

    Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program. PMID:25561716

  5. A metalloprotease secreted by the type II secretion system links Vibrio cholerae with collagen.

    PubMed

    Park, Bo R; Zielke, Ryszard A; Wierzbicki, Igor H; Mitchell, Kristie C; Withey, Jeffrey H; Sikora, Aleksandra E

    2015-03-01

    Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program. PMID:25561716

  6. Leishmania infantum chagasi: A genome-based approach to identification of excreted/secreted proteins

    PubMed Central

    DebRoy, Sruti; Keenan, Alexandra B.; Ueno, Norikiyo; Jeronimo, Selma M. B.; Donelson, John E.; Wilson, Mary E.

    2010-01-01

    The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite’s interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity. PMID:20542033

  7. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    PubMed Central

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.; Hallström, Björn M.; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N.; Andersson-Svahn, Helene; Nielsen, Jens

    2015-01-01

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories. PMID:26261321

  8. Structural models of intrinsically disordered and calcium-bound folded states of a protein adapted for secretion

    PubMed Central

    O’Brien, Darragh P.; Hernandez, Belen; Durand, Dominique; Hourdel, Véronique; Sotomayor-Pérez, Ana-Cristina; Vachette, Patrice; Ghomi, Mahmoud; Chamot-Rooke, Julia; Ladant, Daniel; Brier, Sébastien; Chenal, Alexandre

    2015-01-01

    Many Gram-negative bacteria use Type I secretion systems, T1SS, to secrete virulence factors that contain calcium-binding Repeat-in-ToXin (RTX) motifs. Here, we present structural models of an RTX protein, RD, in both its intrinsically disordered calcium-free Apo-state and its folded calcium-bound Holo-state. Apo-RD behaves as a disordered polymer chain comprising several statistical elements that exhibit local rigidity with residual secondary structure. Holo-RD is a folded multi-domain protein with an anisometric shape. RTX motifs thus appear remarkably adapted to the structural and mechanistic constraints of the secretion process. In the low calcium environment of the bacterial cytosol, Apo-RD is an elongated disordered coil appropriately sized for transport through the narrow secretion machinery. The progressive folding of Holo-RD in the extracellular calcium-rich environment as it emerges form the T1SS may then favor its unidirectional export through the secretory channel. This process is relevant for hundreds of bacterial species producing virulent RTX proteins. PMID:26374675

  9. Isoprenoid biosynthetic pathway inhibition disrupts monoclonal protein secretion and induces the unfolded protein response pathway in multiple myeloma cells

    PubMed Central

    Holstein, Sarah A.; Hohl, Raymond J.

    2010-01-01

    Myeloma is characterized by the overproduction and secretion of monoclonal protein. Inhibitors of the isoprenoid biosynthetic pathway (IBP) have pleiotropic effects in myeloma cells. To investigate whether IBP inhibition interferes with monoclonal protein secretion, human myeloma cells were treated with specific inhibitors of the IBP or prenyltransferases. These studies demonstrate that agents that inhibit Rab geranylgeranylation disrupt light chain trafficking, lead to accumulation of light chain in the endoplasmic reticulum, activate the unfolded protein response pathway and induce apoptosis. These studies provide a novel mechanism of action for IBP inhibitors and suggest that further exploration of Rab-targeted agents in myeloma is warranted. PMID:20828814

  10. Molecular Characterization of a Functional Type VI Secretion System from a Clinical Isolate of Aeromonas hydrophila

    PubMed Central

    Suarez, Giovanni; Sierra, Johanna C.; Sha, Jian; Wang, Shaofei; Erova, Tatiana E.; Fadl, Amin A.; Foltz, Sheri M.; Horneman, Amy J.; Chopra, Ashok K.

    2008-01-01

    Our laboratory recently molecularly characterized the type II secretion system (T2SS)- associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type 6 secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT

  11. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    PubMed Central

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  12. Platelet secretion and hemostasis require syntaxin-binding protein STXBP5.

    PubMed

    Ye, Shaojing; Huang, Yunjie; Joshi, Smita; Zhang, Jinchao; Yang, Fanmuyi; Zhang, Guoying; Smyth, Susan S; Li, Zhenyu; Takai, Yoshimi; Whiteheart, Sidney W

    2014-10-01

    Genome-wide association studies (GWAS) have linked genes encoding several soluble NSF attachment protein receptor (SNARE) regulators to cardiovascular disease risk factors. Because these regulatory proteins may directly affect platelet secretion, we used SNARE-containing complexes to affinity purify potential regulators from human platelet extracts. Syntaxin-binding protein 5 (STXBP5; also known as tomosyn-1) was identified by mass spectrometry, and its expression in isolated platelets was confirmed by RT-PCR analysis. Coimmunoprecipitation studies showed that STXBP5 interacts with core secretion machinery complexes, such as syntaxin-11/SNAP23 heterodimers, and fractionation studies suggested that STXBP5 also interacts with the platelet cytoskeleton. Platelets from Stxbp5 KO mice had normal expression of other key secretory components; however, stimulation-dependent secretion from each of the 3 granule types was markedly defective. Secretion defects in STXBP5-deficient platelets were confirmed via lumi-aggregometry and FACS analysis for P-selectin and LAMP-1 exposure. Interestingly, STXBP5-deficient platelets had altered granule cargo levels, despite having normal morphology and granule numbers. Consistent with secretion and cargo deficiencies, Stxbp5 KO mice showed dramatic bleeding in the tail transection model and defective hemostasis in the FeCl3-induced carotid injury model. Transplantation experiments indicated that these defects were due to loss of STXBP5 in BM-derived cells. Our data demonstrate that STXBP5 is required for normal arterial hemostasis, due to its contributions to platelet granule cargo packaging and secretion. PMID:25244094

  13. Platelet secretion and hemostasis require syntaxin-binding protein STXBP5

    PubMed Central

    Ye, Shaojing; Huang, Yunjie; Joshi, Smita; Zhang, Jinchao; Yang, Fanmuyi; Zhang, Guoying; Smyth, Susan S.; Li, Zhenyu; Takai, Yoshimi; Whiteheart, Sidney W.

    2014-01-01

    Genome-wide association studies (GWAS) have linked genes encoding several soluble NSF attachment protein receptor (SNARE) regulators to cardiovascular disease risk factors. Because these regulatory proteins may directly affect platelet secretion, we used SNARE-containing complexes to affinity purify potential regulators from human platelet extracts. Syntaxin-binding protein 5 (STXBP5; also known as tomosyn-1) was identified by mass spectrometry, and its expression in isolated platelets was confirmed by RT-PCR analysis. Coimmunoprecipitation studies showed that STXBP5 interacts with core secretion machinery complexes, such as syntaxin-11/SNAP23 heterodimers, and fractionation studies suggested that STXBP5 also interacts with the platelet cytoskeleton. Platelets from Stxbp5 KO mice had normal expression of other key secretory components; however, stimulation-dependent secretion from each of the 3 granule types was markedly defective. Secretion defects in STXBP5-deficient platelets were confirmed via lumi-aggregometry and FACS analysis for P-selectin and LAMP-1 exposure. Interestingly, STXBP5-deficient platelets had altered granule cargo levels, despite having normal morphology and granule numbers. Consistent with secretion and cargo deficiencies, Stxbp5 KO mice showed dramatic bleeding in the tail transection model and defective hemostasis in the FeCl3-induced carotid injury model. Transplantation experiments indicated that these defects were due to loss of STXBP5 in BM-derived cells. Our data demonstrate that STXBP5 is required for normal arterial hemostasis, due to its contributions to platelet granule cargo packaging and secretion. PMID:25244094

  14. Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling procedure

    PubMed Central

    2011-01-01

    Background The budding yeast Pichia pastoris is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intracellular protein formation, degradation and secretion defines the major bottleneck of the production system. Because these parameters are different for unlimited growth (shake flask) and carbon-limited growth (bioreactor) conditions, they should be determined under "production like" conditions. Thus labeling procedures must be compatible with minimal production media and the usage of bioreactors. The inorganic and non-radioactive 34S labeled sodium sulfate meets both demands. Results We used a novel labeling method with the stable sulfur isotope 34S, administered as sodium sulfate, which is performed during chemostat culivations. The intra- and extracellular sulfur 32 to 34 ratios of purified recombinant protein, the antibody fragment Fab3H6, are measured by HPLC-ICP-MS. The kinetic model described here is necessary to calculate the kinetic parameters from sulfur ratios of consecutive samples as well as for sensitivity analysis. From the total amount of protein produced intracellularly (143.1 μg g-1 h-1 protein per yeast dry mass and time) about 58% are degraded within the cell, 35% are secreted to the exterior and 7% are inherited to the daughter cells. Conclusions A novel 34S labeling procedure that enables in vivo quantification of intracellular fluxes of recombinant protein under "production like" conditions is described. Subsequent sensitivity analysis of the fluxes by using MATLAB, indicate the most promising approaches for strain improvement towards increased secretion. PMID:21703020

  15. Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle.

    PubMed

    Sharma, V K; Sacco, R E; Kunkle, R A; Bearson, S M D; Palmquist, D E

    2012-04-01

    The locus of enterocyte effacement (LEE) of Escherichia coli O157:H7 (O157) encodes a type III secretion system (T3SS) for secreting LEE-encoded and non-LEE-encoded virulence proteins that promote the adherence of O157 to intestinal epithelial cells and the persistence of this food-borne human pathogen in bovine intestines. In this study, we compared hha sepB and hha mutants of O157 for LEE transcription, T3SS activity, adherence to HEp-2 cells, persistence in bovine intestines, and the ability to induce changes in the expression of proinflammatory cytokines. LEE transcription was upregulated in the hha sepB and hha mutant strains compared to that in the wild-type strain, but the secretion of virulence proteins in the hha sepB mutant was severely compromised. This reduced secretion resulted in reduced adherence of the hha sepB mutant to Hep-2 cells, correlating with a significantly shorter duration and lower magnitude of fecal shedding in feces of weaned (n = 4 per group) calves inoculated with this mutant strain. The levels of LEE transcription, T3SS activity, and adherence to HEp-2 cells were much lower in the wild-type strain than in the hha mutant, but no significant differences were observed in the duration or the magnitude of fecal shedding in calves inoculated with these strains. Examination of the rectoanal junction (RAJ) tissues from three groups of calves showed no adherent O157 bacteria and similar proinflammatory cytokine gene expression, irrespective of the inoculated strain, with the exception that interleukin-1β was upregulated in calves inoculated with the hha sepB mutant. These results indicate that the T3SS is essential for intestinal colonization and prolonged shedding, but increased secretion of virulence proteins did not enhance the duration and magnitude of fecal shedding of O157 in cattle or have any significant impact on the cytokine gene expression in RAJ tissue compared with that in small intestinal tissue from the same calves. PMID

  16. Conserved type III secretion system exerts important roles in Chlamydia trachomatis

    PubMed Central

    Dai, Wenting; Li, Zhongyu

    2014-01-01

    Upon infection, Chlamydiae alter host cellular functions in a variety of ways. Chlamydial infection prevents host cell apoptosis, induces re-organization of the actin cytoskeleton and alters host cellular signaling mechanisms. Chlamydia is among the many pathogenic Gram-negative bacteria that employ the type III secretion system (T3SS) to overcome host defenses and exploit available resources. T3SS are used by many Gram-negative bacterial pathogens to manipulate eukaryotic host cells through the delivery of effector proteins into their cytosol and membranes. T3SS is an evolutionarily refined, virulence determinant of Gram-negative bacteria where more than 20 proteins form an apparatus, generally termed injectisome, to achieve the vectorial secretion and translocation of anti-host effector proteins. This review describes challenges and recent advances that have revealed how Chlamydia trachomatis utilizes diversification to produce a conserved T3SS that exerts an important role in Chlamydia trachomatis. PMID:25337183

  17. Genetically distinct pathways guide effector export through the type VI secretion system

    PubMed Central

    Whitney, John C.; Beck, Christina M.; Goo, Young Ah; Russell, Alistair B.; Harding, Brittany; De Leon, Justin A.; Cunningham, David A.; Tran, Bao Q.; Low, David A.; Goodlett, David R.; Hayes, Christopher S.; Mougous, Joseph D.

    2014-01-01

    Summary Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), hemolysin co-regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone-like quality of Hcp. Application of this approach to the Hcp secretion island I-encoded T6SS (H1-T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (type VI secretion exported 4), subsequently shown to act as a potent intra-specific H1-T6SS-delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1-T6SS effectors, Tse5 and Tse6, which differ from Hcp-stabilized substrates by the presence of toxin-associated PAAR-repeat motifs and genetic linkage to members of the valine-glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp-stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1-T6SS-exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export. PMID:24589350

  18. Miraculin, a taste-modifying protein is secreted into intercellular spaces in plant cells.

    PubMed

    Hirai, Tadayoshi; Sato, Mayuko; Toyooka, Kiminari; Sun, Hyeon-Jin; Yano, Megumu; Ezura, Hiroshi

    2010-02-15

    A taste-modifying protein, miraculin, is highly accumulated in ripe fruit of miracle fruit (Richadella dulcifica) and the content can reach up to 10% of the total soluble protein in these fruits. Although speculated for decades that miraculin is secreted into intercellular spaces in miracle fruit, no evidence exists of its cellular localization. To study the cellular localization of miraculin in plant cells, using miracle fruit and transgenic tomato that constitutively express miraculin, immunoelectron microscopy, imaging GFP fusion proteins, and immunological detection of secreted proteins in culture medium of transgenic tomato were carried out. Immunoelectron microscopy showed the specific accumulation of miraculin in the intercellular layers of both miracle fruit and transgenic tomato. Imaging GFP fusion protein demonstrated that the miraculin-GFP fusion protein was accumulated in the intercellular spaces of tomato epidermal cells. Immunological detection of secreted proteins in culture medium of transgenic tomato indicated that miraculin was secreted from the roots of transgenic tomato expressing miraculin. This study firstly showed the evidences of the intercellular localization of miraculin, and provided a new insight of biological roles of miraculin in plants. PMID:19712996

  19. A model system for the study of stimulus - enzyme secretion coupling in rat pancreatic acinar cells.

    PubMed

    Guderley, H; Heisler, S

    1980-08-01

    A superfusion technique was developed as a model system for the study of stimulus-secretion coupling in collagenase-dispersed rat pancreatic acinar cells. Cells (10(7)) were combined with a slurry of Biogel P-4 beads and the mixture was decanted into a plastic column (1.5 cm X 8.5 cm) and perfused with Krebs-Ringer. Amylase activity was determined in sequentially collected effusate fractions and used to estimate the secretory rate. Carbachol, carbachol plus dibutyryl cyclic AMP, cholecystokinin-pancreozymin, and the ionophore A-23187 all stimulated a rapid increase in the rate of secretion. Cell integrity was unaffected by these stimulants as evidenced microscopically and by the lack of lactate dehydrogenase activity in the effusates. Enzymes secreted in response to secretagogues were collected, concentrated, and isoelectrofocused on polyacrylamide gels. A film detection technique was developed to localize amylase activity. The model system has the following advantages: (1) secreted proteolytic products are removed from the vicinity of cells, thereby preventing direct cellular damage and hydrolysis of peptide agonist; (2) the need to add trypsin inhibitors is eliminated and only a minimal addition of albumin (0.001%) is required, thus allowing the separation and distortion-free analysis of secreted proteins; (3) the perfusion conditions can be changed rapidly without disturbing the cells. The model described is therefore well suited to the study of both molecular and kinetic events involved in the enzyme secretory phenomenon in exocrine pancreas. PMID:6164455

  20. hESC-secreted proteins can be enriched for multiple regenerative therapies by heparin-binding.

    PubMed

    Yousef, Hanadie; Conboy, Michael J; Li, Ju; Zeiderman, Matthew; Vazin, Tandis; Schlesinger, Christina; Schaffer, David V; Conboy, Irina M

    2013-05-01

    This work builds upon our findings that proteins secreted by hESCs exhibit pro-regenerative activity, and determines that hESC-conditioned medium robustly enhances the proliferation of both muscle and neural progenitor cells. Importantly, this work establishes that it is the proteins that bind heparin which are responsible for the pro-myogenic effects of hESC-conditioned medium, and indicates that this strategy is suitable for enriching the potentially therapeutic factors. Additionally, this work shows that hESC-secreted proteins act independently of the mitogen FGF-2, and suggests that FGF-2 is unlikely to be a pro-aging molecule in the physiological decline of old muscle repair. Moreover, hESC-secreted factors improve the viability of human cortical neurons in an Alzheimer's disease (AD) model, suggesting that these factors can enhance the maintenance and regeneration of multiple tissues in the aging body. PMID:23793469

  1. DsbA2 (27 kDa Com1-like protein) of Legionella pneumophila catalyses extracytoplasmic disulphide-bond formation in proteins including the Dot/Icm type IV secretion system.

    PubMed

    Jameson-Lee, Max; Garduño, Rafael A; Hoffman, Paul S

    2011-05-01

    In Gram-negative bacteria, thiol oxidoreductases catalyse the formation of disulphide bonds (DSB) in extracytoplasmic proteins. In this study, we sought to identify DSB-forming proteins required for assembly of macromolecular structures in Legionella pneumophila. Here we describe two DSB-forming proteins, one annotated as dsbA1 and the other annotated as a 27 kDa outer membrane protein similar to Com1 of Coxiella burnetii, which we designate as dsbA2. Both proteins are predicted to be periplasmic, and while dsbA1 mutants were readily isolated and without phenotype, dsbA2 mutants were not obtained. To advance studies of DsbA2, a cis-proline residue at position 198 was replaced with threonine that enables formation of stable disulphide-bond complexes with substrate proteins. Expression of DsbA2 P198T mutant protein from an inducible promoter produced dominant-negative effects on DsbA2 function that resulted in loss of infectivity for amoeba and HeLa cells and loss of Dot/Icm T4SS-mediated contact haemolysis of erythrocytes. Analysis of captured DsbA2 P198T-substrate complexes from L. pneumophila by mass spectrometry identified periplasmic and outer membrane proteins that included components of the Dot/Icm T4SS. More broadly, our studies establish a DSB oxidoreductase function for the Com1 lineage of DsbA2-like proteins which appear to be conserved among those bacteria also expressing T4SS. PMID:21375592

  2. DsbA2 (27-kDa Com1-Like Protein) of Legionella pneumophila Catalyses Extracytoplasmic Disulfide-Bond Formation in Proteins Including the Dot/Icm Type IV Secretion System

    PubMed Central

    Jameson-Lee, Max; Garduno, Rafael A.; Hoffman, Paul S.

    2011-01-01

    Summary In Gram negative bacteria, thiol oxidoreductases catalyze the formation of disulfide bonds (DSB) in extracytoplasmic proteins. In this study, we sought to identify DSB-forming proteins required for assembly of macromolecular structures in Legionella pneumophila. Here we describe two DSB forming proteins, one annotated as dsbA1 and the other annotated as a 27-kDa outer membrane protein similar to Com1 of Coxiella burnetii, which we designate as dsbA2. Both proteins are predicted to be periplasmic, and while dsbA1 mutants were readily isolated and without phenotype, dsbA2 mutants were not obtained. To advance studies of DsbA2, a cis-proline residue at position 198 was replaced with threonine that enables formation of stable disulfide-bond complexes with substrate proteins. Expression of DsbA2 P198T-mutant protein from an inducible promoter produced dominant-negative effects on DsbA2 function that resulted in loss of infectivity for amoeba and HeLa cells and loss of Dot/Icm T4SS-mediated contact hemolysis of erythrocytes. Analysis of captured DsbA2 P198T-substrate complexes from L. pneumophila by mass spectrometry identified periplasmic and outer membrane proteins that included components of the Dot/Icm T4SS. More broadly, our studies establish a DSB oxidoreductase function for the Com1 lineage of DsbA2-like proteins which appear to be conserved among those bacteria also expressing T4SS. PMID:21375592

  3. γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

    PubMed Central

    Grieder, Nicole C.; Caussinus, Emmanuel; Parker, David S.; Cadigan, Kenneth; Affolter, Markus; Luschnig, Stefan

    2008-01-01

    Background There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex. Principal Findings We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis. Conclusions/Significance These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein. PMID:18802472

  4. Aim, Load, Fire: The Type VI Secretion System, a Bacterial Nanoweapon.

    PubMed

    Cianfanelli, Francesca R; Monlezun, Laura; Coulthurst, Sarah J

    2016-01-01

    Bacteria utilise specialised protein secretion systems to interact with host organisms, competitor bacteria, and the environment. The Type VI secretion system (T6SS) is a versatile weapon deployed by many bacterial species to target either host cells or rival bacteria. The widespread occurrence and significance of the T6SS is becoming increasingly appreciated, as is its intriguing mode of action. The T6SS delivers multiple, diverse effector proteins directly into target cells using a dynamic 'firing' mechanism related to the action of contractile bacteriophage tails. Here, we summarise the contribution of recent findings to our developing picture of how the T6SS assembles and fires, how it is loaded with different types of effectors, and how it can be aimed towards an incoming assault. PMID:26549582

  5. Regulation of follistatin-like protein 1 expression and secretion in primary human skeletal muscle cells.

    PubMed

    Görgens, Sven W; Raschke, Silja; Holven, Kirsten Bjørklund; Jensen, Jørgen; Eckardt, Kristin; Eckel, Jürgen

    2013-05-01

    Follistatin-like protein 1 (Fstl1) is a secreted glycoprotein of the follistatin family. Fstl1 is secreted by C2C12 cells, and Akt1 over-expression in skeletal muscle leads to its induction in muscle and increased circulating levels. So far, secretion of Fstl1 by human myotubes and the effect of exercise on its circulating levels have not been investigated. Here, we examined both the regulation of Fstl1 expression and secretion in primary human skeletal muscle cells and the effect of acute exercise on Fstl1 serum concentrations in humans. We show that human myotubes express and secrete Fstl1 in a differentiation-dependent manner. Furthermore, IFNγ and IL-1β significantly increase Fstl1 secretion. Electrical pulse stimulation (EPS)-induced contractile activity of myotubes did not regulate Fstl1. Interestingly, we observed that 60 min cycling increased serum Fstl1 level by 22%. In conclusion, we demonstrate that Fstl1 is expressed and secreted by human myotubes and plasma Fstl1 levels are increased after exercise. PMID:23419164

  6. Broadly protective Shigella vaccine based on type III secretion apparatus proteins.

    PubMed

    Martinez-Becerra, Francisco J; Kissmann, Julian M; Diaz-McNair, Jovita; Choudhari, Shyamal P; Quick, Amy M; Mellado-Sanchez, Gabriela; Clements, John D; Pasetti, Marcela F; Picking, Wendy L

    2012-03-01

    Shigella spp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei. We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine. PMID:22202122

  7. Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris.

    PubMed

    Whyteside, Graham; Alcocer, Marcos J C; Kumita, Janet R; Dobson, Christopher M; Lazarou, Maria; Pleass, Richard J; Archer, David B

    2011-01-01

    We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general. PMID:21818368

  8. Native-State Stability Determines the Extent of Degradation Relative to Secretion of Protein Variants from Pichia pastoris

    PubMed Central

    Whyteside, Graham; Alcocer, Marcos J. C.; Kumita, Janet R.; Dobson, Christopher M.; Lazarou, Maria; Pleass, Richard J.; Archer, David B.

    2011-01-01

    We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general. PMID:21818368

  9. Diffusely Adhering Escherichia coli Strains Induce Attaching and Effacing Phenotypes and Secrete Homologs of Esp Proteins

    PubMed Central

    Beinke, Christina; Laarmann, Sven; Wachter, Clemens; Karch, Helge; Greune, Lilo; Schmidt, M. Alexander

    1998-01-01

    Recent epidemiological studies indicate that Escherichia coli strains which exhibit the diffuse-adherence phenotype (DAEC strains) represent a potential cause of diarrhea in infants. We investigated the interaction of DAEC strains isolated from diarrhea patients in Brazil and in Germany with epithelial cells in tissue culture. The investigated strains were identified as DAEC strains by (i) their attachment pattern, (ii) presence of genes associated with the Dr family of adhesins, and (iii) lack of genetic markers for other diarrhea-associated E. coli categories. Several clinical DAEC isolates were shown to secrete similar patterns of proteins into tissue culture medium. Protein secretion was found to be regulated by environmental parameters, namely, medium, temperature, pH, and iron concentration. DAEC strains secreting these proteins induced accumulation of actin and tyrosine-phosphorylated proteins at sites of bacterial attachment, leading to the formation of pedestals and/or extended surface structures. These changes were phenotypically similar to the attaching and effacing (A/E) lesions observed with enteropathogenic and some enterohemorrhagic E. coli strains carrying the locus of enterocyte effacement (LEE) pathogenicity island. Proteins homologous to the EspA, EspB, and EspD proteins, necessary for signal transduction events inducing A/E lesions, were identified by sequence analysis and cross-reaction of specific antibodies. However, initially nonadhering strains secreting these proteins induced signal transduction events only after prolonged infection. These results indicate that secretion of the Esp proteins alone is not sufficient for efficient signal transduction. This study further shows that some DAEC strains are likely to contain a homolog(s) of the LEE locus which may contribute to the pathogenic potential of DAEC. PMID:9453606

  10. Adiporedoxin, an upstream regulator of ER oxidative folding and protein secretion in adipocytes

    PubMed Central

    Jedrychowski, Mark P.; Liu, Libin; Laflamme, Collette J.; Karastergiou, Kalypso; Meshulam, Tova; Ding, Shi-Ying; Wu, Yuanyuan; Lee, Mi-Jeong; Gygi, Steven P.; Fried, Susan K.; Pilch, Paul F.

    2015-01-01

    Objective Adipocytes are robust protein secretors, most notably of adipokines, hormone-like polypeptides, which act in an endocrine and paracrine fashion to affect numerous physiological processes such as energy balance and insulin sensitivity. To understand how such proteins are assembled for secretion we describe the function of a novel endoplasmic reticulum oxidoreductase, adiporedoxin (Adrx). Methods Adrx knockdown and overexpressing 3T3-L1 murine adipocyte cell lines and a knockout mouse model were used to assess the influence of Adrx on secreted proteins as well as the redox state of ER resident chaperones. The metabolic phenotypes of Adrx null mice were characterized and compared to WT mice. The correlation of Adrx levels BMI, adiponectin levels, and other inflammatory markers from adipose tissue of human subjects was also studied. Results Adiporedoxin functions via a CXXC active site, and is upstream of protein disulfide isomerase whose direct function is disulfide bond formation, and ultimately protein secretion. Over and under expression of Adrx in vitro enhances and reduces, respectively, the secretion of the disulfide-bonded proteins including adiponectin and collagen isoforms. On a chow diet, Adrx null mice have normal body weights, and glucose tolerance, are moderately hyperinsulinemic, have reduced levels of circulating adiponectin and are virtually free of adipocyte fibrosis resulting in a complex phenotype tending towards insulin resistance. Adrx protein levels in human adipose tissue correlate positively with adiponectin levels and negatively with the inflammatory marker phospho-Jun kinase. Conclusion These data support the notion that Adrx plays a critical role in adipocyte biology and in the regulation of mouse and human metabolism via its modulation of adipocyte protein secretion. PMID:26629401

  11. Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems.

    PubMed

    Roelofs, Kevin G; Jones, Christopher J; Helman, Sarah R; Shang, Xiaoran; Orr, Mona W; Goodson, Jonathan R; Galperin, Michael Y; Yildiz, Fitnat H; Lee, Vincent T

    2015-10-01

    Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP. PMID:26506097

  12. Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems

    PubMed Central

    Shang, Xiaoran; Orr, Mona W.; Goodson, Jonathan R.; Galperin, Michael Y.; Yildiz, Fitnat H.; Lee, Vincent T.

    2015-01-01

    Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP. PMID:26506097

  13. Mechanical Unfolding of an Autotransporter Passenger Protein Reveals the Secretion Starting Point and Processive Transport Intermediates.

    PubMed

    Baclayon, Marian; Ulsen, Peter van; Mouhib, Halima; Shabestari, Maryam Hashemi; Verzijden, Timo; Abeln, Sanne; Roos, Wouter H; Wuite, Gijs J L

    2016-06-28

    The backbone of secreted autotransporter passenger proteins generally attains a stable β-helical structure. The secretion of passengers across the outer membrane was proposed to be driven by sequential folding of this structure at the cell surface. This mechanism would require a relatively stable intermediate as starting point. Here, we investigated the mechanics of secreted truncated versions of the autotransporter hemoglobin protease (Hbp) of Escherichia coli using atomic force microscopy. The data obtained reveal a β-helical structure at the C terminus that is very stable. In addition, several other distinct metastable intermediates are found which are connected during unfolding by multiroute pathways. Computational analysis indicates that these intermediates correlate to the β-helical rungs in the Hbp structure which are clamped by stacked aromatic residues. Our results suggest a secretion mechanism that is initiated by a stable C-terminal structure and driven forward by several folding intermediates that build up the β-helical backbone. PMID:27219538

  14. Ectosomes: a new mechanism for non-exosomal secretion of tau protein.

    PubMed

    Dujardin, Simon; Bégard, Séverine; Caillierez, Raphaëlle; Lachaud, Cédrick; Delattre, Lucie; Carrier, Sébastien; Loyens, Anne; Galas, Marie-Christine; Bousset, Luc; Melki, Ronald; Aurégan, Gwennaëlle; Hantraye, Philippe; Brouillet, Emmanuel; Buée, Luc; Colin, Morvane

    2014-01-01

    Tau is a microtubule-associated protein that aggregates in neurodegenerative disorders known as tauopathies. Recently, studies have suggested that Tau may be secreted and play a role in neural network signalling. However, once deregulated, secreted Tau may also participate in the spreading of Tau pathology in hierarchical pathways of neurodegeneration. The mechanisms underlying neuron-to-neuron Tau transfer are still unknown; given the known role of extra-cellular vesicles in cell-to-cell communication, we wondered whether these vesicles could carry secreted Tau. We found, among vesicles, that Tau is predominately secreted in ectosomes, which are plasma membrane-originating vesicles, and when it accumulates, the exosomal pathway is activated. PMID:24971751

  15. Expression and secretion of a biologically active mouse sonic hedgehog protein by the methylotrophic yeast Pichia pastoris.

    PubMed

    Sakuma, Y; Kimura, M; Takabatake, T; Takeshima, K; Fujimura, H

    1999-09-01

    We have successfully secreted the amino-terminal functional domain of mouse sonic hedgehog protein (SHH) into culture fluid using a yeast Pichia pastoris expression system. A cDNA fragment encoding the amino-terminal domain of mouse SHH was inserted downstream of the Saccharomyces cerevisiae alpha-mating factor secretion signal. The DNA fragment was introduced into the host genome by the spheroplast transformation method. Transformants were selected based on their resistance to G418: His+ transformants which showed resistance to over 8 mg G418/ml were selected and analyzed for determination of the plasmid copy number. One His+ clone which has eight copies of the expression cassette per genome was cultured in minimal medium deficient for histidine, and further cultured in buffered medium supplemented with methanol which activates the AOX1 promoter. SDS-PAGE analysis indicated efficient expression and secretion of mouse SHH into culture fluid. The yield of secreted SHH was estimated to be 50 micrograms/ml. Purified protein was assayed for biological activity and found to activate the transcription of the Patched genes (Ptc-1 and Ptc-2) encoding receptors for SHH. PMID:10531654

  16. Repair of hair bundles in sea anemones by secreted proteins.

    PubMed

    Watson, G M; Mire, P; Hudson, R R

    1998-01-01

    Sea anemones are sessile invertebrates that detect movements of prey using numerous hair bundles located on tentacles surrounding their mouth. Previously we found that hair bundles of anemones are structurally and functionally similar to those of vertebrates. After 10-15 min exposure to calcium depleted buffers, hair bundles in chickens suffer moderate damage from which they recover in 12 h without requiring new protein synthesis [Zhao, Yamoah and Gillespie, Proc. Natl. Acad. Sci. USA 94 (1996) 15469-15474]. We find that after 1 h exposure to calcium free seawater, hair bundles of anemones suffer extensive damage from which they recover in 4 h, apparently because of newly synthesized, secretory proteins called 'repair proteins'. Recovery is delayed in a dose dependent fashion by cycloheximide. In the presence of exogenously added repair proteins, recovery occurs within 8 min and is cycloheximide insensitive. Recovery is ascertained by a bioassay performed on intact specimens, by electrophysiology, and by timelapse video microscopy. Fraction beta, a chromatographic fraction with bioactivity comparable to the complete mixture of repair proteins, consists of complexes having an estimated mass of 2000 kDa. Avidin based cytochemistry suggests that biotinylated fraction beta binds to damaged hair bundles. SDS-PAGE gel electrophoresis demonstrates that fraction beta contains 8-10 polypeptides of 90 kDa or smaller. At least four of these polypeptides apparently are consumed during the repair process. Negatively stained samples of fraction beta are shown by transmission electron microscopy to include filamentous structures similar in length (150 nm) and width (6 nm) to linkages between stereocilia. The filamentous structures can be associated with globular structures (20 nm in diameter). A model is presented wherein repair proteins comprise replacement linkages and enzymes that attach linkages to appropriate membrane proteins. PMID:9472741

  17. Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes

    PubMed Central

    Nelson, Matthew S.; Sadowsky, Michael J.

    2015-01-01

    The formation of symbiotic nitrogen-fixing nodules on the roots and/or stem of leguminous plants involves a complex signal exchange between both partners. Since many microorganisms are present in the soil, legumes and rhizobia must recognize and initiate communication with each other to establish symbioses. This results in the formation of nodules. Rhizobia within nodules exchange fixed nitrogen for carbon from the legume. Symbiotic relationships can become non-beneficial if one partner ceases to provide support to the other. As a result, complex signal exchange mechanisms have evolved to ensure continued, beneficial symbioses. Proper recognition and signal exchange is also the basis for host specificity. Nodule formation always provides a fitness benefit to rhizobia, but does not always provide a fitness benefit to legumes. Therefore, legumes have evolved a mechanism to regulate the number of nodules that are formed, this is called autoregulation of nodulation. Sequencing of many different rhizobia have revealed the presence of several secretion systems - and the Type III, Type IV, and Type VI secretion systems are known to be used by pathogens to transport effector proteins. These secretion systems are also known to have an effect on host specificity and are a determinant of overall nodule number on legumes. This review focuses on signal exchange between rhizobia and legumes, particularly focusing on the role of secretion systems involved in nodule formation and host specificity. PMID:26191069

  18. Protein secretion and outer membrane assembly in Alphaproteobacteria

    PubMed Central

    Gatsos, Xenia; Perry, Andrew J; Anwari, Khatira; Dolezal, Pavel; Wolynec, P Peter; Likić, Vladimir A; Purcell, Anthony W; Buchanan, Susan K; Lithgow, Trevor

    2008-01-01

    The assembly of β-barrel proteins into membranes is a fundamental process that is essential in Gram-negative bacteria, mitochondria and plastids. Our understanding of the mechanism of β-barrel assembly is progressing from studies carried out in Escherichia coli and Neisseria meningitidis. Comparative sequence analysis suggests that while many components mediating β-barrel protein assembly are conserved in all groups of bacteria with outer membranes, some components are notably absent. The Alphaproteobacteria in particular seem prone to gene loss and show the presence or absence of specific components mediating the assembly of β-barrels: some components of the pathway appear to be missing from whole groups of bacteria (e.g. Skp, YfgL and NlpB), other proteins are conserved but are missing characteristic domains (e.g. SurA). This comparative analysis is also revealing important structural signatures that are vague unless multiple members from a protein family are considered as a group (e.g. tetratricopeptide repeat (TPR) motifs in YfiO, β-propeller signatures in YfgL). Given that the process of the β-barrel assembly is conserved, analysis of outer membrane biogenesis in Alphaproteobacteria, the bacterial group that gave rise to mitochondria, also promises insight into the assembly of β-barrel proteins in eukaryotes. PMID:18759741

  19. Granule Protein Processing and Regulated Secretion in Neutrophils

    PubMed Central

    Sheshachalam, Avinash; Srivastava, Nutan; Mitchell, Troy; Lacy, Paige; Eitzen, Gary

    2014-01-01

    Neutrophils are part of a family of granulocytes that, together with eosinophils and basophils, play an essential role in innate immunity. Neutrophils are the most abundant circulating leukocytes and are vital for rapid immune responses, being recruited to sites of injury or infection within minutes, where they can act as specialized phagocytic cells. However, another prominent function of neutrophils is the release of pro-inflammatory compounds, including cytokines, chemokines, and digestive enzymes, which are stored in intracellular compartments and released through regulated exocytosis. Hence, an important feature that contributes to rapid immune responses is capacity of neutrophils to synthesize and store pre-formed pro-inflammatory mediators in specialized intracellular vesicles and thus no new synthesis is required. This review will focus on advancement in three topics relevant to neutrophil secretion. First, we will examine what is known about basal level pro-inflammatory mediator synthesis, trafficking, and storage in secretory compartments. Second, we will review recent advancements in the mechanisms that control vesicle mobilization and the release of pre-formed mediators. Third, we will examine the upregulation and de novo synthesis of pro-inflammatory mediators by neutrophils engaged at sites of infection. PMID:25285096

  20. Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns.

    PubMed

    Arcinas, Arthur; Yen, Ten-Yang; Kebebew, Electron; Macher, Bruce A

    2009-08-01

    Cell surface proteins have been shown to be effective therapeutic targets. In addition, shed forms of these proteins and secreted proteins can serve as biomarkers for diseases, including cancer. Thus, identification of cell surface and secreted proteins has been a prime area of interest in the proteomics field. Most cell surface and secreted proteins are known to be glycosylated, and therefore, a proteomics strategy targeting these proteins was applied to obtain proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using two-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), Hurthle cell carcinoma (XTC-1), and anaplastic thyroid cancer (ARO and DRO-1) were evaluated. An average of 150 glycoproteins were identified per cell line, of which more than 57% are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g., CD44, galectin 3 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in the more well-differentiated thyroid cancer cell lines (follicular, Hurthle cell and papillary), or by cell lines derived from undifferentiated tumors that are uniformly fatal forms of thyroid cancer (i.e., anaplastic). On the basis of the results obtained, a

  1. Cyclophilin C-associated protein: A normal secreted glycoprotein that down-modulates endotoxin and proinflammatory responses in vivo

    PubMed Central

    Trahey, Meg; Weissman, Irving L.

    1999-01-01

    Mouse cyclophilin C-associated protein (CyCAP) is a member of the scavenger-receptor cysteine-rich domain superfamily and is 69% identical to the human Mac-2 binding protein. Here, we show that CyCAP is a widely expressed secreted glycoprotein that modulates the host response to endotoxin. Gene-targeted CyCAP-deficient mice are more sensitive to the lethal effects of endotoxin. In response to endotoxin, CyCAP-deficient mice overproduced interleukin 12 and interferon-γ systemically and tumor necrosis factor α locally; these are proinflammatory molecules that also promote T helper 1 responses. Furthermore, macrophages stimulated in vitro with endotoxin in serum deficient in CyCAP secreted more tumor necrosis factor α, supporting the proposal that CyCAP specifically down-modulates endotoxin signaling. PMID:10077627

  2. EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model.

    PubMed

    Brothers, Kimberly M; Stella, Nicholas A; Romanowski, Eric G; Kowalski, Regis P; Shanks, Robert M Q

    2015-11-01

    Serratia marcescens is a soil- and water-derived bacterium that secretes several host-directed factors and causes hospital infections and community-acquired ocular infections. The putative two-component regulatory system composed of EepR and EepS regulates hemolysis and swarming motility through transcriptional control of the swrW gene and pigment production through control of the pigA-pigN operon. Here, we identify and characterize a role for EepR in regulation of exoenzyme production, stress survival, cytotoxicity to human epithelial cells, and virulence. Genetic analysis supports the model that EepR is in a common pathway with the widely conserved cyclic-AMP receptor protein that regulates protease production. Together, these data introduce a novel regulator of host-pathogen interactions and secreted-protein production. PMID:26324535

  3. EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model

    PubMed Central

    Brothers, Kimberly M.; Stella, Nicholas A.; Romanowski, Eric G.; Kowalski, Regis P.

    2015-01-01

    Serratia marcescens is a soil- and water-derived bacterium that secretes several host-directed factors and causes hospital infections and community-acquired ocular infections. The putative two-component regulatory system composed of EepR and EepS regulates hemolysis and swarming motility through transcriptional control of the swrW gene and pigment production through control of the pigA-pigN operon. Here, we identify and characterize a role for EepR in regulation of exoenzyme production, stress survival, cytotoxicity to human epithelial cells, and virulence. Genetic analysis supports the model that EepR is in a common pathway with the widely conserved cyclic-AMP receptor protein that regulates protease production. Together, these data introduce a novel regulator of host-pathogen interactions and secreted-protein production. PMID:26324535

  4. Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein.

    PubMed

    Xu, Juan; Zhang, Chunhua

    2016-05-01

    Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EV71. High level expression and secretion of VP1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP1 DNA vaccines, including wt-VP1, tPA-VP1, VP1-d, VP1-hFc and VP1-mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P<0.05). In this study, we further investigated the protein levels of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VP1-hFc protein for additional studies. PMID:27533931

  5. Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

    PubMed Central

    Xu, Juan; Zhang, Chunhua

    2016-01-01

    Abstract Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EV71. High level expression and secretion of VP1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP1 DNA vaccines, including wt-VP1, tPA-VP1, VP1-d, VP1-hFc and VP1-mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P<0.05). In this study, we further investigated the protein levels of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VP1-hFc protein for additional studies.

  6. Crystal Structure of Hcp from Acinetobacter baumannii: A Component of the Type VI Secretion System

    PubMed Central

    Ruiz, Federico M.; Santillana, Elena; Spínola-Amilibia, Mercedes; Torreira, Eva; Culebras, Esther; Romero, Antonio

    2015-01-01

    The type VI secretion system (T6SS) is a bacterial macromolecular machine widely distributed in Gram-negative bacteria, which transports effector proteins into eukaryotic host cells or other bacteria. Membrane complexes and a central tubular structure, which resembles the tail of contractile bacteriophages, compose the T6SS. One of the proteins forming this tube is the hemolysin co-regulated protein (Hcp), which acts as virulence factor, as transporter of effectors and as a chaperone. In this study, we present the structure of Hcp from Acinetobacter baumannii, together with functional and oligomerization studies. The structure of this protein exhibits a tight β barrel formed by two β sheets and flanked at one side by a short α-helix. Six Hcp molecules associate to form a donut-shaped hexamer, as observed in both the crystal structure and solution. These results emphasize the importance of this oligomerization state in this family of proteins, despite the low similarity of sequence among them. The structure presented in this study is the first one for a protein forming part of a functional T6SS from A. baumannii. These results will help us to understand the mechanism and function of this secretion system in this opportunistic nosocomial pathogen. PMID:26079269

  7. Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation.

    PubMed

    Menkhorst, Ellen Melaleuca; Lane, Natalie; Winship, Amy Louise; Li, Priscilla; Yap, Joanne; Meehan, Katie; Rainczuk, Adam; Stephens, Andrew; Dimitriadis, Evdokia

    2012-01-01

    Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8) M), medroxyprogesterone acetate (10(-7) M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition

  8. Decidual-Secreted Factors Alter Invasive Trophoblast Membrane and Secreted Proteins Implying a Role for Decidual Cell Regulation of Placentation

    PubMed Central

    Menkhorst, Ellen Melaleuca; Lane, Natalie; Winship, Amy Louise; Li, Priscilla; Yap, Joanne; Meehan, Katie; Rainczuk, Adam; Stephens, Andrew; Dimitriadis, Evdokia

    2012-01-01

    Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the ‘extravillous trophoblast’ (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10−8 M), medroxyprogesterone acetate (10−7 M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition

  9. Artemisinin Induces Calcium-Dependent Protein Secretion in the Protozoan Parasite Toxoplasma gondii▿ †

    PubMed Central

    Nagamune, Kisaburo; Beatty, Wandy L.; Sibley, L. David

    2007-01-01

    Intracellular calcium controls several crucial cellular events in apicomplexan parasites, including protein secretion, motility, and invasion into and egress from host cells. The plant compound thapsigargin inhibits the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), resulting in elevated calcium and induction of protein secretion in Toxoplasma gondii. Artemisinins are natural products that show potent and selective activity against parasites, making them useful for the treatment of malaria. While the mechanism of action is uncertain, previous studies have suggested that artemisinin may inhibit SERCA, thus disrupting calcium homeostasis. We cloned the single-copy gene encoding SERCA in T. gondii (TgSERCA) and demonstrate that the protein localizes to the endoplasmic reticulum in the parasite. In extracellular parasites, TgSERCA partially relocalized to the apical pole, a highly active site for regulated secretion of micronemes. TgSERCA complemented a calcium ATPase-defective yeast mutant, and this activity was inhibited by either thapsigargin or artemisinin. Treatment of T. gondii with artemisinin triggered calcium-dependent secretion of microneme proteins, similar to the SERCA inhibitor thapsigargin. Artemisinin treatment also altered intracellular calcium in parasites by increasing the periodicity of calcium oscillations and inducing recurrent, strong calcium spikes, as imaged using Fluo-4 labeling. Collectively, these results demonstrate that artemisinin perturbs calcium homeostasis in T. gondii, supporting the idea that Ca2+-ATPases are potential drug targets in parasites. PMID:17766463

  10. Stimulus-dependent secretion of plasma proteins from human neutrophils.

    PubMed Central

    Borregaard, N; Kjeldsen, L; Rygaard, K; Bastholm, L; Nielsen, M H; Sengeløv, H; Bjerrum, O W; Johnsen, A H

    1992-01-01

    In search for matrix proteins released from secretory vesicles of human neutrophils, a prominent 67-kD protein was identified in the extracellular medium of neutrophils stimulated by the chemotactic peptide, FMLP. The protein was purified to apparent homogeneity and partially sequenced. The sequence of the first 32 NH2-terminal amino acids was identical to the sequence of albumin. mRNA for human albumin could not be detected in bone marrow cells, nor could biosynthetic labeling of albumin be demonstrated in bone marrow cells during incubation with [14C]leucine. Immunofluorescence studies on single cells demonstrated the presence of intracellular albumin in fixed permeabilized neutrophils. Light microscopy of immunogold-silver-stained cryosections visualized albumin in cytoplasmic "granules." The morphology of these was determined by immunoelectron microscopy as vesicles of varying form and size. Subcellular fractionation studies on unstimulated neutrophils demonstrated the presence of albumin in the low density pre-gamma and gamma-regions that contain secretory vesicles, but are devoid of specific granules and azurophil granules. Albumin was readily released from these structures during activation of neutrophils with inflammatory mediators. Immunoblotting demonstrated the presence of immunoglobulin and transferrin along with albumin in exocytosed material from stimulated neutrophils. This indicates that secretory vesicles are unique endocytic vesicles that can be triggered to exocytose by inflammatory stimuli. Images PMID:1378856

  11. Closing the circle on the discovery of genes encoding Hrp regulon members and type III secretion system effectors in the genomes of three model Pseudomonas syringae strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas syringae strains translocate large and distinct collections of effector proteins into plant cells via the type III secretion system (T3SS). P. syringae effectors are designated Hop (Hrp outer protein) or Avr (avirulence) proteins. Some Hop proteins are considered to be extracellular T3...

  12. Cell Wall and Secreted Proteins of Candida albicans: Identification, Function, and Expression

    PubMed Central

    Chaffin, W. Lajean; López-Ribot, José Luis; Casanova, Manuel; Gozalbo, Daniel; Martínez, José P.

    1998-01-01

    The cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was intially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis. PMID:9529890

  13. Proteomic Profiling of Cereal Aphid Saliva Reveals Both Ubiquitous and Adaptive Secreted Proteins

    PubMed Central

    Wilkinson, Tom L.

    2013-01-01

    The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113. PMID:23460852

  14. Improving Protein Production on the Level of Regulation of both Expression and Secretion Pathways in Bacillus subtilis.

    PubMed

    Song, Yafeng; Nikoloff, Jonas M; Zhang, Dawei

    2015-07-01

    The well-characterized gram-positive bacterium Bacillus subtilis is an outstanding industrial candidate for protein expression owing to its single membrane and high capacity of secretion, simplifying the downstream processing of secretory proteins. During the last few years, there has been continuous progress in the illustration of secretion mechanisms and application of this robust host in various fields of life science, such as enzyme production, feed additives, and food and pharmaceutical industries. Here, we review the developments of Bacillus subtilis as a highly promising expression system illuminating strong chemical- and temperatureinducible and other types of promoters, strategies for ribosome-binding-site utilization, and the novel approach of signal peptide selection. Furthermore, we outline the main steps of the Sec pathway and the relevant elements as well as their interactions. In addition, we introduce the latest discoveries of Tat-related complex structures and functions and the countless applications of this full-folded protein secretion pathway. This review also lists some of the current understandings of ATP-binding cassette transporters. According to the extensive knowledge on the genetic modification strategies and molecular biology of Bacillus subtilis, we propose some suggestions and strategies for improving the yield of intended productions. We expect this to promote striking future developments in the optimization and application of this bacterium. PMID:25737123

  15. Apocrine Secretion in Drosophila Salivary Glands: Subcellular Origin, Dynamics, and Identification of Secretory Proteins

    PubMed Central

    Farkaš, Robert; Ďatková, Zuzana; Mentelová, Lucia; Löw, Péter; Beňová-Liszeková, Denisa; Beňo, Milan; Sass, Miklós; Řehulka, Pavel; Řehulková, Helena; Raška, Otakar; Kováčik, Lubomír; Šmigová, Jana; Raška, Ivan; Mechler, Bernard M.

    2014-01-01

    In contrast to the well defined mechanism of merocrine exocytosis, the mechanism of apocrine secretion, which was first described over 180 years ago, remains relatively uncharacterized. We identified apocrine secretory activity in the late prepupal salivary glands of Drosophila melanogaster just prior to the execution of programmed cell death (PCD). The excellent genetic tools available in Drosophila provide an opportunity to dissect for the first time the molecular and mechanistic aspects of this process. A prerequisite for such an analysis is to have pivotal immunohistochemical, ultrastructural, biochemical and proteomic data that fully characterize the process. Here we present data showing that the Drosophila salivary glands release all kinds of cellular proteins by an apocrine mechanism including cytoskeletal, cytosolic, mitochondrial, nuclear and nucleolar components. Surprisingly, the apocrine release of these proteins displays a temporal pattern with the sequential release of some proteins (e.g. transcription factor BR-C, tumor suppressor p127, cytoskeletal β-tubulin, non-muscle myosin) earlier than others (e.g. filamentous actin, nuclear lamin, mitochondrial pyruvate dehydrogenase). Although the apocrine release of proteins takes place just prior to the execution of an apoptotic program, the nuclear DNA is never released. Western blotting indicates that the secreted proteins remain undegraded in the lumen. Following apocrine secretion, the salivary gland cells remain quite vital, as they retain highly active transcriptional and protein synthetic activity. PMID:24732043

  16. Identifying mutated proteins secreted by colon cancer cell lines using mass spectrometry.

    PubMed

    Mathivanan, Suresh; Ji, Hong; Tauro, Bow J; Chen, Yuan-Shou; Simpson, Richard J

    2012-12-01

    Secreted proteins encoded by mutated genes (mutant proteins) are a particularly rich source of biomarkers being not only components of the cancer secretome but also actually implicated in tumorigenesis. One of the challenges of proteomics-driven biomarker discovery research is that the bulk of secreted mutant proteins cannot be identified directly and quantified by mass spectrometry due to the lack of mutated peptide information in extant proteomics databases. Here we identify, using an integrated genomics and proteomics strategy (referred to iMASp - identification of Mutated And Secreted proteins), 112 putative mutated tryptic peptides (corresponding to 57 proteins) in the collective secretomes derived from a panel of 18 human colorectal cancer (CRC) cell lines. Central to this iMASp was the creation of Human Protein Mutant Database (HPMD), against which experimentally-derived secretome peptide spectra were searched. Eight of the identified mutated tryptic peptides were confirmed by RT-PCR and cDNA sequencing of RNA extracted from those CRC cells from which the mutation was identified by mass spectrometry. The iMASp technology promises to improve the link between proteomics and genomic mutation data thereby providing an effective tool for targeting tryptic peptides with mutated amino acids as potential cancer biomarker candidates. This article is part of a Special Issue entitled: Integrated omics. PMID:22796352

  17. Fibroblastic synoviocytes secrete plasma proteins via α2 -macroglobulins serving as intracellular and extracellular chaperones.

    PubMed

    Zhao, Ke-Wei; Murray, Elsa J Brochmann; Murray, Samuel S

    2015-11-01

    Changes in plasma protein levels in synovial fluid (SF) have been implicated in osteoarthritis and rheumatoid arthritis. It was previously thought that the presence of plasma proteins in SF reflected ultrafiltration or extravasation from the vasculature, possibly due to retraction of inflamed endothelial cells. Recent proteomic analyses have confirmed the abundant presence of plasma proteins in SF from control and arthritic patients. Systematic depletion of high-abundance plasma proteins from SF and conditioned media from synoviocytes cultured in serum, and protein analysis under denaturing/reducing conditions have limited our understanding of sources and the native structures of "plasma protein" complexes in SF. Using Western blotting, qPCR, and mass spectrometry, we found that Hig-82 lapine fibroblastic synovicytes cultured under serum-free conditions expressed and secreted plasma proteins, including the cytokine-binding protein secreted phosphoprotein 24 kDa (Spp24) and many of the proteases and protease inhibitors found in SF. Treating synoviocytes with TGF-β1 or BMP-2 for 24 h upregulated the expression of plasma proteins, including Spp24, α2 -HS-glycoprotein, α1 -antitrypsin, IGF-1, and C-reactive protein. Furthermore, many of the plasma proteins of mass <151 kDa were secreted as disulfide-bound complexes with members of the α2 -macroglobulin (A2M) family, which serve as intracellular and extracellular chaperones, not protease inhibitors. Using brefeldin A to block vesicular traffic and protease inhibitors to inhibit endogenous activation of naïve A2M, we demonstrated that the complexes were formed in the endoplasmic reticulum lumen and that Ca(2+) cysteine protease-dependent processes are involved. PMID:25900303

  18. Neuroprotective Secreted Amyloid Precursor Protein Acts by Disrupting Amyloid Precursor Protein Dimers*S⃞

    PubMed Central

    Gralle, Matthias; Botelho, Michelle Gralle; Wouters, Fred S.

    2009-01-01

    The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPPα and the neurotoxic β-amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of Förster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPPα, as sAPPα binding disrupts APP dimers, and this disruption of APP dimers by sAPPα is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPPα. These findings suggest a potentially beneficial effect of increasing sAPPα production or disrupting APP dimers for neuronal survival. PMID:19336403

  19. Secretome Analysis of Vibrio cholerae Type VI Secretion System Reveals a New Effector-Immunity Pair

    PubMed Central

    Altindis, Emrah; Dong, Tao; Catalano, Christy

    2015-01-01

    ABSTRACT The type VI secretion system (T6SS) is a dynamic macromolecular organelle that many Gram-negative bacteria use to inhibit or kill other prokaryotic or eukaryotic cells. The toxic effectors of T6SS are delivered to the prey cells in a contact-dependent manner. In Vibrio cholerae, the etiologic agent of cholera, T6SS is active during intestinal infection. Here, we describe the use of comparative proteomics coupled with bioinformatics to identify a new T6SS effector-immunity pair. This analysis was able to identify all previously identified secreted substrates of T6SS except PAAR (proline, alanine, alanine, arginine) motif-containing proteins. Additionally, this approach led to the identification of a new secreted protein encoded by VCA0285 (TseH) that carries a predicted hydrolase domain. We confirmed that TseH is toxic when expressed in the periplasm of Escherichia coli and V. cholerae cells. The toxicity observed in V. cholerae was suppressed by coexpression of the protein encoded by VCA0286 (TsiH), indicating that this protein is the cognate immunity protein of TseH. Furthermore, exogenous addition of purified recombinant TseH to permeabilized E. coli cells caused cell lysis. Bioinformatics analysis of the TseH protein sequence suggest that it is a member of a new family of cell wall-degrading enzymes that include proteins belonging to the YD repeat and Rhs superfamilies and that orthologs of TseH are likely expressed by species belonging to phyla as diverse as Bacteroidetes and Proteobacteria. PMID:25759499

  20. The Effect of α-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris

    PubMed Central

    Lin-Cereghino, Geoff P.; Stark, Carolyn M.; Kim, Daniel; Chang, Jennifer; Shaheen, Nadia; Poerwanto, Hansel; Agari, Kimiko; Moua, Pachai; Low, Lauren K.; Tran, Namphuong; Huang, Amy D.; Nattestad, Maria; Oshiro, Kristin T.; Chang, John William; Chavan, Archana; Tsai, Jerry W.; Lin-Cereghino, Joan

    2013-01-01

    The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. PMID:23454485

  1. Combination of hydrogel nanoparticles and proteomics to reveal secreted proteins associated with decidualization of human uterine stromal cells

    PubMed Central

    2011-01-01

    Background Identification of secreted proteins of low abundance is often limited by abundant and high molecular weight (MW) proteins. We have optimised a procedure to overcome this limitation. Results Low MW proteins in the conditioned media of cultured cells were first captured using dual-size exclusion/affinity hydrogel nanoparticles and their identities were then revealed by proteomics. Conclusions This technique enables the analysis of secreted proteins of cultured cells low MW and low abundance. PMID:21884602

  2. Lack of a surface layer in Tannerella forsythia mutants deficient in the type IX secretion system

    PubMed Central

    Narita, Yuka; Sato, Keiko; Yukitake, Hideharu; Shoji, Mikio; Nakane, Daisuke; Nagano, Keiji; Yoshimura, Fuminobu; Naito, Mariko

    2014-01-01

    Tannerella forsythia, a Gram-negative anaerobic bacterium, is an important pathogen in periodontal disease. This bacterium possesses genes encoding all known components of the type IX secretion system (T9SS). T. forsythia mutants deficient in genes orthologous to the T9SS-encoding genes porK, porT and sov were constructed. All porK, porT and sov single mutants lacked the surface layer (S-layer) and expressed less-glycosylated versions of the S-layer glycoproteins TfsA and TfsB. In addition, these mutants exhibited decreased haemagglutination and increased biofilm formation. Comparison of the proteins secreted by the porK and WT strains revealed that the secretion of several proteins containing C-terminal domain (CTD)-like sequences is dependent on the porK gene. These results indicate that the T9SS is functional in T. forsythia and contributes to the translocation of CTD proteins to the cell surface or into the extracellular milieu. PMID:25023245

  3. Kin of IRRE-like Protein 2 Is a Phosphorylated Glycoprotein That Regulates Basal Insulin Secretion.

    PubMed

    Yesildag, Burcak; Bock, Thomas; Herrmanns, Karolin; Wollscheid, Bernd; Stoffel, Markus

    2015-10-23

    Direct interactions among pancreatic β-cells via cell surface proteins inhibit basal and enhance stimulated insulin secretion. Here, we functionally and biochemically characterized Kirrel2, an immunoglobulin superfamily protein with β-cell-specific expression in the pancreas. Our results show that Kirrel2 is a phosphorylated glycoprotein that co-localizes and interacts with the adherens junction proteins E-cadherin and β-catenin in MIN6 cells. We further demonstrate that the phosphosites Tyr(595-596) are functionally relevant for the regulation of Kirrel2 stability and localization. Analysis of the extracellular and intracellular domains of Kirrel2 revealed that it is cleaved and shed from MIN6 cells and that the remaining membrane spanning cytoplasmic domain is processed by γ-secretase complex. Kirrel2 knockdown with RNA interference in MIN6 cells and ablation of Kirrel2 from mice with genetic deletion resulted in increased basal insulin secretion from β-cells, with no immediate influence on stimulated insulin secretion, total insulin content, or whole body glucose metabolism. Our results show that in pancreatic β-cells Kirrel2 localizes to adherens junctions, is regulated by multiple post-translational events, including glycosylation, extracellular cleavage, and phosphorylation, and engages in the regulation of basal insulin secretion. PMID:26324709

  4. Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase

    PubMed Central

    Kato, Junya; Lefebre, Matthew

    2015-01-01

    ABSTRACT Many bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with a Salmonella enterica serovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines. IMPORTANCE Type III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases from Salmonella enterica serovar Typhimurium that help to explain important aspects of its function. PMID:26170413

  5. The Coxiella burnetii Cryptic Plasmid Is Enriched in Genes Encoding Type IV Secretion System Substrates▿ †

    PubMed Central

    Voth, Daniel E.; Beare, Paul A.; Howe, Dale; Sharma, Uma M.; Samoilis, Georgios; Cockrell, Diane C.; Omsland, Anders; Heinzen, Robert A.

    2011-01-01

    The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a phagolysosome-like parasitophorous vacuole (PV), in which it replicates. The organism encodes a Dot/Icm type IV secretion system (T4SS) predicted to deliver to the host cytosol effector proteins that mediate PV formation and other cellular events. All C. burnetii isolates carry a large, autonomously replicating plasmid or have chromosomally integrated plasmid-like sequences (IPS), suggesting that plasmid and IPS genes are critical for infection. Bioinformatic analyses revealed two candidate Dot/Icm substrates with eukaryotic-like motifs uniquely encoded by the QpH1 plasmid from the Nine Mile reference isolate. CpeC, containing an F-box domain, and CpeD, possessing kinesin-related and coiled-coil regions, were secreted by the closely related Legionella pneumophila Dot/Icm T4SS. An additional QpH1-specific gene, cpeE, situated in a predicted operon with cpeD, also encoded a secreted effector. Further screening revealed that three hypothetical proteins (CpeA, CpeB, and CpeF) encoded by all C. burnetii plasmids and IPS are Dot/Icm substrates. By use of new genetic tools, secretion of plasmid effectors by C. burnetii during host cell infection was confirmed using β-lactamase and adenylate cyclase translocation assays, and a C-terminal secretion signal was identified. When ectopically expressed in HeLa cells, plasmid effectors trafficked to different subcellular sites, including autophagosomes (CpeB), ubiquitin-rich compartments (CpeC), and the endoplasmic reticulum (CpeD). Collectively, these results suggest that C. burnetii plasmid-encoded T4SS substrates play important roles in subversion of host cell functions, providing a plausible explanation for the absolute maintenance of plasmid genes by this pathogen. PMID:21216993

  6. Lipids and proteins in the Rathke's gland secretions of the North American mud turtle (Kinosternon subrubrum)

    USGS Publications Warehouse

    Seifert, W.E., Jr.; Gotte, S.W.; Leto, T.L.; Weldon, P.J.

    1994-01-01

    Lipids and proteins in the Rathke's gland secretions of the North American mud turtle (Kinosternon subrubrum, Kinosternidae) were analyzed by gas chromatography-mass spectrometry (GC-MS) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Analysis by GC-MS indicates 2,3-dihydroxypropanal and C3?C24 free or esterified fatty acids. Analysis by SDS-PAGE indicates a major protein component with an approximate molecular mass of 60 kDa and minor components ranging from ca. 23 to 34 kDa. The major component of K. subrubrum glandular secretions exhibits a mobility that matches that of the Kemp's ridley sea turtle (Lepidochelys kempi, Cheloniidae), suggesting that these proteins are evolutionarily conserved.

  7. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

    PubMed Central

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-01-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives. PMID:26508080

  8. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins.

    PubMed

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N; Patil, Navinkumar J; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-01-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives. PMID:26508080

  9. Proteomics informed by transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva

    SciTech Connect

    Mudenda, Lwiindi; Aguilar Pierle, Sebastian; Turse, Joshua E.; Scoles, Glen A.; Purvine, Samuel O.; Nicora, Carrie D.; Clauss, Therese RW; Ueti, Massaro W.; Brown, Wendy C.; Brayton, Kelly A.

    2014-08-07

    Dermacentor andersoni, known as the Rocky Mountain wood tick, is found in the western United States and transmits pathogens that cause diseases of veterinary and public health importance including Rocky Mountain spotted fever, tularemia, Colorado tick fever and bovine anaplasmosis. Tick saliva is known to modulate both innate and acquired immune responses, enabling ticks to feed for several days without detection. During feeding ticks subvert host defences such as hemostasis and inflammation, which would otherwise result in coagulation, wound repair and rejection of the tick. Molecular characterization of the proteins and pharmacological molecules secreted in tick saliva offers an opportunity to develop tick vaccines as an alternative to the use of acaricides, as well as new anti-inflammatory drugs. We performed proteomics informed by transcriptomics to identify D. andersoni saliva proteins that are secreted during feeding. The transcript data generated a database of 21,797 consensus sequences, which we used to identify 677 proteins secreted in the saliva of D. andersoni ticks fed for 2 and 5 days, following proteomic investigations of whole saliva using mass spectrometry. Salivary gland transcript levels of unfed ticks were compared with 2 and 5 day fed ticks to identify genes upregulated early during tick feeding. We cross-referenced the proteomic data with the transcriptomic data to identify 157 proteins of interest for immunomodulation and blood feeding. Proteins of unknown function as well as known immunomodulators were identified.

  10. In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response

    PubMed Central

    Rebnegger, Corinna; Graf, Alexandra B; Valli, Minoska; Steiger, Matthias G; Gasser, Brigitte; Maurer, Michael; Mattanovich, Diethard

    2014-01-01

    Protein production in yeasts is related to the specific growth rate μ. To elucidate on this correlation, we studied the transcriptome of Pichia pastoris at different specific growth rates by cultivating a strain secreting human serum albumin at μ = 0.015 to 0.15 h–1 in glucose-limited chemostats. Genome-wide regulation revealed that translation-related as well as mitochondrial genes were upregulated with increasing μ, while autophagy and other proteolytic processes, carbon source-responsive genes and other targets of the TOR pathway as well as many transcriptional regulators were downregulated at higher μ. Mating and sporulation genes were most active at intermediate μ of 0.05 and 0.075 h–1. At very slow growth (μ = 0.015 h–1) gene regulation differs significantly, affecting many transporters and glucose sensing. Analysis of a subset of genes related to protein folding and secretion reveals that unfolded protein response targets such as translocation, endoplasmic reticulum genes, and cytosolic chaperones are upregulated with increasing growth rate while proteolytic degradation of secretory proteins is downregulated. We conclude that a high μ positively affects specific protein secretion rates by acting on multiple cellular processes. PMID:24323948

  11. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

    NASA Astrophysics Data System (ADS)

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-10-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives.

  12. Proteomics informed by transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva.

    PubMed

    Mudenda, Lwiindi; Pierlé, Sebastián Aguilar; Turse, Joshua E; Scoles, Glen A; Purvine, Samuel O; Nicora, Carrie D; Clauss, Therese R W; Ueti, Massaro W; Brown, Wendy C; Brayton, Kelly A

    2014-11-01

    Dermacentor andersoni, known as the Rocky Mountain wood tick, is found in the western United States and transmits pathogens that cause diseases of veterinary and public health importance including Rocky Mountain spotted fever, tularemia, Colorado tick fever and bovine anaplasmosis. Tick saliva is known to modulate both innate and acquired immune responses, enabling ticks to feed for several days without detection. During feeding ticks subvert host defences such as hemostasis and inflammation, which would otherwise result in coagulation, wound repair and rejection of the tick. Molecular characterization of the proteins and pharmacological molecules secreted in tick saliva offers an opportunity to develop tick vaccines as an alternative to the use of acaricides, as well as new anti-inflammatory drugs. We performed proteomics informed by transcriptomics to identify D. andersoni saliva proteins that are secreted during feeding. The transcript data generated a database of 21,797 consensus sequences, which we used to identify 677 proteins secreted in the saliva of D. andersoni ticks fed for 2 and 5days, following proteomic investigations of whole saliva using mass spectrometry. Salivary gland transcript levels of unfed ticks were compared with 2 and 5day fed ticks to identify genes upregulated early during tick feeding. We cross-referenced the proteomic data with the transcriptomic data to identify 157 proteins of interest for immunomodulation and blood feeding. Proteins of unknown function as well as known immunomodulators were identified. PMID:25110293

  13. Cag Type IV Secretion System: CagI Independent Bacterial Surface Localization of CagA

    PubMed Central

    Kumar, Navin; Shariq, Mohd; Kumari, Rajesh; Tyagi, Rakesh K.; Mukhopadhyay, Gauranga

    2013-01-01

    Helicobacter pylori Cag type IV secretion system (Cag-T4SS) is a multi-component transporter of oncoprotein CagA across the bacterial membranes into the host epithelial cells. To understand the role of unique Cag-T4SS component CagI in CagA translocation, we have characterized it by biochemical and microscopic approaches. We observed that CagI is a predominantly membrane attached periplasmic protein partially exposed to the bacterial surface especially on the pili. The association of the protein with membrane appeared to be loose as it could be easily recovered in soluble fraction. We documented that the stability of the protein is dependent on several key components of the secretion system and it has multiple interacting partners including a non-cag-PAI protein HP1489. Translocation of CagA across the bacterial membranes to cell surface is CagI-independent process. The observations made herein are expected to assist in providing an insight into the substrate translocation by the Cag-T4SS system and Helicobacter pylori pathogenesis. PMID:24040297

  14. Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia.

    PubMed

    Konrad, Christian; Spycher, Cornelia; Hehl, Adrian B

    2010-04-01

    Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1-3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this "minimal Golgi" hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting

  15. Selective Condensation Drives Partitioning and Sequential Secretion of Cyst Wall Proteins in Differentiating Giardia lamblia

    PubMed Central

    Konrad, Christian; Spycher, Cornelia; Hehl, Adrian B.

    2010-01-01

    Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1–3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this “minimal Golgi” hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi

  16. A genome-wide RNA interference screen uncovers two p24 proteins as regulators of Wingless secretion.

    PubMed

    Port, Fillip; Hausmann, George; Basler, Konrad

    2011-11-01

    Wnt proteins are secreted, lipid-modified glycoproteins that control animal development and adult tissue homeostasis. Secretion of Wnt proteins is at least partly regulated by a dedicated machinery. Here, we report a genome-wide RNA interference screen for genes involved in the secretion of Wingless (Wg), a Drosophila Wnt. We identify three new genes required for Wg secretion. Of these, Emp24 and Eclair are required for proper export of Wg from the endoplasmic reticulum (ER). We propose that Emp24 and Eca act as specific cargo receptors for Wg to concentrate it in forming vesicles at sites of ER export. PMID:21886182

  17. Exercise, but not acute sleep loss, increases salivary antimicrobial protein secretion.

    PubMed

    Gillum, Trevor L; Kuennen, Matthew R; Castillo, Micaela N; Williams, Nicole L; Jordan-Patterson, Alex T

    2015-05-01

    Sleep deficiencies may play a role in depressing immune parameters. Little is known about the impact of exercise after sleep deprivation on mucosal immunity. The purpose of this study was to quantify salivary antimicrobial proteins (AMPs) in response to sleep loss before and after exercise. Four men and 4 women (age: 22.8 ± 2; : 49.1 ± 7.1 ml · kg(-1) · min(-1)) completed 2 exercise trials consisting of 45 minutes of running at 75% VO2peak after a normal night of sleep (CON) and after a night without sleep (WS). Exercise trials were separated by 10 ± 3 days. Saliva was collected before, immediately after, and 1 hour after exercise. LL-37, HNP1-3, Lactoferrin (Lac), and Lysozyme (Lys) were measured. Sleep loss did not affect the concentration or secretion rate of AMPs before or in response to exercise. However, exercise increased the concentration from pre- to post-exercise of LL-37 (pre: 15.5 ± 8.7; post: 22.3 ± 16.2 ng · ml(-1)), HNP1-3 (pre: 2.2 ± 2.3; post: 3.3 ± 2.5 µg · ml(-1)), Lac (pre: 5,234 ± 4,202; post: 12,283 ± 10,995 ng · ml(-1)), and Lys (pre: 5,831 ± 4,465; post: 12,542 ± 10,755 ng · ml(-1)), p <= 0.05. The secretion rates were higher immediately after and 1 hour after exercise compared with before exercise for LL-37 (pre: 3.1 ± 2.1; post: 5.1 ± 3.7; +1: 6.9 ± 8.4 ng · min(-1)), HNP1-3 (pre: 0.38 ± 0.38; post: 0.80 ± 0.75; +1: 0.84 ± 0.67 µg · min(-1)), Lac (pre: 1,096 ± 829; post: 2,948 ± 2,923; +1: 2,464 ± 3,785 ng · min(-1)), and Lys (pre: 1,534 ± 1,790; post: 3,042 ± 2,773; +1: 1,916 ± 1,682 ng · min-(1)), p <= 0.05. These data suggest that the major constituents of the mucosal immune system are unaffected by acute sleep loss and by exercise after acute sleep loss. Exercise increased the concentration and secretion rate of each AMP suggesting enhanced immunity and control of inflammation, despite limited sleep. PMID:25915527

  18. Comparison of different signal peptides for secretion of heterologous proteins in fission yeast

    SciTech Connect

    Kjaerulff, Soren

    2005-10-28

    In the fission yeast Schizosaccharomyces pombe, there are relatively few signal peptides available and most reports of their activity have not been comparative. Using sequence information from the S. pombe genome database we have identified three putative signal peptides, designated Cpy, Amy and Dpp, and compared their ability to support secretion of green fluorescent protein (GFP). In the comparison we also included the two well-described secretion signals derived from the precursors of, respectively, the Saccharomyces cerevisiae {alpha}-factor and the S. pombe P-factor. The capability of the tested signal peptides to direct secretion of GFP varied greatly. The {alpha}-factor signal did not confer secretion to GFP and all the produced GFP was trapped intracellular. In contrast, the Cpy signal peptide supported efficient secretion of GFP with yields approximating 10 mg/L. We also found that the use of an attenuated version of the S. cerevisiae URA3 marker substantially increases vector copy number and expression yield in fission yeast.

  19. Genetic analysis of an MDR-like export system: the secretion of colicin V.

    PubMed

    Gilson, L; Mahanty, H K; Kolter, R

    1990-12-01

    The extracellular secretion of the antibacterial toxin colicin V is mediated via a signal sequence independent process which requires the products of two linked genes: cvaA and cvaB. The nucleotide sequence of cvaB reveals that its product is a member of a subfamily of proteins, involved in the export of diverse molecules, found in both eukaryotes and prokaryotes. This group of proteins, here referred to as the 'MDR-like' subfamily, is characterized by the presence of a hydrophobic region followed by a highly conserved ATP binding fold. By constructing fusions between the structural gene for colicin V, cvaC, and a gene for alkaline phosphatase, phoA, lacking its signal sequence, it was determined that 39 codons in the N-terminus of cvaC contained the structural information to allow CvaC-PhoA fusion proteins to be efficiently translocated across the plasma membrane of Escherichia coli in a CvaA/CvaB dependent fashion. This result is consistent with the location of point mutations in the cvaC gene which yielded export deficient colicin V. The presence of the export signal at the N-terminus of CvaC contrasts with the observed C-terminal location of the export signal for hemolysin, which also utilizes an MDR-like protein for its secretion. It was also found that the CvaA component of the colicin V export system shows amino acid sequence similarities with another component involved in hemolysin export, HlyD. The role of the second component in these systems and the possibility that other members of the MDR-like subfamily will also have corresponding second components are discussed. A third component used in both colicin V and hemolysin extracellular secretion is the E. coli host outer membrane protein, TolC. PMID:2249654

  20. The marine bacteria Shewanella frigidimarina NCIMB400 upregulates the type VI secretion system during early biofilm formation.

    PubMed

    Linares, Denis; Jean, Natacha; Van Overtvelt, Perrine; Ouidir, Tassadit; Hardouin, Julie; Blache, Yves; Molmeret, Maëlle

    2016-02-01

    Shewanella sp. are facultative anaerobic Gram-negative bacteria, extensively studied for their electron transfer ability. Shewanella frigidimarina has been detected and isolated from marine environments, and in particular, from biofilms. However, its ability to adhere to surfaces and form a biofilm is poorly understood. In this study, we show that the ability to adhere and to form a biofilm of S. frigidimarina NCIMB400 is significantly higher than that of Shewanella oneidensis in our conditions. We also show that this strain forms a biofilm in artificial seawater, whereas in Luria-Bertani, this capacity is reduced. To identify proteins involved in early biofilm formation, a proteomic analysis of sessile versus planktonic membrane-enriched fractions allowed the identification of several components of the same type VI secretion system gene cluster: putative Hcp1 and ImpB proteins as well as a forkhead-associated domain-containing protein. The upregulation of Hcp1 a marker of active translocation has been confirmed using quantitative reverse transcription polymerase chain reaction. Our data demonstrated the presence of a single and complete type VI secretion system in S. frigidimarina NCIMB400 genome, upregulated in sessile compared with planktonic conditions. The fact that three proteins including the secreted protein Hcp1 have been identified may suggest that this type VI secretion system is functional. PMID:26617163

  1. Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways.

    PubMed

    Perera, Erick; Rodríguez-Viera, Leandro; Rodríguez-Casariego, Javier; Fraga, Iliana; Carrillo, Olimpia; Martínez-Rodríguez, Gonzalo; Mancera, Juan M

    2012-03-01

    The effects of pelleted diets with different protein composition (fish, squid or soybean meals as main protein sources) on trypsin secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4 h after ingestion. At this time, fish- and squid-based diets induced trypsin secretion, as well as up-regulation of the major trypsin isoform at the transcription level. While fish- and squid-based diets elicited a prandial response, soybean-based diet failed to stimulate the digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters. In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However, the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free amino acids in the digestive gland, suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting appropriated protein sources. However, other factors like the poor solubility of dietary proteins in dry diets could hamper further enhancement of digestion efficiency. PMID:22323208

  2. Apparent inhibition of. beta. -fructosidase secretion by tunicamycin may be explained by breakdown of the unglycosylated protein during secretion. [Daucus carota

    SciTech Connect

    Faye, L. ); Chrispeels, M.J. )

    1989-03-01

    Suspension-cultured carrot (Daucus carota) cells synthesize and secrete {beta}-fructosidase, a glycoprotein with asparagine-linked glycans. Treatment of the cells with tunicamycin completely inhibits the apparent secretion of {beta}-fructosidase as measured by the accumulation of the {sup 35}S-labelled protein in the cell wall or the culture medium. In the past, such a result has been interpreted as an inhibition of secretion by tunicamycin, but we suggest another explanation based on the following results. In the presence of tunicamycin, unglycosylated {beta}-fructosidase is synthesized and is associated with an endoplasmic-reticulum-rich microsomal fraction. Pulse-chase experiments show that the unglycosylated {beta}-fructosidase does not remain in the cells and appears to be secreted in the same way as glycosylated {beta}-fructosidase; however, no radioactive, unglycosylated {beta}-fructosidase accumulates extracellularly (cell wall or medium). Protoplasts obtained from carrot cells secrete {beta}-fructosidase protein and activity, and treatment of the protoplasts with tunicamycin results in the synthesis of unglycosylated {beta}-fructosidase. In the presence of tunicamycin, there is no accumulation of {beta}-fructosidase activity or unglycosylated {beta}-fructosidase polypeptide in the protoplast incubation medium. These results are consistent with the interpretation that the glycans of {beta}-fructosidase are necessary for its stability, and that in these suspension-cultured cells, the unglycosylated enzyme is degraded during the last stage(s) of secretion, or immediately after its arrival in the wall.

  3. Differential distributions of the Ca2+ -dependent activator protein for secretion family proteins (CAPS2 and CAPS1) in the mouse brain.

    PubMed

    Sadakata, Tetsushi; Itakura, Makoto; Kozaki, Shunji; Sekine, Yukiko; Takahashi, Masami; Furuichi, Teiichi

    2006-04-20

    The Ca(2+)-dependent activator protein for secretion (CAPS/Cadps) family consists of two members, CAPS1 and CAPS2, and plays an important role in secretory granule exocytosis. It has been shown that CAPS1 regulates catecholamine release from neuroendocrine cells, whereas CAPS2 is involved in the release of two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), from parallel fibers of cerebellar granule cells. Although both CAPS proteins are expressed predominantly in the brain, their cellular and regional distributions in the brain are largely unknown. In this study we analyzed the immunohistochemical distributions of the CAPS family proteins in the mouse brain. In most areas of the embryonic nervous system CAPS1 and CAPS2 proteins were complementarily expressed. In the postnatal brain, CAPS1 was widespread at different levels. On the other hand, CAPS2 was localized to distinct cell types and fibers of various brain regions, including the olfactory bulb, cerebrum, hippocampal formation, thalamus, mesencephalic tegmentum, cerebellum, medulla, and spinal cord, except for some regions that overlapped with CAPS1. These CAPS2 cellular distribution patterns had the marked feature of coinciding with those of BDNF in various brain regions. Immunolabels for CAPS2 were also colocalized with those for some proteins related to exocytosis (VAMP and SNAP-25) and endocytosis (Dynamin I) in the cell soma and processes of the mesencephalic tegmentum and cerebellum, suggesting that these proteins might be involved in the dynamics of CAPS2-associated vesicles, although their colocalization on vesicles remains elusive. These results demonstrate that the CAPS family proteins are involved in the secretion of different secretory substances in developing and postnatal brains, and that CAPS2 is probably involved in BDNF secretion in many brain areas. PMID:16506193

  4. Comparative analysis of secretomes from Ectomycorrhizal fungi with an emphasis on small-secreted proteins

    DOE PAGESBeta

    Pellegrin, Clement; Morin, Emmanuelle; Martin, Francis M.; Veneault-Fourrey, Claire

    2015-11-18

    Fungi are major players in the carbon cycle in forest ecosystems due to the wide range of interactions they have with plants either through soil degradation processes by litter decayers or biotrophic interactions with pathogenic and ectomycorrhizal symbionts. Secretion of fungal proteins mediates these interactions by allowing the fungus to interact with its environment and/or host. Ectomycorrhizal (ECM) symbiosis independently appeared several times throughout evolution and involves approximately 80% of trees. Despite extensive physiological studies on ECM symbionts, little is known about the composition and specificities of their secretomes. In this study, we used a bioinformatics pipeline to predict andmore » analyze the secretomes of 49 fungal species, including 11 ECM fungi, wood and soil decayers and pathogenic fungi to tackle the following questions: (1) Are there differences between the secretomes of saprophytic and ECM fungi? (2) Are small-secreted proteins (SSPs) more abundant in biotrophic fungi than in saprophytic fungi? and (3) Are there SSPs shared between ECM, saprotrophic and pathogenic fungi? We showed that the number of predicted secreted proteins is similar in the surveyed species, independently of their lifestyle. The secretome from ECM fungi is characterized by a restricted number of secreted CAZymes, but their repertoires of secreted proteases and lipases are similar to those of saprotrophic fungi. Focusing on SSPs, we showed that the secretome of ECM fungi is enriched in SSPs compared with other species. Most of the SSPs are coded by orphan genes with no known PFAM domain or similarities to known sequences in databases. Finally, based on the clustering analysis, we identified shared- and lifestyle-specific SSPs between saprotrophic and ECM fungi. The presence of SSPs is not limited to fungi interacting with living plants as the genome of saprotrophic fungi also code for numerous SSPs. As a result, ECM fungi shared lifestyle-specific SSPs

  5. Comparative analysis of secretomes from Ectomycorrhizal fungi with an emphasis on small-secreted proteins

    SciTech Connect

    Pellegrin, Clement; Morin, Emmanuelle; Martin, Francis M.; Veneault-Fourrey, Claire

    2015-11-18

    Fungi are major players in the carbon cycle in forest ecosystems due to the wide range of interactions they have with plants either through soil degradation processes by litter decayers or biotrophic interactions with pathogenic and ectomycorrhizal symbionts. Secretion of fungal proteins mediates these interactions by allowing the fungus to interact with its environment and/or host. Ectomycorrhizal (ECM) symbiosis independently appeared several times throughout evolution and involves approximately 80% of trees. Despite extensive physiological studies on ECM symbionts, little is known about the composition and specificities of their secretomes. In this study, we used a bioinformatics pipeline to predict and analyze the secretomes of 49 fungal species, including 11 ECM fungi, wood and soil decayers and pathogenic fungi to tackle the following questions: (1) Are there differences between the secretomes of saprophytic and ECM fungi? (2) Are small-secreted proteins (SSPs) more abundant in biotrophic fungi than in saprophytic fungi? and (3) Are there SSPs shared between ECM, saprotrophic and pathogenic fungi? We showed that the number of predicted secreted proteins is similar in the surveyed species, independently of their lifestyle. The secretome from ECM fungi is characterized by a restricted number of secreted CAZymes, but their repertoires of secreted proteases and lipases are similar to those of saprotrophic fungi. Focusing on SSPs, we showed that the secretome of ECM fungi is enriched in SSPs compared with other species. Most of the SSPs are coded by orphan genes with no known PFAM domain or similarities to known sequences in databases. Finally, based on the clustering analysis, we identified shared- and lifestyle-specific SSPs between saprotrophic and ECM fungi. The presence of SSPs is not limited to fungi interacting with living plants as the genome of saprotrophic fungi also code for numerous SSPs. As a result, ECM fungi shared lifestyle-specific SSPs likely

  6. Comparative Analysis of Secretomes from Ectomycorrhizal Fungi with an Emphasis on Small-Secreted Proteins.

    PubMed

    Pellegrin, Clement; Morin, Emmanuelle; Martin, Francis M; Veneault-Fourrey, Claire

    2015-01-01

    Fungi are major players in the carbon cycle in forest ecosystems due to the wide range of interactions they have with plants either through soil degradation processes by litter decayers or biotrophic interactions with pathogenic and ectomycorrhizal symbionts. Secretion of fungal proteins mediates these interactions by allowing the fungus to interact with its environment and/or host. Ectomycorrhizal (ECM) symbiosis independently appeared several times throughout evolution and involves approximately 80% of trees. Despite extensive physiological studies on ECM symbionts, little is known about the composition and specificities of their secretomes. In this study, we used a bioinformatics pipeline to predict and analyze the secretomes of 49 fungal species, including 11 ECM fungi, wood and soil decayers and pathogenic fungi to tackle the following questions: (1) Are there differences between the secretomes of saprophytic and ECM fungi? (2) Are small-secreted proteins (SSPs) more abundant in biotrophic fungi than in saprophytic fungi? and (3) Are there SSPs shared between ECM, saprotrophic and pathogenic fungi? We showed that the number of predicted secreted proteins is similar in the surveyed species, independently of their lifestyle. The secretome from ECM fungi is characterized by a restricted number of secreted CAZymes, but their repertoires of secreted proteases and lipases are similar to those of saprotrophic fungi. Focusing on SSPs, we showed that the secretome of ECM fungi is enriched in SSPs compared with other species. Most of the SSPs are coded by orphan genes with no known PFAM domain or similarities to known sequences in databases. Finally, based on the clustering analysis, we identified shared- and lifestyle-specific SSPs between saprotrophic and ECM fungi. The presence of SSPs is not limited to fungi interacting with living plants as the genome of saprotrophic fungi also code for numerous SSPs. ECM fungi shared lifestyle-specific SSPs likely involved in

  7. Comparative Analysis of Secretomes from Ectomycorrhizal Fungi with an Emphasis on Small-Secreted Proteins

    PubMed Central

    Pellegrin, Clement; Morin, Emmanuelle; Martin, Francis M.; Veneault-Fourrey, Claire

    2015-01-01

    Fungi are major players in the carbon cycle in forest ecosystems due to the wide range of interactions they have with plants either through soil degradation processes by litter decayers or biotrophic interactions with pathogenic and ectomycorrhizal symbionts. Secretion of fungal proteins mediates these interactions by allowing the fungus to interact with its environment and/or host. Ectomycorrhizal (ECM) symbiosis independently appeared several times throughout evolution and involves approximately 80% of trees. Despite extensive physiological studies on ECM symbionts, little is known about the composition and specificities of their secretomes. In this study, we used a bioinformatics pipeline to predict and analyze the secretomes of 49 fungal species, including 11 ECM fungi, wood and soil decayers and pathogenic fungi to tackle the following questions: (1) Are there differences between the secretomes of saprophytic and ECM fungi? (2) Are small-secreted proteins (SSPs) more abundant in biotrophic fungi than in saprophytic fungi? and (3) Are there SSPs shared between ECM, saprotrophic and pathogenic fungi? We showed that the number of predicted secreted proteins is similar in the surveyed species, independently of their lifestyle. The secretome from ECM fungi is characterized by a restricted number of secreted CAZymes, but their repertoires of secreted proteases and lipases are similar to those of saprotrophic fungi. Focusing on SSPs, we showed that the secretome of ECM fungi is enriched in SSPs compared with other species. Most of the SSPs are coded by orphan genes with no known PFAM domain or similarities to known sequences in databases. Finally, based on the clustering analysis, we identified shared- and lifestyle-specific SSPs between saprotrophic and ECM fungi. The presence of SSPs is not limited to fungi interacting with living plants as the genome of saprotrophic fungi also code for numerous SSPs. ECM fungi shared lifestyle-specific SSPs likely involved in

  8. Use of a Novel Report Protein to Study the Secretion Signal of Flagellin in Bacillus subtilis.

    PubMed

    Wang, Guangqiang; Xia, Yongjun; Xiong, Zhiqiang; Zhang, Hui; Ai, Lianzhong

    2016-08-01

    Flagellin (also called Hag) is the main component of bacterial flagellum and is transported across the cytoplasmic membrane by flagellar secretion apparatus. Because flagella play an essential role in the pathogenesis of numerous pathogens, the flagellins of Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Campylobacter jejuni, and Vibrio cholerae have been intensively studied; however, very few studies have focused on the flagellin of Bacillus subtilis, which is considered to be a model organism with which to study the secretion of bacteria and is used on an industrial scale for the secretion of proteins. The signal of B. subtilis flagellin is still debated. This study was performed to seek the export signals of flagellin from B. subtilis. The naturally nonsecretory, intrinsically disordered domain of nucleoskeletal-like protein (Nsp) was used as the reporter protein. Our results demonstrate that the export signal is contained within the first 50 amino acids of B. subtilis flagellin. Nsp is easily degraded inside the cell and can be exported into culture medium with the aid of the signal of flagellin. This method provides a new potential strategy for the expression of proteins with high proteolytic susceptibility via fusion to export signals. PMID:27154466

  9. Bacteria may induce the secretion of mucin-like proteins by the diatom Phaeodactylum tricornutum.

    PubMed

    Buhmann, Matthias T; Schulze, Birgit; Förderer, Alexander; Schleheck, David; Kroth, Peter G

    2016-06-01

    Benthic diatoms live in photoautotrophic/heterotrophic biofilm communities embedded in a matrix of secreted extracellular polymeric substances. Closely associated bacteria influence their growth, aggregation, and secretion of exopolymers. We have studied a diatom/bacteria model community, in which a marine Roseobacter strain is able to grow with secreted diatom exopolymers as a sole source of carbon. The strain influences the aggregation of Phaeodactylum tricornutum by inducing a morphotypic transition from planktonic, fusiform cells to benthic, oval cells. Analysis of the extracellular soluble proteome of P. tricornutum in the presence and absence of bacteria revealed constitutively expressed newly identified proteins with mucin-like domains that appear to be typical for extracellular diatom proteins. In contrast to mucins, the proline-, serine-, threonine-rich (PST) domains in these proteins were also found in combination with protease-, glucosidase- and leucine-rich repeat-domains. Bioinformatic functional predictions indicate that several of these newly identified diatom-specific proteins may be involved in algal defense, intercellular signaling, and aggregation. PMID:26993172

  10. Detection of secreted and temporarily inducible heat shock responsive proteins in mouse testicular tissue

    SciTech Connect

    Lemaire, L.; Heinlein, U.A.O. )

    1991-01-01

    Temperature-induced effects on the synthesis of murine testicular proteins were investigated by one- and two-dimensional SDS polyacrylamide gel electrophoresis. Newly synthesized proteins were monitored by incorporation of {sup 35}S-methionine and autoradiography. Three heat shock responsive proteins, which are differently affected by elevated temperatures, are described. These proteins represent special examples for how testicular cells respond to environmental stress. One of these proteins, HS136, is synthesized and secreted at 38{degree}C, whereas at lower, scrotal temperatures it is not detectable. HSID74 protein is synthesized at elevated temperatures, but only in prepuberal testis, not in adult. Synthesis of the third example, HSR28, is decreased within the seminiferous tubules, but only in those regions which bear cell associations of the elongation stage. These results indicate that the use of DNA probes of the heat shock-gene family might not be sufficient to describe the molecular reasons for impaired spermatogenesis following hyperthermia.

  11. Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant path...

  12. NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip-translocon protein-protein interactions.

    PubMed

    McShan, Andrew C; Kaur, Kawaljit; Chatterjee, Srirupa; Knight, Kevin M; De Guzman, Roberto N

    2016-08-01

    The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein-protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097-1107. © 2016 Wiley Periodicals, Inc. PMID:27093649

  13. Multiple Driving Forces Required for Efficient Secretion of Autotransporter Virulence Proteins*

    PubMed Central

    Drobnak, Igor; Braselmann, Esther; Clark, Patricia L.

    2015-01-01

    Autotransporter (AT) proteins are a broad class of virulence proteins from Gram-negative bacterial pathogens that require their own C-terminal transmembrane domain to translocate their N-terminal passenger across the bacterial outer membrane (OM). But given the unavailability of ATP or a proton gradient across the OM, it is unknown what energy source(s) drives this process. Here we used a combination of computational and experimental approaches to quantitatively compare proposed AT OM translocation mechanisms. We show directly for the first time that when translocation was blocked an AT passenger remained unfolded in the periplasm. We demonstrate that AT secretion is a kinetically controlled, non-equilibrium process coupled to folding of the passenger and propose a model connecting passenger conformation to secretion kinetics. These results reconcile seemingly contradictory reports regarding the importance of passenger folding as a driving force for OM translocation but also reveal that another energy source is required to initiate translocation. PMID:25670852

  14. A Gram-Negative Bacterial Secreted Protein Types Prediction Method Based on PSI-BLAST Profile

    PubMed Central

    2016-01-01

    Prediction of secreted protein types based solely on sequence data remains to be a challenging problem. In this study, we extract the long-range correlation information and linear correlation information from position-specific score matrix (PSSM). A total of 6800 features are extracted at 17 different gaps; then, 309 features are selected by a filter feature selection method based on the training set. To verify the performance of our method, jackknife and independent dataset tests are performed on the test set and the reported overall accuracies are 93.60% and 100%, respectively. Comparison of our results with the existing method shows that our method provides the favorable performance for secreted protein type prediction. PMID:27563663

  15. Enhancement of protein secretion in Saccharomyces cerevisiae by overproduction of Sso protein, a late-acting component of the secretory machinery.

    PubMed

    Ruohonen, L; Toikkanen, J; Tieaho, V; Outola, M; Soderlund, H; Keranen, S

    1997-03-30

    Increased production of secreted proteins in Saccharomyces cerevisiae was achieved by overexpressing the yeast syntaxins. Sso1 or Sso2 protein, the t-SNAREs functioning at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane. Up to four- or six-fold yields of a heterologous secreted protein, Bacillus alpha-amylase, or an endogenous secreted protein, invertase, were obtained respectively when expressing either one of the SSO genes, SSO1 or SSO2, from the ADH1 promoter on a multicopy plasmid. Direct correlation between the Sso protein level and the amount of secreted alpha-amylase was demonstrated by modulating the expression level of the SSO2 gene. Quantitation of the alpha-amylase activity in the culture medium, periplasmic space and cytoplasm suggests that secretion into the periplasmic space is the primary stage at which the SSO genes exert the secretion-enhancing function. Pulse-chase data also support enhanced secretion efficiently obtained by SSO overexpression. Our data suggest that the Sso proteins may be rate-limiting components of the protein secretion machinery at the exocytosis step in yeast. PMID:9133737

  16. Virulence Plasmid of Rhodococcus equi Contains Inducible Gene Family Encoding Secreted Proteins

    PubMed Central

    Byrne, Barbara A.; Prescott, John F.; Palmer, Guy H.; Takai, Shinji; Nicholson, Vivian M.; Alperin, Debra C.; Hines, Stephen A.

    2001-01-01

    Rhodococcus equi causes severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains of R. equi bear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15- to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the same R. equi isolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid, vapC, -D, and -E, that are tandemly arranged downstream of vapA. These new genes are members of a gene family and encode proteins that are approximately 50% homologous to VapA, VapB, and each other. vapC, -D, and -E are found only in R. equi strains that express VapA and are highly conserved in VapA-positive isolates from both horses and humans. VapC, -D, and -E are secreted proteins coordinately regulated by temperature with VapA; the proteins are expressed when R. equi is cultured at 37°C but not at 30°C, a finding that is compatible with a role in virulence. As secreted proteins, VapC, -D, and -E may represent targets for the prevention of rhodococcal pneumonia. An immunologic study using VapA-specific antibodies and recombinant Vap proteins revealed no evidence of cross-reactivity despite extensive sequence similarity over the carboxy terminus of all four proteins. PMID:11159951

  17. Secretion of curli fibre subunits is mediated by the outer membrane-localized CsgG protein

    PubMed Central

    Robinson, Lloyd S.; Ashman, Elisabeth M.; Hultgren, Scott J.; Chapman, Matthew R.

    2010-01-01

    Summary Produced by many Enterobacteriaceae spp., curli are biologically important amyloid fibres that have been associated with biofilm formation, host cell adhesion and invasion, and immune system activation. CsgA is the major fibre subunit and CsgE, CsgF and CsgG are non-structural proteins involved in curli biogenesis. We have characterized the role of CsgG in curli subunit secretion across the outer membrane. Directed mutagenesis of CsgG confirmed that its activity is dependent on localization to the outer membrane. Rotary Shadow electron microscopy of purified CsgG suggested that this protein assembles into an oligomeric complex with an apparent central pore. Oligomeric CsgG complexes were confirmed using co-purification experiments. Antibiotic sensitivity assays demonstrated that overexpression of CsgG rendered Escherichia coli susceptible to the antibiotic erythromycin. A 22-amino-acid sequence at the N-terminus of CsgA was sufficient to direct heterologous proteins to the CsgG secretion apparatus. Finally, we determined that CsgG participates in an outer membrane complex with two other curli assembly proteins, CsgE and CsgF. PMID:16420357

  18. Temperature- and medium-dependent secretion of proteins by Shiga toxin-producing Escherichia coli.

    PubMed Central

    Ebel, F; Deibel, C; Kresse, A U; Guzmán, C A; Chakraborty, T

    1996-01-01

    Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal disease in humans and livestock, and these bacteria have recently emerged as a leading cause of renal failure in children. In this study, we have examined medium- and temperature-dependent production of secreted proteins from a STEC O26 serotype strain. Growth of bacteria in Luria broth led to the detection of secreted polypeptides of 104, 55, 54, and 37 kDa (p104, p55, p54, and p37, respectively). When grown in serum-free tissue culture medium, only p104, p37 and two additional polypeptides of 25 and 22 kDa (p25 and p22) were present in supernatant fluids. Production of these polypeptides was growth temperature dependent and induced in cultures grown at 37 degrees C. N-terminal amino acid sequencing revealed that p104 was homologous to the secreted p110 of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a family of secreted proteins in pathogenic bacteria of which the immunoglobulin A protease of Neisseria gonorrhoeae is the prototype. The N-terminal amino acid sequences of p55 and p54 were unique to the STEC strain, while p37 and p25 were found to be highly homologous to the similarly sized EspA and EspB proteins, previously detected in culture supernatants of EPEC. Molecular cloning and sequencing of STEC espB alleles from two different serotypes showed that the encoded polypeptides were about 80% homologous. A monoclonal antibody raised against STEC EspB also cross-reacted with its EPEC analog and allowed us to demonstrate medium- and temperature-dependent production of this important virulence factor in STEC and EPEC strains of differing serotypes. PMID:8890194

  19. Keratinocyte-Secreted Heat Shock Protein-90alpha: Leading Wound Reepithelialization and Closure

    PubMed Central

    Bhatia, Ayesha; O'Brien, Kathryn; Chen, Mei; Woodley, David T.; Li, Wei

    2016-01-01

    Significance: Delayed and nonhealing wounds pose a health, economic, and social problem worldwide. For decades, the conventional wisdom pointed to growth factors as the driving force of wound healing and granted them a center stage for therapeutic development. To date, few have obtained US FDA approvals or shown clinical effectiveness and safety. Critical Issue: Wound closure is the initial and most critical step during wound healing. Closing chronic wounds to shut down continued infection is the primary and likely the only achievable goal at the clinic in the foreseeable future. The critical question here is to identify the factor(s) in wounded tissues that drives the initial wound closure. Recent Advances: We made an unexpected discovery of the secreted form of heat shock protein-90alpha (Hsp90α) for promoting skin cell motility, reepithelialization, and wound closure. Secreted Hsp90α possesses unique properties to remain functional under the hostile wound environment that compromises conventional growth factors' effectiveness. Through the common lipoprotein receptor-related protein-1 cell surface receptor and activation of the Akt signaling pathway, topical application of human recombinant Hsp90α protein greatly accelerates excision, burn, and diabetic skin wound closure in rodent and porcine models. Future Directions: In almost all cells, the 2–3% of their total proteins (∼7,000 per cell) are Hsp90 (α and β), a long unraveled puzzle. Our new finding of Hsp90 secretion in wounded tissues suggests that the stockpile of Hsp90α by all cells is to rapidly supply the need for extracellular Hsp90α to repair damaged tissues. We propose that keratinocytes at the wound edge secrete Hsp90α that leads the reepithelialization process to close the wound. PMID:27076995

  20. Small G proteins as key regulators of pancreatic digestive enzyme secretion

    PubMed Central

    Williams, John A.; Chen, Xuequn; Sabbatini, Maria E.

    2009-01-01

    Small GTP-binding (G) proteins act as molecular switches to regulate a number of cellular processes, including vesicular transport. Emerging evidence indicates that small G proteins regulate a number of steps in the secretion of pancreatic acinar cells. Diverse small G proteins have been localized at discrete compartments along the secretory pathway and particularly on the secretory granule. Rab3D, Rab27B, and Rap1 are present on the granule membrane and play a role in the steps leading up to exocytosis. Whether the function of these G proteins is simply to ensure appropriate targeting or if they are involved as regulatory molecules is discussed. Most evidence suggests that Rab3D and Rab27B play a role in tethering the secretory granule to its target membrane. Other Rabs have been identified on the secretory granule that are associated with different steps in the secretory pathway. The Rho family small G proteins RhoA and Rac1 also regulate secretion through remodeling of the actin cytoskeleton. Possible mechanisms for regulation of these G proteins and their effector molecules are considered. PMID:19088252

  1. Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate

    PubMed Central

    Cooley, Richard B.; Smith, T. Jarrod; Leung, Wilfred; Tierney, Valerie; Borlee, Bradley R.; O'Toole, George A.

    2015-01-01

    ABSTRACT We previously identified a second-messenger-regulated signaling system in the environmental bacterium Pseudomonas fluorescens which controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogen Pseudomonas aeruginosa harbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identified P. aeruginosa CdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen. IMPORTANCE Here, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen Pseudomonas aeruginosa is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger. PMID:26100041

  2. Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata.

    PubMed

    Hatano, Naoya; Hamada, Tatsuro

    2012-08-01

    The Nepenthes species are carnivorous plants that have evolved a specialized leaf organ, the 'pitcher', to attract, capture, and digest insects. The digested insects provide nutrients for growth, allowing these plants to grow even in poor soil. Several proteins have been identified in the pitcher fluid, including aspartic proteases (nepenthesin I and II) and pathogenesis-related (PR) proteins (β-1,3-glucanase, class IV chitinase, and thaumatin-like protein). In this study, we collected and concentrated pitcher fluid to identify minor proteins. In addition, we tried to identify the protein secreted in response to trapping the insect. To make a similar situation in which the insect falls into the pitcher, chitin which was a major component of the insect exoskeleton was added to the fluid in the pitcher. Three PR proteins, class III peroxidase (Prx), β-1,3-glucanase, and class III chitinase, were newly identified. Prx was induced after the addition of chitin to the pitcher fluid. Proteins in the pitcher fluid of the carnivorous plant Nepenthes alata probably have two roles in nutrient supply: digestion of prey and the antibacterial effect. These results suggest that the system for digesting prey has evolved from the defense system against pathogens in the carnivorous plant Nepenthes. PMID:22705321

  3. Crystal structure of Spa40, the specificity switch for the Shigella flexneri type III secretion system

    PubMed Central

    Deane, Janet E; Graham, Stephen C; Mitchell, Edward P; Flot, David; Johnson, Steven; Lea, Susan M

    2008-01-01

    The pathogenic bacterium Shigella flexneri uses a type III secretion system to inject virulence factors from the bacterial cytosol directly into host cells. The machinery that identifies secretion substrates and controls the export of extracellular components and effector proteins consists of several inner-membrane and cytoplasmic proteins. One of the inner membrane components, Spa40, belongs to a family of proteins proposed to regulate the switching of substrate specificity of the export apparatus. We show that Spa40 is cleaved within the strictly conserved amino acid sequence NPTH and substitution of the proposed autocatalytic residue abolishes cleavage. Here we also report the crystal structure of the cytoplasmic complex Spa40C and compare it with the recent structures of the homologues from Escherichia coli and Salmonella typhimurium. These structures reveal the tight association of the cleaved fragments and show that the conserved NPTH sequence lies on a loop which, when cleaved, swings away from the catalytic N257 residue, resulting in different surface features in this region. This structural rearrangement suggests a mechanism by which non-cleaving forms of these proteins interfere with correct substrate switching of the apparatus. PMID:18485071

  4. Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots.

    PubMed

    Huet, Yoann; Ekouna, Jean-Pierre Ele; Caron, Aurore; Mezreb, Katiba; Boitel-Conti, Michèle; Guerineau, François

    2014-01-01

    A fully contained and efficient heterologous protein production system was designed using Brassica rapa rapa (turnip) hairy roots. Two expression cassettes containing a cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region, an Arabidopsis thaliana sequence encoding a signal peptide and the CaMV polyadenylation signal were constructed. One cassette was used to express the green fluorescent protein (GFP)-encoding gene in hairy roots grown in flasks. A stable and fast-growing hairy root line secreted GFP at >120 mg/l culture medium. GFP represented 60 % of the total soluble proteins in the culture medium. Turnip hairy roots retained sustainable growth and stable GFP production over 3 years. These results were superior to those obtained using tobacco hairy roots. PMID:24078130

  5. On the presence of prostatic secretion protein in rat seminal fluid

    SciTech Connect

    Borgstroem, E.; Pousette, A.; Bjoerk, P.; Hoegberg, B.; Carlstroem, K.; Sundelin, B.; Gustafsson, J.A.

    1981-01-01

    The copulating plug collected from the tip of the penis from rats immediately after decapitation contains a protein very similar and probably identical to PSP (prostatic secretion protein); this protein has earlier been purified from rat prostatic cytosol and characterized. The protein present in the copulating plug interacts with (3H)estramustine and binds to the antibody raised against rat PSP. The concentration of the protein in the copulating plug is 400 ng/mg of total protein, when measured using the radioimmunoassay technique developed earlier for measurement of PSP in rat prostate. The (3H)estramustine-protein complex formed in a preparation of the copulating plug has an apparent molecular weight of about 50,000 and a sedimentation coefficient of about 3S when analyzed using sucrose density gradient centrifugation. The complex was retained on Concanavalin-A Sepharose indicating that the protein is a glycoprotein. Binding of the complex was also observed on hydroxylapatite and DEAE-Sephadex columns, from which it was eluted at 0.18 M KCl. Light microscope autoradiograms of rat sperms incubated with 125I-labeled PSP indicated that PSP is bound to all parts of the sperms. A macromolecule interacting with the PSP-antibodies is also present in human seminal fluid but at a concentration considerably lower than in rat seminal fluid. The present study shows that a macromolecule probably identical to prostatic secretion protein is present in the copulating plug from the rat. The biological role of this protein in normal male fertility is discussed.

  6. Isolation and identification of histone H3 protein enriched in microvesicles secreted from cultured sebocytes.

    PubMed

    Nagai, Ayako; Sato, Takashi; Akimoto, Noriko; Ito, Akira; Sumida, Michihiro

    2005-06-01

    Secretion of microvesicles, defined as sebosomes, containing lipid particles were discovered for the first time in cultured sebocytes. After reaching confluency, hamster-cloned sebocytes released bubble-like microvesicles with a diameter range of 0.5-5.0 microm. They had a complex structure containing multiple Oil Red O-stainable particles. The lipid components of the microvesicles were large amounts of squalene both of hamster-cloned and rat primary cultured sebocytes. The microvesicles contained a concentrated 17-kDa cationic protein, which was soluble in sulfate buffer including Nonidet P-40 at pH 1.5. As the protein bound tightly to heparin-Sepharose and eluted with 1.5 M NaCl, it was further purified from a SDS-PAGE gel. Peptide sequencing identified the protein to be histone H3. Polyclonal antibodies against the purified protein detected the antigen in the microvesicles both in the hamster-cloned and rat primary cultured sebocytes. The antibodies demonstrated a distribution of the protein within the nucleus, cytoplasm, and precursor microvesicles. When a gene construct encoding histone H3-enhanced green fluorescent protein was transfected to the sebocytes, fluorescence of the fusion proteins was detected within both the nucleus and the precursor microvesicles of the cytoplasm. The distribution of heparan sulfate was evident in the microvesicles, and it suggested the possibility that the histone H3 protein was recruited and then condensed to the secreted microvesicles by the molecules. In addition, the 14-3-3 protein, which was detected in the microvesicles, also may help incorporate the histone H3 protein in the microvesicles because it can bind to both histone and lipid particles. PMID:15746254

  7. The Signal Peptide of a Vacuolar Protein Is Necessary and Sufficient for the Efficient Secretion of a Cytosolic Protein 1

    PubMed Central

    Hunt, Dale C.; Chrispeels, Maarten J.

    1991-01-01

    A cytosolic pea (Pisum sativum) seed albumin (ALB) and a chimeric protein (PHALB) consisting of the signal peptide and first three amino acids of phytohemagglutinin (PHA) and the amino acid sequence of ALB were expressed in parallel suspension cultures of tobacco (Nicotiana tabacum) cells and their intracellular fates examined. PHALB was efficiently secreted by the cells whereas ALB remained intracellular. These experiments show that the information contained in the signal peptide of a vacuolar protein is both necessary and sufficient for efficient secretion, and define secretion as a default or bulk-flow pathway. Entry into the secretory pathway was accompanied by glycosylation and the efficient conversion of the high mannose glycans into complex glycans indicating that transported glycoproteins do not need specific recognition domains for the modifying enzymes in the Golgi. Tunicamycin depressed the accumulation of the unglycosylated polypeptide in the culture medium much less than the accumulation of other glycoproteins. We interpret this as evidence that glycans on proteins that are not normally glycosylated do not have the same function of stabilizing and protecting the polypeptide as on natural glycoproteins. ImagesFigure 2Figure 3Figure 5Figure 6Figure 7Figure 8 PMID:16668149

  8. Unveiling Cell Surface and Type IV Secretion Proteins Responsible for Archaeal Rudivirus Entry

    PubMed Central

    Deng, Ling; He, Fei; Bhoobalan-Chitty, Yuvaraj; Martinez-Alvarez, Laura; Guo, Yang

    2014-01-01

    Sulfolobus mutants resistant to archaeal lytic virus Sulfolobus islandicus rod-shaped virus 2 (SIRV2) were isolated, and mutations were identified in two gene clusters, cluster sso3138 to sso3141 and cluster sso2386 and sso2387, encoding cell surface and type IV secretion proteins, respectively. The involvement of the mutations in the resistance was confirmed by genetic complementation. Blocking of virus entry into the mutants was demonstrated by the lack of early gene transcription, strongly supporting the idea of a role of the proteins in SIRV2 entry. PMID:24965447

  9. Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain

    PubMed Central

    Cooley, Christina B.; Ryno, Lisa M.; Plate, Lars; Morgan, Gareth J.; Hulleman, John D.; Kelly, Jeffery W.; Wiseman, R. Luke

    2014-01-01

    Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation. PMID:25157167

  10. Effect of honokiol on exotoxin proteins listeriolysin O and p60 secreted by Listeria monocytogenes.

    PubMed

    Meng, Rizeng; Zhao, Ziwen; Guo, Na; Liu, Zonghui; Zhao, Xingchen; Li, Wenli; Li, Xiaoxu; Shi, Ce; Nie, Dandan; Wang, Weilin; Liu, Tao; Ma, Wenchen; Yu, Lu; Li, Juan

    2015-12-01

    Listeria monocytogenes is considered one of the most important foodborne pathogens. The virulence-related proteins listeriolysin O (LLO) and p60 are critical factors involved in Listeria pathogenesis. In the present study, we investigated the effect of honokiol on LLO and p60 secreted from L. monocytogenes. A listeriolysin assay was used to investigate the haemolytic activities of L. monocytogenes exposed to honokiol, and the secretion of LLO and p60 was detected by immunoblot analysis. Additionally, the influence of honokiol on the transcription of LLO and p60 genes (hly and iap, respectively) was analysed by real-time reverse transcription PCR. TNF-α release assays were performed to elucidate the biological relevance of changes in LLO and p60 secretion induced by honokiol. According to the data, honokiol showed good anti-L. monocytogenes activity, with MICs of 8-16 μg ml(-1), and the secretion of LLO and p60 was decreased by honokiol. In addition, the transcription of hly and iap was inhibited by honokiol. Our results indicate that TNF-α production by RAW264.7 cells stimulated with L. monocytogenes supernatants was inhibited by honokiol. Based on these data, we propose that honokiol could be used as a promising natural compound against L. monocytogenes and its virulence factors. PMID:26445991

  11. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins.

    PubMed Central

    Andersen, P

    1994-01-01

    An experimental vaccine that was based on secreted proteins of Mycobacterium tuberculosis was investigated in a mouse model of tuberculosis. I used a short-term culture filtrate (ST-CF) containing proteins secreted from actively replicating bacteria grown under defined culture conditions. The immunogenicity of the ST-CF was investigated in combination with different adjuvants, and peak proliferative responses were observed when ST-CF was administered with the surface-active agent dimethyldioctadecylammonium chloride. The immunity induced by this vaccine was dose dependent, and, in the optimal concentration, the vaccine induced a potent T-helper 1 response which efficiently protected the animals against a subsequent challenge with virulent M. tuberculosis. Antigenic targets for the T cells generated were mapped by employing narrow-molecular-weight fractions of ST-CF. The experimental vaccine primed a broadly defined T-cell repertoire directed to multiple secreted antigens present in ST-CF. A vaccination with viable Mycobacterium bovis bacillus Calmette-Guérin (BCG), in contrast, induced a restricted T-cell reactivity directed to two secreted protein fractions with molecular masses of 5 to 12 and 25 to 35 kDa. The protective efficacy of the ST-CF vaccine was compared with that of a BCG standard vaccine, and both induced a highly significant protection of equal magnitude. The vaccination with ST-CF gave rise to a population of long-lived CD4 cells which could be isolated 22 weeks after the vaccination and could adoptively transfer acquired resistance to T-cell-deficient recipients. My results confirm the hypothesis that M. tuberculosis cells release protective antigens during growth. The high efficacy of a subunit vaccine observed in the present study is discussed as a possible alternative to a live recombinant vaccine carrier. Images PMID:7910595

  12. Proteins Secreted By Embryonic Stem Cells Activate Cardiomyocytes Through Ligand Binding Pathways

    PubMed Central

    LaFramboise, W. A.; Petrosko, P.; Krill-Burger, J. M.; Morris, D. R.; McCoy, A. R.; Scalise, D.; Malehorn, D. E.; Guthrie, R. D.; Becich, M. J.; Dhir, R.

    2010-01-01

    Human embryonic stem cells (hESC) underly embryogenesis but paracrine signals associated with the process are unknown. This study was designed to 1) profile native proteins secreted by undifferentiated hESC and 2) determine their biological effects on primary neonatal cardiomyocytes. We utilized multi-analyte, immunochemical assays to characterize media conditioned by undifferentiated hESC versus unconditioned media. Expression profiling was performed on cardiomyocytes subjected to these different media conditions and altered transcripts were mapped to critical pathways. Thirty-two of 109 proteins were significantly elevated in conditioned media ranging in concentration from thrombospondin (57.2 ± 5.0 ng/ml) to nerve growth factor (7.4 ± 1.2 pg/ml) and comprising chemokines, cytokines, growth factors, and proteins involved in cell adhesion and extracellular matrix remodeling. Conditioned media induced karyokinesis, cytokinesis and proliferation in mono- and binucleate cardiomyocytes. Pathway analysis revealed comprehensive activation of the ROCK 1 and 2 G-protein coupled receptor (GPCR) pathway associated with cytokinesis, and the RAS/RAF/MEK/ERK receptor tyrosine kinase (RTK) and JAK/STAT-cytokine pathway involved in cell cycle progression. These results provide a partial database of proteins secreted by pluripotent hESC that potentiate cell division in cardiomyocytes via a paracrine mechanism suggesting a potential role for these stem cell factors in cardiogenesis and cardiac repair. PMID:20045494

  13. Roles of STAT3 in Protein Secretion Pathways during the Acute-Phase Response

    PubMed Central

    Ahyi, Ayele-Nati N.; Quinton, Lee J.; Jones, Matthew R.; Ferrari, Joseph D.; Pepper-Cunningham, Zachary A.; Mella, Juan R.; Remick, Daniel G.

    2013-01-01

    The acute-phase response is characteristic of perhaps all infections, including bacterial pneumonia. In conjunction with the acute-phase response, additional biological pathways are induced in the liver and are dependent on the transcription factors STAT3 and NF-κB, but these responses are poorly understood. Here, we demonstrate that pneumococcal pneumonia and other severe infections increase expression of multiple components of the cellular secretory machinery in the mouse liver, including the endoplasmic reticulum (ER) translocon complex, which mediates protein translation into the ER, and the coat protein complexes (COPI and COPII), which mediate vesicular transport of proteins to and from the ER. Hepatocyte-specific mutation of STAT3 prevented the induction of these secretory pathways during pneumonia, with similar results observed following pharmacological activation of ER stress by using tunicamycin. These findings implicate STAT3 in the unfolded protein response and suggest that STAT3-dependent optimization of secretion may apply broadly. Pneumonia also stimulated the binding of phosphorylated STAT3 to promoter regions of secretion-related genes in the liver, supporting a direct role for STAT3 in their transcription. Altogether, these results identify a novel function of STAT3 during the acute-phase response, namely, the induction of secretory machinery in hepatocytes. This may facilitate the processing and delivery of newly synthesized loads of acute-phase proteins, enhancing innate immunity and preventing liver injury during infection. PMID:23460517

  14. Expression of Cryptosporidium parvum Cpa135/CpCCP1 chimeras in Giardia duodenalis: organization of the protein domains affects the protein secretion pathway.

    PubMed

    Lalle, Marco; Rosati, Maria Adelaide; Bien, Justina; Hehl, Adrian B; Pozio, Edoardo; Tosini, Fabio

    2011-03-01

    Cpa135 is a multidomain antigenic protein secreted at the sporozoite stage of the Apicomplexa protozoan Cryptosporidium parvum. Previous studies have shown that the protozoan flagellate parasite Giardia duodenalis is a suitable system for the heterologous expression of secreted proteins of Apicomplexa. Here, we designed three different Cpa135 variants fused to a C-terminal HA tag in order to test their expression in G. duodenalis under the control of the inducible promoter of the cyst wall protein 1 gene (cwp1). The three Cpa135 chimeras encompassed different portions of the protein; CpaG encodes the entire polypeptide of 1574 amino acids (aa); CpaGΔC includes the first 826 aa at the N-terminus; and CpaGΔN consists in of the final 833 aa at the C-terminus. Immunoblot experiments showed that CpaG and CpaGΔN maintained the epitopes recognized by anti-C. parvum-specific human serum. The intracellular localization and transport of the three Cpa135 variants were studied by immunofluorescence in combination with G. duodenalis-specific antibodies. CpaGΔC was mainly accumulated in the endoplasmic reticulum and the intact form was also excreted in the medium. Differently, the Cpa135 chimeras possessing an intact C-terminus (CpaG and CpaGΔN) were transported towards the forming cyst wall possibly and were not detected in the medium. Furthermore, the full-length CpaG was incorporated into the cyst wall. The data presented suggest that the C-terminus of Cpa135, which includes a cysteine reach domain, could influence the secretion of the chimeric proteins. PMID:21112325

  15. Electrolyte and protein secretion by the perfused rabbit mandibular gland stimulated with acetylcholine or catecholamines

    PubMed Central

    Case, R. M.; Conigrave, A. D.; Novak, I.; Young, J. A.

    1980-01-01

    1. A method is described for the isolation and vascular perfusion in vitro of the mandibular gland of the rabbit. The perfusate is a physiological salt solution containing glucose as the only metabolic substrate. 2. During perfusion with solutions containing acetylcholine, the gland secretes vigorously at a rate and in a manner similar to that seen in vivo. Although the gland becomes oedematous during perfusion, the extent of this oedema appears to have no influence on secretory ability: the perfused glands were capable of functioning for at least 4 h, and often for more than 6 h. 3. Acetylcholine evoked a small secretory response at a concentration of 8 × 10-9 mol l-1 and a maximum response at 8 × 10-7 mol l-1. Eserine (2 × 10-5 mol l-1) evoked secretory responses comparable to those evoked by acetylcholine in a concentration of 8 × 10-9 mol l-1. Secretion, whether unstimulated or evoked by acetylcholine or eserine, could be blocked completely by atropine. 4. During prolonged stimulation with acetylcholine, the fluid secretory response declined rapidly over a period of about 15 min from an initial high value to a much lower plateau value. After 3 or more hours of stimulation, the secretory response began once more to decline, this time towards zero. If, before the second period of decline begins, stimulation is interrupted for about 30 min, the gland recovers its initial responsiveness to further stimulation with acetylcholine. 5. The Na, K, Cl and HCO3 co